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Allosteric targeting of receptor tyrosine kinases
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Frederik De Smet1,2, Arthur Christopoulos3 & Peter Carmeliet4,5 The drug discovery landscape has been transformed over the past decade by the discovery of allosteric modulators of all major mammalian receptor superfamilies. Allosteric ligands are a rich potential source of drugs and drug targets with clear therapeutic advantages. G protein–coupled receptors, ligand-gated ion channels and intracellular nuclear hormone receptors have all been targeted by allosteric modulators. More recently, a receptor tyrosine kinase (RTK) has been targeted by an extracellular small-molecule allosteric modulator. Allosteric mechanisms of structurally distinct molecules that target the various receptor families are more alike than originally anticipated and include selectivity, orthosteric probe dependence and pathway-biased signaling. Receptors are proteins that serve the dual roles of recognition of chemical or environmental stimuli and transduction of these stimuli to cellular responses. They can be embedded in the plasma membrane, or present in the cytosol or nucleus. Because of their widespread involvement in numerous physiological and pathological processes, it is not surprising that receptors have been successfully targeted by multiple drug-discovery programs1,2. Four major receptor classes have been identified; the membrane-bound cell surface G protein–coupled receptors (GPCRs), ligand-gated ion channels, enzyme-linked receptors (including the structurally related RTKs and phosphatases) and intracellular receptors (including the nuclear hormone receptors, which function as ligand-inducible transcription factors) (Fig. 1). About 70% of all currently available targeted drugs target cellular receptors3. Each class of receptors interacts with specific signaling molecules. Upon binding a ligand, the conformation of a receptor changes to activate or inhibit biochemical signaling pathways. This change in conformation was thought to be actively promoted by the ligand (conformational induction), but it is now known that most receptors are present as an ensemble of pre-existing states, and that the ligand preferentially stabilizes a state for which it has the higher affinity (conformational selection). The endogenous ligand-binding region 1The
Switch Laboratory, Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium. 2The Switch Laboratory, Flanders Institute for Biotechnology (VIB), Leuven, Belgium. 3Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia. 4Laboratory of Angiogenesis and Neurovascular link, Vesalius Research Center, Department of Oncology, University of Leuven, Leuven, Belgium. 5Laboratory of Angiogenesis and Neurovascular link, Vesalius Research Center, VIB, Leuven, Belgium. Correspondence should be addressed to P.C. ([email protected]). Received 9 December 2013; accepted 19 August 2014; published online 7 November 2014; doi:10.1038/nbt.3028
in the receptor that has been evolutionarily conserved to mediate this role is the ‘orthosteric site’4. The orthosteric site of some receptors has been identified using structural biophysics and mutational studies, but our understanding of the mechanisms of receptor activation by ligands remains limited. Structure-based design has been successful in the search for kinase inhibitors, but only modest progress has been achieved in designing drugs that target membrane proteins. Indeed, of the 77,000 highresolution crystal structures deposited in databases, less than 1% (~550) have been obtained for membrane proteins3, reflecting the technical limitations of solving structures of integral membrane proteins upon their removal from their native environment. Although three-dimensional structures are now becoming available for membrane-bound receptors5, most of our knowledge of the mechanisms underlying receptor function originates from the identification of various types of agonists (activators) and antagonists (inhibitors), which, when bound to their orthosteric binding sites, activate or inhibit specific downstream pathways. All receptors couple to signaling pathways in a cell context– dependent manner. Historically, it was believed that the differences in the ability of ligands to activate these pathways reflected the differences in the ‘intrinsic efficacy’ of each ligand, that is, the strength of the signal imparted to a given receptor. However, it is now known that the selection and magnitude of receptor-mediated responses is determined by the conformational state of the receptor when bound to a ligand, in addition to receptor concentration, coupling efficiency and the complement of cellular interacting partners. The stabilization of different active states by distinct ligands of a receptor in the same cellular background, leading to different functional outcomes, has been termed ‘biased agonism’ and is a mode of regulation that is being increasingly identified across receptor superfamilies6. Although most studies on receptor biology have focused on how ligands modulate receptor signaling through binding to the orthosteric site, receptor conformation and signaling can also be modified by ligands acting at topographically and spatially distinct allosteric sites (Fig. 1). The ability of one ligand (be it a small molecule or large protein) to change the binding and/or effects of a second ligand (small molecule, protein) at a target protein by means of an interaction with a nonoverlapping and spatially distant site is referred to as allostery. Allosteric interactions serve to fine-tune and regulate receptor function. Recent research has pinpointed allosteric interactions as a useful tool to modulate receptor function in ways that cannot be achieved by ligands that bind to an orthosteric site 5. In this review, we discuss the advantages and challenges of allosterically targeting receptors, focusing on RTKs as the ‘last bastion’ of receptor allostery.
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Figure 1 All superfamilies of cellular receptors can be allosterically modulated. Schematic representations of the activated forms of the four major cellular receptor classes, that is, ligand-gated ion channels, GPCRs, nuclear hormone receptors and RTKs. For each receptor, the nonoverlapping binding sites of the orthosteric ligand (green circle) and an allosteric ligand (orange triangle) are indicated. This comparison suggests that, although these receptors have different cellular functions, they share common features of allosteric modulation.
Allosteric receptor modulation Orthosteric ligands bind to an agonist-binding site, which initiates downstream receptor signaling. By contrast, allosteric ligands are usually structurally different from orthosteric ligands and bind to distinct sites that are spatially distant from the orthosteric site, to modulate the properties of orthosteric ligands (Fig. 2). Three types of allosteric modulators have been documented. First, ‘affinity modulators’ alter the orthosteric binding site by inducing a conformational change that alters the kinetics of ligand binding, and hence its binding affinity without changing the intrinsic signaling properties of the receptor. Second, ‘efficacy modulators’ induce a conformational change that is transmitted to regions of the receptor involved in the transduction of intracellular responses. This alters the signaling ability of the orthosteric ligand independent of, or in addition to, possible effects on orthosteric ligand affinity. Third, ‘allosteric agonists’ or ‘allosteric inverse agonists’ can perturb receptor signaling in the absence of an orthosteric ligand, in addition to any modulatory effects they have in the presence of orthosteric ligand7,8. To date, allosteric drugs are being developed for Orthosteric binding site
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the treatment of muscular, oncologic, immunologic, cardiovascular, psychiatric and neurodegenerative disorders linked to GPCRs5,8,9, ligand-gated ion channels7,8 and nuclear hormone receptors2,10. Recently, in our laboratories, an allosteric, small molecule acting extracellularly was identified for an RTK (F.D.S., A.C. and P.C.)11,12. Most of the initial insights into allosteric modulator function arose from studies of ligand-gated ion channels, for which benzodiazepines were the first allosteric therapeutics (used for the treatment of anxiety and sleeping disorders)13. These molecules potentiate the effect of the neurotransmitter γ-aminobutyric acid (GABA) on the ionotropic GABAA receptor. Orthosteric GABAA agonists can potentially have lethal effects, but benzodiazepines have proven to be clinically safe and effective. Several allosteric modulators have also been described for GPCRs5 and nuclear hormone receptors2,10,14,15. Thanks to the advances in molecular pharmacology and screening technology, allosteric modulators have now also been identified for other ion channels, intracellular kinases and phospholipases13. With the identification of an allosteric modulator of an RTK that acts extracellularly (F.D.S., A.C. and P.C.)11,12, it is becoming clear that although ligands are structurally distinct, there are at least five general properties common to allostery across multiple receptor superfamilies, and these are described next. Advantages and challenges of allosteric receptor modulation There are several features of receptor allostery that confer advantages on allosteric modulators as therapeutic agents as compared with classic orthosteric agents. First, selectivity increases, because unlike orthosteric binding sites, allosteric binding sites are less well conserved. This means that an allosteric compound usually binds to only one receptor subtype, and does not bind to other subtypes from the same receptor family. In some cases, the allosteric site is conserved
Response Orthosteric
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Figure 2 Orthosteric versus allosteric receptor mechanisms. Schematic representation of a cellular receptor (blue), which becomes activated by binding its orthosteric ligand (green), thereby inducing a downstream response. Cellular receptors can be targeted by means of orthosteric inhibitors (left) or allosteric modulators (right). Orthosteric inhibitors contain two major types of molecules, namely (i) ligand traps (gray square), which prevent ligand binding to the receptor or (ii) competitive antagonists (red sphere), which compete for the same binding site on the receptor; both inhibitors eliminate the entire downstream response of the receptor. In contrast, allosteric modulators can modify receptor function in various ways while still allowing the possibility of orthosteric agonist binding: they can change the affinity of the orthosteric ligand for the receptor (‘affinity modulators’), they can induce a structural change in the receptor that alters the cellular response upon orthosteric ligand binding (‘efficacy modulators’), or they can modify a downstream response independent of an orthosteric ligand (‘allosteric agonists/inverse agonists’). Adapted from reference 8.
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Targeting RTKs using allosteric inhibitors In the human genome, 58 RTKs function as high-affinity cell surface receptors for polypeptide growth factors, cytokines and hormones mediating cellular signaling in virtually every normal biological process. As well as regulating normal cellular processes, RTKs have also been implicated in the development and progression of many cancers and other disorders17, for instance, because they are overexpressed, mutated and duplicated. Similar to GPCRs, ligand-gated ion channels and nuclear hormone receptors, RTKs are not simple on/off switches but function as molecular rheostats that can be modulated18. Various drugs have been designed to target RTKs, including small-molecule intracellular tyrosine kinase inhibitors, and extracellularly acting antibodies and peptides. Most known small-molecule RTK inhibitors are
competitive antagonists that impair the tyrosine kinase function by blocking binding of ATP. Most kinase inhibitors that have entered the clinic have multiple additional (nonspecific) off-target effects, thereby increasing the chance of adverse effects. Sometimes drug treatment holidays are required in the regimes associated with these drugs owing to their high toxicity19. In addition, because orthosteric binding sites across diverse members of the RTK family are often highly conserved, it is challenging to achieve sufficiently high selectivity. Several strategies based on allostery exist (or could be developed) to inhibit RTKs. For instance, inhibitors could be designed to bind an allosteric site in the kinase domain. Such molecules have not been developed yet for RTKs, but inhibitors that bind to allosteric sites distinct from the ATP binding site have been described for intracellular nonreceptor type kinases, such as for Janus kinase (JAK)-2 (ref. 20), MAPK or Erk kinase (MEK)-1/2 (ref. 21), breakpoint cluster region-abelson kinase (Bcr/Abl)22–24, pyruvate kinase M2 (PKM2)25,26, v-akt murine thymoma viral oncogene homolog 1 (Akt)27, inducible I kappa-B kinase (IKK)28 and protein kinase C (PKC)29. Some RTK inhibitors not only compete for the binding site of ATP, but also contain an additional side chain that interacts with an allosteric site adjacent to the ATP docking site. Because these ATPadjacent sites are less conserved in the human kinome, they often have increased specificity as compared to broad-spectrum ATP analogs. Although promising, and successfully applied to lapatinib (Tykerb, a clinically approved EGFR/ERBB2 inhibitor21,30), other compounds with such a mechanism of action still lack the desired level of specificity, for example, sorafenib (Nexavar), which targets multiple RTKs, including v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor (VEGFR)-2, as well as the signaling kinase v-raf murine sarcoma viral oncogene homolog B (BRAF)21 (Fig. 3). A structure-based approach using the principle of both ATP and allosteric site binding has been used to develop more specific kinase inhibitors for AXL RTK (AXL), c-mer protooncogene tyrosine kinase (MER) and TYRO3 protein tyrosine kinase (SKY), but the inhibitors still cross-react with other RTKs31. Thus, despite an interaction with less-conserved regions outside the ATP pocket, the steric overlap of such a pocket in other RTKs still results in insufficient specificity. Also, because such drugs inhibit the binding of ATP, they block all RTK signaling pathways, which could induce adverse effects.
Figure 3 Kinase inhibitors with allosteric properties. Schematic representation of the crystal structure of the kinase domain of VEGFR2, bound to the kinase inhibitor sorafenib (blue) (PDB 4ASD)60. The canonical binding site for ATP is highlighted by the green field, whereas an adjacent allosteric binding site that increases the specificity of this inhibitor is indicated by the orange field. VEGF, vascular endothelial growth factor.
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across various receptor family subtypes, but selectivity is maintained because an allosteric effect (positive or negative) occurs only when the allosteric compound binds to the receptor subtype of interest, with no effect occurring when other subtypes are bound16. Second, allosteric modulators can be used in saturating doses. Because there is no competition with the endogenous agonist/ antagonist of the receptor that binds to the orthosteric site, the effective dose of an allosteric drug reaches a ‘saturable’ (ceiling) level. Once every receptor molecule is ligated, there is no additional pharmacological effect even in the presence of increased concentrations of the allosteric modulator. This means that allosteric modulators have a greater potential for on-target safety in overdose situations than orthosteric modulators. This is not the case for inhibitors of orthosteric ligands, which need to be supplied at increased concentrations if the concentration of orthosteric ligands increases. High doses of orthosteric inhibitors might induce on- and off-target toxicity7. Third, in the presence of an allosteric modulator, downstream signaling can be altered, whereby one particular pathway can be stimulated or inhibited, whereas another remains unaffected (or even shows an opposite effect). This is known as ‘biased’ agonism or antagonism and may offer a therapeutic advantage, because a key pathway promoting the progression of a particular disease could be blocked whereas other pathways that are necessary for homeostatic maintenance are not affected. Biased (ant)agonism can however complicate allosteric drug discovery, especially if not all signaling cascades have been identified, and in certain cases, blocking only one pathway may or may not be sufficient to evoke a therapeutic effect (see below). Fourth, some modulators maintain the normal spatiotemporal signaling patterns of the orthosteric ligand, because they only exert their pharmacological effects when an endogenous agonist is released5,7,8. Allosteric modulators can also display ‘probe dependence’, in which the size and/or direction of an allosteric effect can change markedly depending on the assay conditions or on the chemical nature of the ligands that occupy the orthosteric and allosteric site. This can offer an advantage if the goal is to specifically affect one particular liganddriven response, but may also pose a major hurdle to the identification of allosteric modulators, because the allosteric activities of any given compound may be identified in certain but not all screening assays (see below).
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Box 1 Mechanism of allosteric inhibition of FGFRs
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The fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling network plays an important role in cell growth, survival and differentiation of numerous cell types, including endothelial cells, and its dysregulation can lead to cancer development. The FGFR inhibitor, SSR128129E (SSR)11,12, which binds to the extracellular part of the receptor and does not compete with FGF for binding to its receptor, inhibits signaling with allosteric properties, including probe dependence, biased agonism and ceiling (saturability) effects. The molecular mechanism was identified by combining a broad array of biophysical, computational and biological methods, including crystallography, NMR, Fourier transform infrared spectroscopy, molecular dynamics simulations, free-energy calculations, structure-activity relationship analysis and FGFR mutagenesis (Fig. 4). Overall, the allosteric mechanism of inhibition relies on the binding of SSR to a pocket in extracellular domain 3, adjacent to the cell membrane, which is nonoverlapping with the orthosteric binding site of the FGF ligand. This binding pocket becomes exposed through extension of an α-helix (shown in red) and becomes stabilized through high-affinity binding of SSR to this new site. This structural change is sufficient to alter the signaling properties of the receptor. Although the FRS2/ERK1/2 pathway and receptor internalization are inhibited by SSR, the PLC-γ pathway remains unaffected.
Recently, in our laboratories, a novel type of small-molecule allosteric inhibitor was identified, which overcame these limitations by acting on the extracellular domain of an RTK, more specifically, the fibroblast growth factor receptor (FGFR) (F.D.S., A.C. and P.C.). By binding to a distant site, nonoverlapping with the endogenous orthosteric ligand-binding site, this small chemical compound, SSR128129E, induced a conformational change in the receptor that modulated its downstream signaling through biased antagonism. Indeed, SSR128129E inhibited signaling through the Extracellular signal-Regulated Kinase (ERK) but not the phospholipase C (PLC)-γ pathway. This allosteric inhibitor also exhibited signs of saturability and probe dependence (Box 1 and Fig. 4)11,12. Notably, SSR128129E’s ability to inhibit part of FGFR signaling was sufficiently potent to reduce the progression of cancer and inflammatory disorders in preclinical animal models11,12. The binding site for SSR128129E in the FGFR extracellular domain was located in a domain that is conserved only in the FGFR family, resulting in a high degree of specificity. Notably, the inhibitor did not affect orthosteric binding of the FGF ligand or ATP. Overall, SSR128129E induced a conformational change in FGFR that altered signaling in such a way that one pathway was inhibited, whereas the other was still active. Evidence is accruing that indicates that these findings can be extrapolated to other RTKs. For instance, point mutations in the extracellular domain of PDGF-receptor β altered its signaling without affecting orthosteric ligand binding32. A similar approach showed that different extracellular domains of VEGFR-2, which are not involved in ligand binding, are required for signaling33,34, and similar findings were obtained for KIT35. The crystal structure of the extracellular domain of VEGFR-3, another RTK, showed that upon ligand-induced dimerization, additional receptor-receptor interactions are required to generate active signaling complexes36. These findings might indicate that the extracellular domains of RTKs likely contain druggable allosteric sites. Besides the intracellular kinase domain and the extracellular ligand binding domain, the juxta-membrane and transmembrane domains of RTKs are also important for the formation of functional signaling complexes, as illustrated for epidermal growth factor receptor (EGFR) ErbB2 and the ephrin receptor EphA1 (refs. 37–39). Small molecules have been designed to target the juxta-membrane domain of RTKs, which activates signaling by these receptors. For instance, gambogic amide binds to the intracellular juxta-membrane region of the nerve growth factor receptor TrkA, and thereby induces receptor dimerization and activation40 (Fig. 5a). This suggests that allosteric mechanisms of RTK activation are druggable and could be designed to inhibit or activate RTKs in a specific manner. Other RTK inhibitors including peptides and antibodies have been identified that (possibly) function through an allosteric mechanism. 1116
One example is the peptidomimetic cyclotraxin-B, which inhibits the function of the brain-derived neurotrophic factor (BDNF) receptor TrkB through binding of the extracellular domain, without altering the binding properties of BDNF41 (Fig. 5b). Similarly, ‘peptoid drugs’ (a class of peptidomimetics whose side chains are appended to the nitrogen atom of the peptide backbone, rather than to the α-carbons, as they are in amino acids) against VEGFR2 change its signaling activity without affecting VEGF-A ligand binding 42 (Fig. 5c). Monoclonal antibodies have also been obtained that act by a putative allosteric mechanism. For instance, antibodies that interfere with the structural change of the extracellular domain of EGFR upon ligand binding prevent the formation of an active signaling complex (Fig. 5d)43–46. Other examples include anti-VEGFR3 antibodies binding to the extracellular domain of the receptor, which change the dimerization properties of this RTK and disturb downstream signaling without affecting ligand binding (Fig. 5e)47. Allosteric modulation of RTKs by other mechanisms Many RTKs interact with each other, co-receptors and/or intracellular adaptors to generate a functional signaling complex. It has been suggested that the assembly of such ‘quaternary’ structures is highly dependent on the conformation of the different partner proteins and may have profound effects on the intrinsic behavior of the receptor and its downstream signaling pathways48. If the interacting partner induces a conformational change in the RTK and thereby alters its function, allosteric mechanisms may be involved, though in most cases features of biased (ant)agonism, probe dependence and saturability have not been characterized for such interactions. For instance, the interaction of VEGF-A with VEGFR2 induces signaling through PLCγ, ERK and Akt, whereas the presence of the neuropilin 1 (Nrp1) co-receptor is required for the induction of the p38 MAPK pathway49. Subsequent research has shown that the presence of Nrp1 modulates the signaling kinetics and activation of a specific signal output of VEGFR2 (ref. 49). Future work is needed to establish whether this observation fits with an allosteric mechanism. Given that most RTKs have numerous co-receptors49,50, additional opportunities to modulate RTK function and perhaps to target these RTKs therapeutically await further identification. An example of an intracellular scaffold protein that might function as an allosteric modulator of an RTK is PKCε. This protein interacts with the cytosolic domain of EGFR, and thereby inhibits PLCγ sig naling in response to activation of EGFR by EGF, without altering the EGF-induced activation of the ERK and Akt pathways51. Moreover, the binding of PKCε to EGFR alters the sensitivity of EGFR toward kinase inhibitors, probably by inducing a structural change52. Finally, differential modulation of RTKs can also occur by allosterically modifying
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Perspective Allosteric mechanism of FGF receptor inhibition Figure 4 Mechanism of a small-molecule allosteric FGFR inhibitor. Left, scheme Native structure H2 conformation illustrating the native structure of the fibroblast FGF binding site or ‘orthosteric’ site D1 growth factor receptor (FGFR), containing three FGFR remains unaffected by SSR extracelluar domains (blue) and the intracellular SSR binds to a distinct, nonoverlapping D2 tyrosine kinase domain (brown). The orthosteric ‘allosteric’ site and stabilizes an +SSR extracellular structural change in D3, binding site for the FGF ligand (green) is FGF which affects downstream TK activation located between domain 2 (D2) and domain D3 and FGFR signaling Plasma SSR 3 (D3). Ligand binding activates downstream H2 conformation membrane Native structure signaling through ERK1/2 and PLCγ, and leads FGF FGF to receptor internalization. The inset shows a Tyrosine higher magnification of the orthosteric ligand TK activity kinase affected binding site. The α helix and adjacent β-helix Helix (TK) SSR (prone to extending into an α-helix) in domain α1 D3 D3 D3 is also shown. Right, scheme illustrating the β-sheet FGFR bound to the small-molecule allosteric PLCγ PLCγ ERK1/2 ERK1/2 structure Elongated internalization inhibitor SSR129128E (SSR; red), binding to a internalization helix distant site from the orthosteric site in domain D3. This stabilizes a structural change in A β-sheet in domain D3 undergoes a domain D3, which selectively inhibits signaling β-to-α helical change, thereby forming a through ERK1/2 and internalization, without cavity in which SSR binds affecting PLCγ signaling. More in particular, binding of SSR induces a β-to-α helical change (i.e., the α-helix extends by two twists and is therefore referred to as the ‘H2-conformation’) of the labile β-sheet in domain D3, which forms a cavity, that is, binding pocket with high affinity, for SSR that stabilizes a different receptor conformation, which affects downstream signaling. 3D structures were retrieved from PDB 1E0O and from molecular dynamics calculations in reference 12.
their ligands, as shown for hepatocyte growth factor (HGF), which activates the MET proto-oncogene (MET) receptor53. By using peptides that bind to an allosteric site in the HGF ligand, a structural change in HGF enhances receptor binding and signaling, and might offer opportunities to modulate receptor function. Discovery of new allosteric modulators of RTKs The development of allosteric drugs modulating RTK function is attractive and might be more feasible than was originally expected. Nonetheless, most of the currently known allosteric RTK modulators were discovered serendipitously and, to the best of our knowledge, no extracellularly acting allosteric small molecules have thus far been identified using screens specifically designed to detect such compounds. Based on current knowledge (deduced from previous discovery programs of allosteric modulators of enzymes, GPCRs and soluble kinases), we outline below possible strategies, recommendations and challenges for future allosteric RTK drug discovery programs. One possible strategy to identify novel allosteric sites in RTKs could involve an in silico analysis of high-resolution, three-dimensional structures and virtual screening of small molecules to identify putative allosteric pockets, ideally combined with empirical testing of the ability of lead components to modulate the enzymatic activity of the target RTK. Such a strategy was indeed successfully used to identify allosteric small-molecule inhibitors that interact with an intracellular pocket of CD45, one of the receptor protein tyrosine phosphatases, which share a high structural analogy to RTKs54. Small molecules preferentially bind to active sites, typically located in cavities, but can also bind to sites in other regions55. Using such an approach for enzymes, two types of allosteric sites have been proposed: ‘orphan allosteric sites’, which are in use by undiscovered natural (i.e., endogenous) modulators but can bind small molecules, and ‘serendipitous allosteric sites’, which may not have any natural ligand but none theless can be targeted by exogenous and/or synthetic substances55. The limitation of this approach is that it requires high-resolution structures of the target RTK. Unfortunately, such information is available only for isolated kinase domains or for parts of the extracellular domains of RTKs, but not for the entire protein. Nonetheless, purified kinase domains of RTKs could perhaps be used to discover
allosteric molecules by using Förster resonance energy transfer–based assays and fragment-based crystallographic screening, in analogy to how allosteric modulators of soluble kinases have been successfully identified26,56. An alternative approach may involve the evolutionary analysis of structural regions, as sites that are less well conserved may point toward allosteric regulatory regions57. Because structures of RTKs are in short supply, a combination of cell-based binding and/or signaling assays typically represents the mainstay for compound-screening campaigns. Cellular binding assays can be used to identify allosteric compounds that influence binding of the orthosteric reference agonist (i.e., growth factor) to the RTK. In instances where the binding is problematic, binding assays can be performed on isolated regions of the RTK of interest, with the caveat that the effects of a small molecule upon binding an isolated portion of the receptor could be different to that of the same smallmolecule binding the complete protein. The general characteristics that need to be pursued in any such screening assay are the properties of saturability, probe dependence and biased agonism/antagonism. In this context, one would look for a limit to the change in the potency (e.g., half maximal effective concentration (EC 50)) of an orthosteric reference agonist (i.e., growth factor) induced by increasing concentrations of a putative allosteric modulator as an index of saturability of effect. In addition, one could look for a change in the maximal orthosteric agonist response, which should not be observed with simple competitive inhibitors, as an indicator of efficacy-based allosteric modulators. Provided multiple downstream signaling pathways of the RTK have been mapped in sufficient detail, informed comparisons between the effects of test compounds on these pathways can be used to detect the potential of modulator-induced biased potentiation/antagonism. Ideally, the effect of the allosteric compound on both the signaling activity of the RTK (efficacy) and the binding affinity of the orthosteric agonist to the RTK should be determined. The combination of binding and functional assays resulted in the successful identification of peptide-mimetics (peptoids) with allosteric properties, able to bind and inhibit VEGFR2, a potent angiogenic RTK41. Once pharmacologically identified, allosteric compounds can be further characterized by structural and biophysical methods that
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Perspective Figure 5 Allosteric targeting strategies for RTKs Schematic representations of RTKs that are targeted by allosteric inhibitors. (a) The NGF receptor TrkA is targeted by the small-molecule allosteric inhibitor gambogic amide, which binds to the intracellular juxtamembrane region and thereby inhibits downstream signaling upon ligand binding. (b,c) Similarly, the BDNF receptor TrkB (b) and the VEGF receptor VEGFR2 (c) are targeted allosterically by the peptide inhibitor cyclotraxin-B or a ‘peptoid’, respectively, through binding to a nonoverlapping region in the extracellular region of the receptor, thereby not interfering with orthosteric ligand binding while still affecting downstream signaling. (d,e) The antibody, matuzumab, has been developed to inhibit EGFR signaling by binding to a region that is nonoverlapping with the orthosteric site, thereby trapping the receptor in a noninducible conformation (d), whereas the anti-VEGFR3 antibody binds to domain 5 of the extracellular domain of VEGFR3 (e), a site distant from the orthosteric binding site, and thereby changes the structure of this domain that prevents signaling complex upon ligand binding. VEGF: vascular endothelial growth factor; VEGFR: VEGF receptor; NGF: nerve growth factor; BDNF: brain-derived growth factor; EGF: epidermal growth factor; EGFR: EGF receptor.
more directly assess the binding of reference and test compounds to the RTK, which will also give an indication of the binding site for the modulator and its effects (if any) on the reference orthosteric ligand. Assays monitoring the binding kinetics of the orthosteric probe to the RTK in the presence of the modulator (for instance, by surface plasmon resonance), as opposed to only monitoring binding events that reach an equilibrium state, allow for the identification of allosteric compounds that modulate orthosteric ligand binding affinity. Various types of biophysical and computational modeling and simulation techniques can in addition be used to further characterize the allosteric binding site and properties, and eventually, the compound can be fully characterized for its biological effectiveness in cell culture and in various preclinical in vivo models, as was done for the allosteric FGFR inhibitor SSR128129 (refs. 11,12). Not only small molecules but also antibodies could be considered as putative allosteric modulators47. Antibodies raised against domains of the RTK not involved in ligand binding could be assessed for their utility as allosteric modulators or could be used to identify possible druggable allosteric sites. Although most allosteric kinase modulators investigated to date are inhibitors, it should be noted that the above approaches could also be used to detect activators. For instance, an allosteric antibody has been described that increases the affinity of insulin for its receptor, thereby enhancing signaling by this RTK58. Programs for allosteric drug discovery might be frustrated by limitations and challenges. For instance, binding or functional screens are normally performed using one orthosteric ligand (the most relevant endogenous ligand), but probe-dependent effects can influence the properties of an allosteric compound when using different orthosteric ligands. This is relevant to RTKs, as they often bind multiple members or isoforms of the same growth factor family, which can be further processed post-translationally. For instance, the FGF family has 23 members, whereas the VEGF family has 5 members, which exist in various (>10) isoforms that can be further proteolytically cleaved into several additional ligands49,59. It is also challenging to account for all 1118
possible biased signaling effects of allosteric compounds, given the multitude of signaling pathways downstream of RTKs 17. The lack of knowledge of RTK structures, and of how ligands interact with RTKs and change their function, limits optimization of lead candidates and guidance of structure-activity relationships, and hampers the discovery of new compound scaffolds by molecular docking and related approaches. It should also be remembered that, because an allosteric ligand may not alter the binding of the orthosteric ligand, but change RTK signaling, binding assays may not identify allosteric RTK modulators detected in functional assays. Finally, as exemplified by Louis Pasteur’s quote “le hasard ne favorise que les esprits préparés” (“chance favors the prepared mind”), seemingly strange results, not fitting expectations, during orthosteric inhibitor screens should not be overlooked as they might represent possible signs of allostery. For instance, a high-throughput screen to identify molecules inhibiting 125I-labeled FGF binding to the extracellular domain of FGFR2 coupled to a Fc fragment coated on plastic yielded a low affinity (µM) compound (SSR128129). This compound did not block binding of 125I-labeled FGF to the native FGFRs in intact cells, but was capable of inhibiting cellular responses to FGFs at nanomolar concentrations11,12. These pharmacological signs of probe dependence led to consideration of SSR128129 as a possible allosteric inhibitor of FGFRs, and subsequent multidisciplinary approaches confirmed this function. Conclusion From a therapeutic perspective, allosteric modulation offers advantages over classical orthosteric activation or inhibition, not only in terms of safety, but also of efficacy and reduced potential for drug resistance. Indeed, promising data suggest that the use of an allosteric inhibitor of a nonreceptor kinase in combination with an orthosteric
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inhibitor of the same target can improve efficacy and may help to reduce the likelihood of resistance to either compound alone 56. Saturability and biased antagonism of allosteric modulators can also reduce the problem of toxicity that is often an issue with orthosteric inhibitors that block all RTK signaling. Even though development of allosteric therapeutics remains challenging, the identification of the first extracellularly acting small molecule that can allosterically modulate FGFR signaling is a promising proof-of-concept discovery, warranting further consideration of other RTKs as targets of allosteric drugs. Although insights into allosteric regulation of RTKs remain limited, the opportunities for therapeutic discovery are substantial. A better understanding of the structural and molecular mechanisms of RTK function, and the development of adequate screening strategies will greatly aid in the discovery of new allosteric RTK modulators. Acknowledgments F.D.S. is supported by the Fund for Scientific Research (FWO), Flanders. A.C. is a principal research fellow of the National Health and Medical Research Council of Australia. This work is supported by grant #G.0789.11 and #G.00764.10 from the FWO, Belgium; the Belgian Science Policy (IAP #P7/03); Leducq Network of Excellence; and long-term structural Methusalem funding by the Flemish Government to P.C. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Hopkins, A.L. & Groom, C.R. The druggable genome. Nat. Rev. Drug Discov. 1, 727–730 (2002). 2. Overington, J.P., Al-Lazikani, B. & Hopkins, A.L. How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996 (2006). 3. Lundstrom, K. An overview on GPCRs and drug discovery: structure-based drug design and structural biology on GPCRs. Methods Mol. Biol. 552, 51–66 (2009). 4. Neubig, R.R., Spedding, M., Kenakin, T. & Christopoulos, A. International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology. Pharmacol. Rev. 55, 597–606 (2003). 5. Wootten, D., Christopoulos, A. & Sexton, P.M. Emerging paradigms in GPCR allostery: implications for drug discovery. Nat. Rev. Drug Discov. 12, 630–644 (2013). 6. Kenakin, T. & Christopoulos, A. Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat. Rev. Drug Discov. 12, 205–216 (2013). 7. Christopoulos, A. Allosteric binding sites on cell-surface receptors: novel targets for drug discovery. Nat. Rev. Drug Discov. 1, 198–210 (2002). 8. Conn, P.J., Christopoulos, A. & Lindsley, C.W. Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders. Nat. Rev. Drug Discov. 8, 41–54 (2009). 9. De Amici, M., Dallanoce, C., Holzgrabe, U., Trankle, C. & Mohr, K. Allosteric ligands for G protein-coupled receptors: a novel strategy with attractive therapeutic opportunities. Med. Res. Rev. 30, 463–549 (2010). 10. Moore, T.W., Mayne, C.G. & Katzenellenbogen, J.A. Minireview: Not picking pockets: nuclear receptor alternate-site modulators (NRAMs). Mol. Endocrinol. 24, 683–695 (2010). 11. Bono, F. et al. Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties. Cancer Cell 23, 477–488 (2013). 12. Herbert, C. et al. Molecular mechanism of SSR128129E, an extracellularly acting, small-molecule, allosteric inhibitor of FGF receptor signaling. Cancer Cell 23, 489–501 (2013). 13. Melancon, B.J. et al. Allosteric modulation of seven transmembrane spanning receptors: theory, practice, and opportunities for central nervous system drug discovery. J. Med. Chem. 55, 1445–1464 (2012). 14. McEwan, I.J. Nuclear hormone receptors: allosteric switches. Mol. Cell. Endocrinol. 348, 345–347 (2012). 15. Estébanez-Perpiñá, E. et al. A surface on the androgen receptor that allosterically regulates coactivator binding. Proc. Natl. Acad. Sci. USA 104, 16074–16079 (2007). 16. Birdsall, N.J. & Lazareno, S. Allosterism at muscarinic receptors: ligands and mechanisms. Mini Rev. Med. Chem. 5, 523–543 (2005). 17. Lemmon, M.A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010).
18. O′Hayre, M. et al. The emerging mutational landscape of G proteins and G-proteincoupled receptors in cancer. Nat. Rev. Cancer 13, 412–424 (2013). 19. Shah, D.R., Shah, R.R. & Morganroth, J. Tyrosine kinase inhibitors: their on-target toxicities as potential indicators of efficacy. Drug Safety 36, 413–426 (2013). 20. He, K. et al. Blockade of glioma proliferation through allosteric inhibition of JAK2. Sci. Signal. 6, ra55 (2013). 21. Zhang, J., Yang, P.L. & Gray, N.S. Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer 9, 28–39 (2009). 22. Grebien, F. et al. Targeting the SH2-kinase interface in Bcr-Abl inhibits leukemogenesis. Cell 147, 306–319 (2011). 23. Hantschel, O., Grebien, F. & Superti-Furga, G. The growing arsenal of ATPcompetitive and allosteric inhibitors of BCR-ABL. Cancer Res. 72, 4890–4895 (2012). 24. Zhang, J. et al. Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors. Nature 463, 501–506 (2010). 25. Gui, D.Y., Lewis, C.A. & Vander Heiden, M.G. Allosteric regulation of PKM2 allows cellular adaptation to different physiological states. Sci. Signal. 6, pe7 (2013). 26. Chaneton, B. et al. Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature 491, 458–462 (2012). 27. Cherrin, C. et al. An allosteric Akt inhibitor effectively blocks Akt signaling and tumor growth with only transient effects on glucose and insulin levels in vivo. Cancer Biol. Ther. 9, 493–503 (2010). 28. Burke, J.R. et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J. Biol. Chem. 278, 1450–1456 (2003). 29. Leonard, T.A., Rozycki, B., Saidi, L.F., Hummer, G. & Hurley, J.H. Crystal structure and allosteric activation of protein kinase C betaII. Cell 144, 55–66 (2011). 30. Karaman, M.W. et al. A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 26, 127–132 (2008). 31. Suárez, R.M. et al. Inhibitors of the TAM subfamily of tyrosine kinases: synthesis and biological evaluation. Eur. J. Med. Chem. 61, 2–25 (2013). 32. Yang, Y., Yuzawa, S. & Schlessinger, J. Contacts between membrane proximal regions of the PDGF receptor ectodomain are required for receptor activation but not for receptor dimerization. Proc. Natl. Acad. Sci. USA 105, 7681–7686 (2008). 33. Hyde, C.A. et al. Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites. Mol. Cell. Biol. 32, 3802–3813 (2012). 34. Yang, Y., Xie, P., Opatowsky, Y. & Schlessinger, J. Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signaling. Proc. Natl. Acad. Sci. USA 107, 1906–1911 (2010). 35. Laine, E., Auclair, C. & Tchertanov, L. Allosteric communication across the native and mutated KIT receptor tyrosine kinase. PLoS Comput. Biol. 8, e1002661 (2012). 36. Leppänen, V.M. et al. Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation. Proc. Natl. Acad. Sci. USA 110, 12960–12965 (2013). 37. Macdonald-Obermann, J.L. & Pike, L.J. The intracellular juxtamembrane domain of the epidermal growth factor (EGF) receptor is responsible for the allosteric regulation of EGF binding. J. Biol. Chem. 284, 13570–13576 (2009). 38. Thiel, K.W. & Carpenter, G. Epidermal growth factor receptor juxtamembrane region regulates allosteric tyrosine kinase activation. Proc. Natl. Acad. Sci. USA 104, 19238–19243 (2007). 39. Li, E. & Hristova, K. Receptor tyrosine kinase transmembrane domains: Function, dimer structure and dimerization energetics. Cell Adh. Migr. 4, 249–254 (2010). 40. Jang, S.W. et al. Gambogic amide, a selective agonist for TrkA receptor that possesses robust neurotrophic activity, prevents neuronal cell death. Proc. Natl. Acad. Sci. USA 104, 16329–16334 (2007). 41. Cazorla, M. et al. Cyclotraxin-B, the first highly potent and selective TrkB inhibitor, has anxiolytic properties in mice. PLoS ONE 5, e9777 (2010). 42. Udugamasooriya, D.G., Dineen, S.P., Brekken, R.A. & Kodadek, T. A peptoid “antibody surrogate” that antagonizes VEGF receptor 2 activity. J. Am. Chem. Soc. 130, 5744–5752 (2008). 43. Endres, N.F., Engel, K., Das, R., Kovacs, E. & Kuriyan, J. Regulation of the catalytic activity of the EGF receptor. Curr. Opin. Struct. Biol. 21, 777–784 (2011). 44. Fleishman, S.J., Schlessinger, J. & Ben-Tal, N. A putative molecular-activation switch in the transmembrane domain of erbB2. Proc. Natl. Acad. Sci. USA 99, 15937–15940 (2002). 45. Leahy, D.J. A molecular view of anti-ErbB monoclonal antibody therapy. Cancer Cell 13, 291–293 (2008). 46. Jura, N. et al. Catalytic control in the EGF receptor and its connection to general kinase regulatory mechanisms. Mol. Cell 42, 9–22 (2011). 47. Tvorogov, D. et al. Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization. Cancer Cell 18, 630–640 (2010). 48. Jaffe, E.K. Impact of quaternary structure dynamics on allosteric drug discovery. Curr. Top. Med. Chem. 13, 55–63 (2013). 49. Grünewald, F.S., Prota, A.E., Giese, A. & Ballmer-Hofer, K. Structure-function analysis of VEGF receptor activation and the role of coreceptors in angiogenic signaling. Biochim. Biophys. Acta 1804, 567–580 (2010).
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50. Murakami, M., Elfenbein, A. & Simons, M. Non-canonical fibroblast growth factor signalling in angiogenesis. Cardiovasc. Res. 78, 223–231 (2008). 51. Weisheit, S., Schafer, C., Mertens, C., Berndt, A. & Liebmann, C. PKCepsilon acts as negative allosteric modulator of EGF receptor signalling. Cell. Signal. 23, 436–448 (2011). 52. Weisheit, S. & Liebmann, C. Allosteric modulation by protein kinase Cepsilon leads to modified responses of EGF receptor towards tyrosine kinase inhibitors. Cell. Signal. 24, 422–434 (2012). 53. Landgraf, K.E. et al. Allosteric peptide activators of pro-hepatocyte growth factor stimulate Met signaling. J. Biol. Chem. 285, 40362–40372 (2010). 54. Perron, M. et al. Allosteric non-competitive small molecule selective inhibitors of CD45 tyrosine phosphatase suppress T-cell receptor signals and inflammation in vivo. Mol. Pharmacol. 85, 553–563 (2014). 55. Hardy, J.A. & Wells, J.A. Searching for new allosteric sites in enzymes. Curr. Opin. Struct. Biol. 14, 706–715 (2004).
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Allosteric targeting of receptor tyrosine kinases
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Frederik De Smet1,2, Arthur Christopoulos3 & Peter Carmeliet4,5 The drug discovery landscape has been transformed over the past decade by the discovery of allosteric modulators of all major mammalian receptor superfamilies. Allosteric ligands are a rich potential source of drugs and drug targets with clear therapeutic advantages. G protein–coupled receptors, ligand-gated ion channels and intracellular nuclear hormone receptors have all been targeted by allosteric modulators. More recently, a receptor tyrosine kinase (RTK) has been targeted by an extracellular small-molecule allosteric modulator. Allosteric mechanisms of structurally distinct molecules that target the various receptor families are more alike than originally anticipated and include selectivity, orthosteric probe dependence and pathway-biased signaling. Receptors are proteins that serve the dual roles of recognition of chemical or environmental stimuli and transduction of these stimuli to cellular responses. They can be embedded in the plasma membrane, or present in the cytosol or nucleus. Because of their widespread involvement in numerous physiological and pathological processes, it is not surprising that receptors have been successfully targeted by multiple drug-discovery programs1,2. Four major receptor classes have been identified; the membrane-bound cell surface G protein–coupled receptors (GPCRs), ligand-gated ion channels, enzyme-linked receptors (including the structurally related RTKs and phosphatases) and intracellular receptors (including the nuclear hormone receptors, which function as ligand-inducible transcription factors) (Fig. 1). About 70% of all currently available targeted drugs target cellular receptors3. Each class of receptors interacts with specific signaling molecules. Upon binding a ligand, the conformation of a receptor changes to activate or inhibit biochemical signaling pathways. This change in conformation was thought to be actively promoted by the ligand (conformational induction), but it is now known that most receptors are present as an ensemble of pre-existing states, and that the ligand preferentially stabilizes a state for which it has the higher affinity (conformational selection). The endogenous ligand-binding region 1The
Switch Laboratory, Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium. 2The Switch Laboratory, Flanders Institute for Biotechnology (VIB), Leuven, Belgium. 3Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia. 4Laboratory of Angiogenesis and Neurovascular link, Vesalius Research Center, Department of Oncology, University of Leuven, Leuven, Belgium. 5Laboratory of Angiogenesis and Neurovascular link, Vesalius Research Center, VIB, Leuven, Belgium. Correspondence should be addressed to P.C. ([email protected]). Received 9 December 2013; accepted 19 August 2014; published online 7 November 2014; doi:10.1038/nbt.3028
in the receptor that has been evolutionarily conserved to mediate this role is the ‘orthosteric site’4. The orthosteric site of some receptors has been identified using structural biophysics and mutational studies, but our understanding of the mechanisms of receptor activation by ligands remains limited. Structure-based design has been successful in the search for kinase inhibitors, but only modest progress has been achieved in designing drugs that target membrane proteins. Indeed, of the 77,000 highresolution crystal structures deposited in databases, less than 1% (~550) have been obtained for membrane proteins3, reflecting the technical limitations of solving structures of integral membrane proteins upon their removal from their native environment. Although three-dimensional structures are now becoming available for membrane-bound receptors5, most of our knowledge of the mechanisms underlying receptor function originates from the identification of various types of agonists (activators) and antagonists (inhibitors), which, when bound to their orthosteric binding sites, activate or inhibit specific downstream pathways. All receptors couple to signaling pathways in a cell context– dependent manner. Historically, it was believed that the differences in the ability of ligands to activate these pathways reflected the differences in the ‘intrinsic efficacy’ of each ligand, that is, the strength of the signal imparted to a given receptor. However, it is now known that the selection and magnitude of receptor-mediated responses is determined by the conformational state of the receptor when bound to a ligand, in addition to receptor concentration, coupling efficiency and the complement of cellular interacting partners. The stabilization of different active states by distinct ligands of a receptor in the same cellular background, leading to different functional outcomes, has been termed ‘biased agonism’ and is a mode of regulation that is being increasingly identified across receptor superfamilies6. Although most studies on receptor biology have focused on how ligands modulate receptor signaling through binding to the orthosteric site, receptor conformation and signaling can also be modified by ligands acting at topographically and spatially distinct allosteric sites (Fig. 1). The ability of one ligand (be it a small molecule or large protein) to change the binding and/or effects of a second ligand (small molecule, protein) at a target protein by means of an interaction with a nonoverlapping and spatially distant site is referred to as allostery. Allosteric interactions serve to fine-tune and regulate receptor function. Recent research has pinpointed allosteric interactions as a useful tool to modulate receptor function in ways that cannot be achieved by ligands that bind to an orthosteric site 5. In this review, we discuss the advantages and challenges of allosterically targeting receptors, focusing on RTKs as the ‘last bastion’ of receptor allostery.
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Figure 1 All superfamilies of cellular receptors can be allosterically modulated. Schematic representations of the activated forms of the four major cellular receptor classes, that is, ligand-gated ion channels, GPCRs, nuclear hormone receptors and RTKs. For each receptor, the nonoverlapping binding sites of the orthosteric ligand (green circle) and an allosteric ligand (orange triangle) are indicated. This comparison suggests that, although these receptors have different cellular functions, they share common features of allosteric modulation.
Allosteric receptor modulation Orthosteric ligands bind to an agonist-binding site, which initiates downstream receptor signaling. By contrast, allosteric ligands are usually structurally different from orthosteric ligands and bind to distinct sites that are spatially distant from the orthosteric site, to modulate the properties of orthosteric ligands (Fig. 2). Three types of allosteric modulators have been documented. First, ‘affinity modulators’ alter the orthosteric binding site by inducing a conformational change that alters the kinetics of ligand binding, and hence its binding affinity without changing the intrinsic signaling properties of the receptor. Second, ‘efficacy modulators’ induce a conformational change that is transmitted to regions of the receptor involved in the transduction of intracellular responses. This alters the signaling ability of the orthosteric ligand independent of, or in addition to, possible effects on orthosteric ligand affinity. Third, ‘allosteric agonists’ or ‘allosteric inverse agonists’ can perturb receptor signaling in the absence of an orthosteric ligand, in addition to any modulatory effects they have in the presence of orthosteric ligand7,8. To date, allosteric drugs are being developed for Orthosteric binding site
Receptor
Allosteric binding site
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Allosteric modulation Orthosteric Affinity modulator
Ligand trap
the treatment of muscular, oncologic, immunologic, cardiovascular, psychiatric and neurodegenerative disorders linked to GPCRs5,8,9, ligand-gated ion channels7,8 and nuclear hormone receptors2,10. Recently, in our laboratories, an allosteric, small molecule acting extracellularly was identified for an RTK (F.D.S., A.C. and P.C.)11,12. Most of the initial insights into allosteric modulator function arose from studies of ligand-gated ion channels, for which benzodiazepines were the first allosteric therapeutics (used for the treatment of anxiety and sleeping disorders)13. These molecules potentiate the effect of the neurotransmitter γ-aminobutyric acid (GABA) on the ionotropic GABAA receptor. Orthosteric GABAA agonists can potentially have lethal effects, but benzodiazepines have proven to be clinically safe and effective. Several allosteric modulators have also been described for GPCRs5 and nuclear hormone receptors2,10,14,15. Thanks to the advances in molecular pharmacology and screening technology, allosteric modulators have now also been identified for other ion channels, intracellular kinases and phospholipases13. With the identification of an allosteric modulator of an RTK that acts extracellularly (F.D.S., A.C. and P.C.)11,12, it is becoming clear that although ligands are structurally distinct, there are at least five general properties common to allostery across multiple receptor superfamilies, and these are described next. Advantages and challenges of allosteric receptor modulation There are several features of receptor allostery that confer advantages on allosteric modulators as therapeutic agents as compared with classic orthosteric agents. First, selectivity increases, because unlike orthosteric binding sites, allosteric binding sites are less well conserved. This means that an allosteric compound usually binds to only one receptor subtype, and does not bind to other subtypes from the same receptor family. In some cases, the allosteric site is conserved
Response Orthosteric
Efficacy modulator
Response Structural change Competitive antagonist Altered response Orthosteric Allosteric (inverse) agonists
Response
Response
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Figure 2 Orthosteric versus allosteric receptor mechanisms. Schematic representation of a cellular receptor (blue), which becomes activated by binding its orthosteric ligand (green), thereby inducing a downstream response. Cellular receptors can be targeted by means of orthosteric inhibitors (left) or allosteric modulators (right). Orthosteric inhibitors contain two major types of molecules, namely (i) ligand traps (gray square), which prevent ligand binding to the receptor or (ii) competitive antagonists (red sphere), which compete for the same binding site on the receptor; both inhibitors eliminate the entire downstream response of the receptor. In contrast, allosteric modulators can modify receptor function in various ways while still allowing the possibility of orthosteric agonist binding: they can change the affinity of the orthosteric ligand for the receptor (‘affinity modulators’), they can induce a structural change in the receptor that alters the cellular response upon orthosteric ligand binding (‘efficacy modulators’), or they can modify a downstream response independent of an orthosteric ligand (‘allosteric agonists/inverse agonists’). Adapted from reference 8.
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Targeting RTKs using allosteric inhibitors In the human genome, 58 RTKs function as high-affinity cell surface receptors for polypeptide growth factors, cytokines and hormones mediating cellular signaling in virtually every normal biological process. As well as regulating normal cellular processes, RTKs have also been implicated in the development and progression of many cancers and other disorders17, for instance, because they are overexpressed, mutated and duplicated. Similar to GPCRs, ligand-gated ion channels and nuclear hormone receptors, RTKs are not simple on/off switches but function as molecular rheostats that can be modulated18. Various drugs have been designed to target RTKs, including small-molecule intracellular tyrosine kinase inhibitors, and extracellularly acting antibodies and peptides. Most known small-molecule RTK inhibitors are
competitive antagonists that impair the tyrosine kinase function by blocking binding of ATP. Most kinase inhibitors that have entered the clinic have multiple additional (nonspecific) off-target effects, thereby increasing the chance of adverse effects. Sometimes drug treatment holidays are required in the regimes associated with these drugs owing to their high toxicity19. In addition, because orthosteric binding sites across diverse members of the RTK family are often highly conserved, it is challenging to achieve sufficiently high selectivity. Several strategies based on allostery exist (or could be developed) to inhibit RTKs. For instance, inhibitors could be designed to bind an allosteric site in the kinase domain. Such molecules have not been developed yet for RTKs, but inhibitors that bind to allosteric sites distinct from the ATP binding site have been described for intracellular nonreceptor type kinases, such as for Janus kinase (JAK)-2 (ref. 20), MAPK or Erk kinase (MEK)-1/2 (ref. 21), breakpoint cluster region-abelson kinase (Bcr/Abl)22–24, pyruvate kinase M2 (PKM2)25,26, v-akt murine thymoma viral oncogene homolog 1 (Akt)27, inducible I kappa-B kinase (IKK)28 and protein kinase C (PKC)29. Some RTK inhibitors not only compete for the binding site of ATP, but also contain an additional side chain that interacts with an allosteric site adjacent to the ATP docking site. Because these ATPadjacent sites are less conserved in the human kinome, they often have increased specificity as compared to broad-spectrum ATP analogs. Although promising, and successfully applied to lapatinib (Tykerb, a clinically approved EGFR/ERBB2 inhibitor21,30), other compounds with such a mechanism of action still lack the desired level of specificity, for example, sorafenib (Nexavar), which targets multiple RTKs, including v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor (VEGFR)-2, as well as the signaling kinase v-raf murine sarcoma viral oncogene homolog B (BRAF)21 (Fig. 3). A structure-based approach using the principle of both ATP and allosteric site binding has been used to develop more specific kinase inhibitors for AXL RTK (AXL), c-mer protooncogene tyrosine kinase (MER) and TYRO3 protein tyrosine kinase (SKY), but the inhibitors still cross-react with other RTKs31. Thus, despite an interaction with less-conserved regions outside the ATP pocket, the steric overlap of such a pocket in other RTKs still results in insufficient specificity. Also, because such drugs inhibit the binding of ATP, they block all RTK signaling pathways, which could induce adverse effects.
Figure 3 Kinase inhibitors with allosteric properties. Schematic representation of the crystal structure of the kinase domain of VEGFR2, bound to the kinase inhibitor sorafenib (blue) (PDB 4ASD)60. The canonical binding site for ATP is highlighted by the green field, whereas an adjacent allosteric binding site that increases the specificity of this inhibitor is indicated by the orange field. VEGF, vascular endothelial growth factor.
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Adjacent allosteric site
Extracellular domain
across various receptor family subtypes, but selectivity is maintained because an allosteric effect (positive or negative) occurs only when the allosteric compound binds to the receptor subtype of interest, with no effect occurring when other subtypes are bound16. Second, allosteric modulators can be used in saturating doses. Because there is no competition with the endogenous agonist/ antagonist of the receptor that binds to the orthosteric site, the effective dose of an allosteric drug reaches a ‘saturable’ (ceiling) level. Once every receptor molecule is ligated, there is no additional pharmacological effect even in the presence of increased concentrations of the allosteric modulator. This means that allosteric modulators have a greater potential for on-target safety in overdose situations than orthosteric modulators. This is not the case for inhibitors of orthosteric ligands, which need to be supplied at increased concentrations if the concentration of orthosteric ligands increases. High doses of orthosteric inhibitors might induce on- and off-target toxicity7. Third, in the presence of an allosteric modulator, downstream signaling can be altered, whereby one particular pathway can be stimulated or inhibited, whereas another remains unaffected (or even shows an opposite effect). This is known as ‘biased’ agonism or antagonism and may offer a therapeutic advantage, because a key pathway promoting the progression of a particular disease could be blocked whereas other pathways that are necessary for homeostatic maintenance are not affected. Biased (ant)agonism can however complicate allosteric drug discovery, especially if not all signaling cascades have been identified, and in certain cases, blocking only one pathway may or may not be sufficient to evoke a therapeutic effect (see below). Fourth, some modulators maintain the normal spatiotemporal signaling patterns of the orthosteric ligand, because they only exert their pharmacological effects when an endogenous agonist is released5,7,8. Allosteric modulators can also display ‘probe dependence’, in which the size and/or direction of an allosteric effect can change markedly depending on the assay conditions or on the chemical nature of the ligands that occupy the orthosteric and allosteric site. This can offer an advantage if the goal is to specifically affect one particular liganddriven response, but may also pose a major hurdle to the identification of allosteric modulators, because the allosteric activities of any given compound may be identified in certain but not all screening assays (see below).
Orthosteric ATP binding site
Plasma membrane
ATP
Sorafenib
Kinase domain
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Box 1 Mechanism of allosteric inhibition of FGFRs
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The fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling network plays an important role in cell growth, survival and differentiation of numerous cell types, including endothelial cells, and its dysregulation can lead to cancer development. The FGFR inhibitor, SSR128129E (SSR)11,12, which binds to the extracellular part of the receptor and does not compete with FGF for binding to its receptor, inhibits signaling with allosteric properties, including probe dependence, biased agonism and ceiling (saturability) effects. The molecular mechanism was identified by combining a broad array of biophysical, computational and biological methods, including crystallography, NMR, Fourier transform infrared spectroscopy, molecular dynamics simulations, free-energy calculations, structure-activity relationship analysis and FGFR mutagenesis (Fig. 4). Overall, the allosteric mechanism of inhibition relies on the binding of SSR to a pocket in extracellular domain 3, adjacent to the cell membrane, which is nonoverlapping with the orthosteric binding site of the FGF ligand. This binding pocket becomes exposed through extension of an α-helix (shown in red) and becomes stabilized through high-affinity binding of SSR to this new site. This structural change is sufficient to alter the signaling properties of the receptor. Although the FRS2/ERK1/2 pathway and receptor internalization are inhibited by SSR, the PLC-γ pathway remains unaffected.
Recently, in our laboratories, a novel type of small-molecule allosteric inhibitor was identified, which overcame these limitations by acting on the extracellular domain of an RTK, more specifically, the fibroblast growth factor receptor (FGFR) (F.D.S., A.C. and P.C.). By binding to a distant site, nonoverlapping with the endogenous orthosteric ligand-binding site, this small chemical compound, SSR128129E, induced a conformational change in the receptor that modulated its downstream signaling through biased antagonism. Indeed, SSR128129E inhibited signaling through the Extracellular signal-Regulated Kinase (ERK) but not the phospholipase C (PLC)-γ pathway. This allosteric inhibitor also exhibited signs of saturability and probe dependence (Box 1 and Fig. 4)11,12. Notably, SSR128129E’s ability to inhibit part of FGFR signaling was sufficiently potent to reduce the progression of cancer and inflammatory disorders in preclinical animal models11,12. The binding site for SSR128129E in the FGFR extracellular domain was located in a domain that is conserved only in the FGFR family, resulting in a high degree of specificity. Notably, the inhibitor did not affect orthosteric binding of the FGF ligand or ATP. Overall, SSR128129E induced a conformational change in FGFR that altered signaling in such a way that one pathway was inhibited, whereas the other was still active. Evidence is accruing that indicates that these findings can be extrapolated to other RTKs. For instance, point mutations in the extracellular domain of PDGF-receptor β altered its signaling without affecting orthosteric ligand binding32. A similar approach showed that different extracellular domains of VEGFR-2, which are not involved in ligand binding, are required for signaling33,34, and similar findings were obtained for KIT35. The crystal structure of the extracellular domain of VEGFR-3, another RTK, showed that upon ligand-induced dimerization, additional receptor-receptor interactions are required to generate active signaling complexes36. These findings might indicate that the extracellular domains of RTKs likely contain druggable allosteric sites. Besides the intracellular kinase domain and the extracellular ligand binding domain, the juxta-membrane and transmembrane domains of RTKs are also important for the formation of functional signaling complexes, as illustrated for epidermal growth factor receptor (EGFR) ErbB2 and the ephrin receptor EphA1 (refs. 37–39). Small molecules have been designed to target the juxta-membrane domain of RTKs, which activates signaling by these receptors. For instance, gambogic amide binds to the intracellular juxta-membrane region of the nerve growth factor receptor TrkA, and thereby induces receptor dimerization and activation40 (Fig. 5a). This suggests that allosteric mechanisms of RTK activation are druggable and could be designed to inhibit or activate RTKs in a specific manner. Other RTK inhibitors including peptides and antibodies have been identified that (possibly) function through an allosteric mechanism. 1116
One example is the peptidomimetic cyclotraxin-B, which inhibits the function of the brain-derived neurotrophic factor (BDNF) receptor TrkB through binding of the extracellular domain, without altering the binding properties of BDNF41 (Fig. 5b). Similarly, ‘peptoid drugs’ (a class of peptidomimetics whose side chains are appended to the nitrogen atom of the peptide backbone, rather than to the α-carbons, as they are in amino acids) against VEGFR2 change its signaling activity without affecting VEGF-A ligand binding 42 (Fig. 5c). Monoclonal antibodies have also been obtained that act by a putative allosteric mechanism. For instance, antibodies that interfere with the structural change of the extracellular domain of EGFR upon ligand binding prevent the formation of an active signaling complex (Fig. 5d)43–46. Other examples include anti-VEGFR3 antibodies binding to the extracellular domain of the receptor, which change the dimerization properties of this RTK and disturb downstream signaling without affecting ligand binding (Fig. 5e)47. Allosteric modulation of RTKs by other mechanisms Many RTKs interact with each other, co-receptors and/or intracellular adaptors to generate a functional signaling complex. It has been suggested that the assembly of such ‘quaternary’ structures is highly dependent on the conformation of the different partner proteins and may have profound effects on the intrinsic behavior of the receptor and its downstream signaling pathways48. If the interacting partner induces a conformational change in the RTK and thereby alters its function, allosteric mechanisms may be involved, though in most cases features of biased (ant)agonism, probe dependence and saturability have not been characterized for such interactions. For instance, the interaction of VEGF-A with VEGFR2 induces signaling through PLCγ, ERK and Akt, whereas the presence of the neuropilin 1 (Nrp1) co-receptor is required for the induction of the p38 MAPK pathway49. Subsequent research has shown that the presence of Nrp1 modulates the signaling kinetics and activation of a specific signal output of VEGFR2 (ref. 49). Future work is needed to establish whether this observation fits with an allosteric mechanism. Given that most RTKs have numerous co-receptors49,50, additional opportunities to modulate RTK function and perhaps to target these RTKs therapeutically await further identification. An example of an intracellular scaffold protein that might function as an allosteric modulator of an RTK is PKCε. This protein interacts with the cytosolic domain of EGFR, and thereby inhibits PLCγ sig naling in response to activation of EGFR by EGF, without altering the EGF-induced activation of the ERK and Akt pathways51. Moreover, the binding of PKCε to EGFR alters the sensitivity of EGFR toward kinase inhibitors, probably by inducing a structural change52. Finally, differential modulation of RTKs can also occur by allosterically modifying
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Perspective Allosteric mechanism of FGF receptor inhibition Figure 4 Mechanism of a small-molecule allosteric FGFR inhibitor. Left, scheme Native structure H2 conformation illustrating the native structure of the fibroblast FGF binding site or ‘orthosteric’ site D1 growth factor receptor (FGFR), containing three FGFR remains unaffected by SSR extracelluar domains (blue) and the intracellular SSR binds to a distinct, nonoverlapping D2 tyrosine kinase domain (brown). The orthosteric ‘allosteric’ site and stabilizes an +SSR extracellular structural change in D3, binding site for the FGF ligand (green) is FGF which affects downstream TK activation located between domain 2 (D2) and domain D3 and FGFR signaling Plasma SSR 3 (D3). Ligand binding activates downstream H2 conformation membrane Native structure signaling through ERK1/2 and PLCγ, and leads FGF FGF to receptor internalization. The inset shows a Tyrosine higher magnification of the orthosteric ligand TK activity kinase affected binding site. The α helix and adjacent β-helix Helix (TK) SSR (prone to extending into an α-helix) in domain α1 D3 D3 D3 is also shown. Right, scheme illustrating the β-sheet FGFR bound to the small-molecule allosteric PLCγ PLCγ ERK1/2 ERK1/2 structure Elongated internalization inhibitor SSR129128E (SSR; red), binding to a internalization helix distant site from the orthosteric site in domain D3. This stabilizes a structural change in A β-sheet in domain D3 undergoes a domain D3, which selectively inhibits signaling β-to-α helical change, thereby forming a through ERK1/2 and internalization, without cavity in which SSR binds affecting PLCγ signaling. More in particular, binding of SSR induces a β-to-α helical change (i.e., the α-helix extends by two twists and is therefore referred to as the ‘H2-conformation’) of the labile β-sheet in domain D3, which forms a cavity, that is, binding pocket with high affinity, for SSR that stabilizes a different receptor conformation, which affects downstream signaling. 3D structures were retrieved from PDB 1E0O and from molecular dynamics calculations in reference 12.
their ligands, as shown for hepatocyte growth factor (HGF), which activates the MET proto-oncogene (MET) receptor53. By using peptides that bind to an allosteric site in the HGF ligand, a structural change in HGF enhances receptor binding and signaling, and might offer opportunities to modulate receptor function. Discovery of new allosteric modulators of RTKs The development of allosteric drugs modulating RTK function is attractive and might be more feasible than was originally expected. Nonetheless, most of the currently known allosteric RTK modulators were discovered serendipitously and, to the best of our knowledge, no extracellularly acting allosteric small molecules have thus far been identified using screens specifically designed to detect such compounds. Based on current knowledge (deduced from previous discovery programs of allosteric modulators of enzymes, GPCRs and soluble kinases), we outline below possible strategies, recommendations and challenges for future allosteric RTK drug discovery programs. One possible strategy to identify novel allosteric sites in RTKs could involve an in silico analysis of high-resolution, three-dimensional structures and virtual screening of small molecules to identify putative allosteric pockets, ideally combined with empirical testing of the ability of lead components to modulate the enzymatic activity of the target RTK. Such a strategy was indeed successfully used to identify allosteric small-molecule inhibitors that interact with an intracellular pocket of CD45, one of the receptor protein tyrosine phosphatases, which share a high structural analogy to RTKs54. Small molecules preferentially bind to active sites, typically located in cavities, but can also bind to sites in other regions55. Using such an approach for enzymes, two types of allosteric sites have been proposed: ‘orphan allosteric sites’, which are in use by undiscovered natural (i.e., endogenous) modulators but can bind small molecules, and ‘serendipitous allosteric sites’, which may not have any natural ligand but none theless can be targeted by exogenous and/or synthetic substances55. The limitation of this approach is that it requires high-resolution structures of the target RTK. Unfortunately, such information is available only for isolated kinase domains or for parts of the extracellular domains of RTKs, but not for the entire protein. Nonetheless, purified kinase domains of RTKs could perhaps be used to discover
allosteric molecules by using Förster resonance energy transfer–based assays and fragment-based crystallographic screening, in analogy to how allosteric modulators of soluble kinases have been successfully identified26,56. An alternative approach may involve the evolutionary analysis of structural regions, as sites that are less well conserved may point toward allosteric regulatory regions57. Because structures of RTKs are in short supply, a combination of cell-based binding and/or signaling assays typically represents the mainstay for compound-screening campaigns. Cellular binding assays can be used to identify allosteric compounds that influence binding of the orthosteric reference agonist (i.e., growth factor) to the RTK. In instances where the binding is problematic, binding assays can be performed on isolated regions of the RTK of interest, with the caveat that the effects of a small molecule upon binding an isolated portion of the receptor could be different to that of the same smallmolecule binding the complete protein. The general characteristics that need to be pursued in any such screening assay are the properties of saturability, probe dependence and biased agonism/antagonism. In this context, one would look for a limit to the change in the potency (e.g., half maximal effective concentration (EC 50)) of an orthosteric reference agonist (i.e., growth factor) induced by increasing concentrations of a putative allosteric modulator as an index of saturability of effect. In addition, one could look for a change in the maximal orthosteric agonist response, which should not be observed with simple competitive inhibitors, as an indicator of efficacy-based allosteric modulators. Provided multiple downstream signaling pathways of the RTK have been mapped in sufficient detail, informed comparisons between the effects of test compounds on these pathways can be used to detect the potential of modulator-induced biased potentiation/antagonism. Ideally, the effect of the allosteric compound on both the signaling activity of the RTK (efficacy) and the binding affinity of the orthosteric agonist to the RTK should be determined. The combination of binding and functional assays resulted in the successful identification of peptide-mimetics (peptoids) with allosteric properties, able to bind and inhibit VEGFR2, a potent angiogenic RTK41. Once pharmacologically identified, allosteric compounds can be further characterized by structural and biophysical methods that
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Perspective Figure 5 Allosteric targeting strategies for RTKs Schematic representations of RTKs that are targeted by allosteric inhibitors. (a) The NGF receptor TrkA is targeted by the small-molecule allosteric inhibitor gambogic amide, which binds to the intracellular juxtamembrane region and thereby inhibits downstream signaling upon ligand binding. (b,c) Similarly, the BDNF receptor TrkB (b) and the VEGF receptor VEGFR2 (c) are targeted allosterically by the peptide inhibitor cyclotraxin-B or a ‘peptoid’, respectively, through binding to a nonoverlapping region in the extracellular region of the receptor, thereby not interfering with orthosteric ligand binding while still affecting downstream signaling. (d,e) The antibody, matuzumab, has been developed to inhibit EGFR signaling by binding to a region that is nonoverlapping with the orthosteric site, thereby trapping the receptor in a noninducible conformation (d), whereas the anti-VEGFR3 antibody binds to domain 5 of the extracellular domain of VEGFR3 (e), a site distant from the orthosteric binding site, and thereby changes the structure of this domain that prevents signaling complex upon ligand binding. VEGF: vascular endothelial growth factor; VEGFR: VEGF receptor; NGF: nerve growth factor; BDNF: brain-derived growth factor; EGF: epidermal growth factor; EGFR: EGF receptor.
more directly assess the binding of reference and test compounds to the RTK, which will also give an indication of the binding site for the modulator and its effects (if any) on the reference orthosteric ligand. Assays monitoring the binding kinetics of the orthosteric probe to the RTK in the presence of the modulator (for instance, by surface plasmon resonance), as opposed to only monitoring binding events that reach an equilibrium state, allow for the identification of allosteric compounds that modulate orthosteric ligand binding affinity. Various types of biophysical and computational modeling and simulation techniques can in addition be used to further characterize the allosteric binding site and properties, and eventually, the compound can be fully characterized for its biological effectiveness in cell culture and in various preclinical in vivo models, as was done for the allosteric FGFR inhibitor SSR128129 (refs. 11,12). Not only small molecules but also antibodies could be considered as putative allosteric modulators47. Antibodies raised against domains of the RTK not involved in ligand binding could be assessed for their utility as allosteric modulators or could be used to identify possible druggable allosteric sites. Although most allosteric kinase modulators investigated to date are inhibitors, it should be noted that the above approaches could also be used to detect activators. For instance, an allosteric antibody has been described that increases the affinity of insulin for its receptor, thereby enhancing signaling by this RTK58. Programs for allosteric drug discovery might be frustrated by limitations and challenges. For instance, binding or functional screens are normally performed using one orthosteric ligand (the most relevant endogenous ligand), but probe-dependent effects can influence the properties of an allosteric compound when using different orthosteric ligands. This is relevant to RTKs, as they often bind multiple members or isoforms of the same growth factor family, which can be further processed post-translationally. For instance, the FGF family has 23 members, whereas the VEGF family has 5 members, which exist in various (>10) isoforms that can be further proteolytically cleaved into several additional ligands49,59. It is also challenging to account for all 1118
possible biased signaling effects of allosteric compounds, given the multitude of signaling pathways downstream of RTKs 17. The lack of knowledge of RTK structures, and of how ligands interact with RTKs and change their function, limits optimization of lead candidates and guidance of structure-activity relationships, and hampers the discovery of new compound scaffolds by molecular docking and related approaches. It should also be remembered that, because an allosteric ligand may not alter the binding of the orthosteric ligand, but change RTK signaling, binding assays may not identify allosteric RTK modulators detected in functional assays. Finally, as exemplified by Louis Pasteur’s quote “le hasard ne favorise que les esprits préparés” (“chance favors the prepared mind”), seemingly strange results, not fitting expectations, during orthosteric inhibitor screens should not be overlooked as they might represent possible signs of allostery. For instance, a high-throughput screen to identify molecules inhibiting 125I-labeled FGF binding to the extracellular domain of FGFR2 coupled to a Fc fragment coated on plastic yielded a low affinity (µM) compound (SSR128129). This compound did not block binding of 125I-labeled FGF to the native FGFRs in intact cells, but was capable of inhibiting cellular responses to FGFs at nanomolar concentrations11,12. These pharmacological signs of probe dependence led to consideration of SSR128129 as a possible allosteric inhibitor of FGFRs, and subsequent multidisciplinary approaches confirmed this function. Conclusion From a therapeutic perspective, allosteric modulation offers advantages over classical orthosteric activation or inhibition, not only in terms of safety, but also of efficacy and reduced potential for drug resistance. Indeed, promising data suggest that the use of an allosteric inhibitor of a nonreceptor kinase in combination with an orthosteric
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inhibitor of the same target can improve efficacy and may help to reduce the likelihood of resistance to either compound alone 56. Saturability and biased antagonism of allosteric modulators can also reduce the problem of toxicity that is often an issue with orthosteric inhibitors that block all RTK signaling. Even though development of allosteric therapeutics remains challenging, the identification of the first extracellularly acting small molecule that can allosterically modulate FGFR signaling is a promising proof-of-concept discovery, warranting further consideration of other RTKs as targets of allosteric drugs. Although insights into allosteric regulation of RTKs remain limited, the opportunities for therapeutic discovery are substantial. A better understanding of the structural and molecular mechanisms of RTK function, and the development of adequate screening strategies will greatly aid in the discovery of new allosteric RTK modulators. Acknowledgments F.D.S. is supported by the Fund for Scientific Research (FWO), Flanders. A.C. is a principal research fellow of the National Health and Medical Research Council of Australia. This work is supported by grant #G.0789.11 and #G.00764.10 from the FWO, Belgium; the Belgian Science Policy (IAP #P7/03); Leducq Network of Excellence; and long-term structural Methusalem funding by the Flemish Government to P.C. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Hopkins, A.L. & Groom, C.R. The druggable genome. Nat. Rev. Drug Discov. 1, 727–730 (2002). 2. Overington, J.P., Al-Lazikani, B. & Hopkins, A.L. How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996 (2006). 3. Lundstrom, K. An overview on GPCRs and drug discovery: structure-based drug design and structural biology on GPCRs. Methods Mol. Biol. 552, 51–66 (2009). 4. Neubig, R.R., Spedding, M., Kenakin, T. & Christopoulos, A. International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology. Pharmacol. Rev. 55, 597–606 (2003). 5. Wootten, D., Christopoulos, A. & Sexton, P.M. Emerging paradigms in GPCR allostery: implications for drug discovery. Nat. Rev. Drug Discov. 12, 630–644 (2013). 6. Kenakin, T. & Christopoulos, A. Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat. Rev. Drug Discov. 12, 205–216 (2013). 7. Christopoulos, A. Allosteric binding sites on cell-surface receptors: novel targets for drug discovery. Nat. Rev. Drug Discov. 1, 198–210 (2002). 8. Conn, P.J., Christopoulos, A. & Lindsley, C.W. Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders. Nat. Rev. Drug Discov. 8, 41–54 (2009). 9. De Amici, M., Dallanoce, C., Holzgrabe, U., Trankle, C. & Mohr, K. Allosteric ligands for G protein-coupled receptors: a novel strategy with attractive therapeutic opportunities. Med. Res. Rev. 30, 463–549 (2010). 10. Moore, T.W., Mayne, C.G. & Katzenellenbogen, J.A. Minireview: Not picking pockets: nuclear receptor alternate-site modulators (NRAMs). Mol. Endocrinol. 24, 683–695 (2010). 11. Bono, F. et al. Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties. Cancer Cell 23, 477–488 (2013). 12. Herbert, C. et al. Molecular mechanism of SSR128129E, an extracellularly acting, small-molecule, allosteric inhibitor of FGF receptor signaling. Cancer Cell 23, 489–501 (2013). 13. Melancon, B.J. et al. Allosteric modulation of seven transmembrane spanning receptors: theory, practice, and opportunities for central nervous system drug discovery. J. Med. Chem. 55, 1445–1464 (2012). 14. McEwan, I.J. Nuclear hormone receptors: allosteric switches. Mol. Cell. Endocrinol. 348, 345–347 (2012). 15. Estébanez-Perpiñá, E. et al. A surface on the androgen receptor that allosterically regulates coactivator binding. Proc. Natl. Acad. Sci. USA 104, 16074–16079 (2007). 16. Birdsall, N.J. & Lazareno, S. Allosterism at muscarinic receptors: ligands and mechanisms. Mini Rev. Med. Chem. 5, 523–543 (2005). 17. Lemmon, M.A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010).
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