Chemokine receptor modeling: an interdisciplinary approach to drug design.

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Chemokine receptor modeling: an interdisciplinary approach to drug design Chemokines and their receptors are integral components of the immune response, regulating lymphocyte development, homing and trafficking, and playing a key role in the pathophysiology of many diseases. Chemokine receptors have, therefore, become the target for both small-molecule, peptide and antibody therapeutics. Chemokine receptors belong to the family of seven transmembrane receptor class A G protein-coupled receptors. The publication of the crystal structure of the archetypal class A seven transmembrane receptor protein rhodopsin, and other G protein-coupled receptors, including C-X-C chemokine receptor 4 and C-C chemokine receptor 5, provided the opportunity to create homology models of chemokine receptors. In this review, we describe an interdisciplinary approach to chemokine receptor modeling and the utility of this approach for structure-based drug design of chemokine receptor inhibitors. Physiology & pathophysiology of chemokine receptors The first chemokines were discovered in the 1980s [1], but it was the discovery of their involvement in the process of HIV infection that provided the stimulus for the investigation of chemokine receptors as therapeutic targets for drug discovery [2–4]. C-X-C chemokine receptor 4 (CXCR4) and C-C chemokine receptor 5 (CCR5) are recognized validated targets for HIV, however, the potential for chemokine receptors as drug targets goes well beyond HIV [5–8]. Their physiological role in immune-system regulation, and the involvement of chemokines in the pathophysiology of multiple inflammatory diseases and cancer progression provides a multitude of opportunities for therapeutic intervention. Chemokines, chemotactic cytokines, are 8–10 kDa proteins. Over 40 chemokines have been identified (Figure 1). They fall into four classes, which are categorized by the number and relative spacing of cysteine residues at the N-terminus. The predominant classes are the CC chemokines, which are characterized by two adjacent cysteines, and the CXC chemokines, where two cysteines are separated by a single amino acid residue. The two minor classes consist of the CX 3C group, which has only one member and where two cysteines are separated by three amino acids, and the C group, which has two members and is characterized by a single cysteine [9–11]. Chemokine receptors belong to the class A family of seven transmembrane (7TM) G protein coupled-receptors (GPCR) [12]. 7TM

proteins span the plasma membrane seven times, as the name suggests, and the TM regions are referred to as TMI–TMVII. In this review, specific amino acid residues are referred either by their residue number, for example Glu288 for the glutamate in TMVII in CXCR4, or their relative position within a TM region for example GluVII:06. The initial step in GPCR signaling is mediated by the interaction of a guanine nucleotide-binding protein (G protein) with the intracellular loops of the 7TM receptor. The G protein consists of three subunits; Ga, Gb and Gg. In the basal state, this heterotrimeric G protein binds the guanine nucleotide GDP to the a subunit. Upon activation by ligand binding to the GPCR, GDP is released and replaced by GTP, resulting in subunit dissociation of the heterotrimer into a bg dimer and the a monomer. The GTP is rapidly hydrolyzed to GDP followed by re-association of the receptor and the trimeric G protein complex. On the basis of sequence similarity, the Ga subunits have been subdivided into four families: Gas,Gai,Gaqand Ga12 . Chemokine receptors are primarily thought to signal through Gai. Signaling is then mediated by several divergent pathways postG protein activation, including phospholipase C, PI3 kinase, small Rho GTPases, JAK/STAT and MAPK. Chemokine receptors are rapidly internalized after ligand activation via recruitment of b-arrestin and clathrin-mediated endocytosis, which is regulated by G protein receptor kinases (GRK) [10,13,14]. In addition, there is a group of chemokine receptors that, although they bind chemokines

Simon P Fricker*1 & Markus Metz2

10.4155/FMC.13.194 © 2014 Future Science Ltd

Future Med. Chem. (2014) 6(1), 91–114

ISSN 1756-8919

Sanofi-Genzyme R&D Center, 49 New York Avenue, Framingham, MA 01701 USA 2 Sanofi LGCR, 153 2nd Avenue, Waltham, MA 02451 USA *Author for correspondence: Tel.: +1 508 271 4598 E-mail: [email protected] 1

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Review | Fricker & Metz Key Terms Chemokines: 8–10 kDa

proteins that are mediators of hematopoiesis and the immune response. They regulate lymphocyte development, homing and trafficking to and from the bone marrow and lymphoid tissues. In addition, they mediate trafficking of immune cells to sites of inflammation. These natural processes have also been usurped by cancer cells and chemokines play a role in various aspects of cancer biology.

HIV: AIDS is caused by

infection with HIV. HIV infects host cells via binding of the viral gp120 protein to CD4 on host cells, such as lymphocytes. This is followed by binding to a co-receptor, which facilitates viral entry. Two co-receptors have been identified, the chemokine receptors CCR5 and CXCR4. The CCR5 inhibitor maraviroc is the only approved chemokine receptor inhibitor for HIV treatment.

Chemokine receptors:

Belong to the class of seven transmembrane G proteincoupled receptors (GPCRs). Ligands for GPCR can consist of small proteins and peptides, such as chemokines and the peptide hormones or small molecules, such as the biogenic amine neurotransmitters. Approximately 50% of marketed drugs target GPCR. Chemokine receptors are therefore attractive drug targets.

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and have the typical 7TM structure, do not produce a signal [15,16]. The members of this group include the Duffy antigen receptor for chemokines (DARC), D6, CXCR7 and CCXCKR1. These receptors lack a specific canonical DRYLAIV motif on the second intracellular loop, which is responsible for G protein interaction. However they can internalize upon ligand binding, presumably via a b-arrestin-mediated mechanism. The role of these receptors is to ‘scavenge’ chemokines and, therefore, modify the chemokine gradient and act as regulators of chemokine-mediated migration. Consequently, they have a different and unique role in the inflammatory and immune response compared with the classical chemokine receptors. Chemokines are mediators of hematopoiesis and inflammation, regulating lymphocyte development, homing and trafficking [17,18] and have been further subdivided by function into homeostatic and inflammatory chemokines [11,19]. The homeostatic chemokines, which are constitutively expressed, and their receptors play a major role in development and lymphoid tissue architecture. Homeostatic chemokines include CXCL12, which is the ligand for the receptor CXCR4, CXCL13 the ligand for the receptor CXCR5, and CCL19 and CCL21 the ligands for the receptor CCR7. This is a somewhat simplistic description as homeostatic chemokines and their receptors have been demonstrated to be involved in inflammatory and immune-mediated diseases. In contrast, the inflammatory cytokines are induced during the inflammatory response. Examples of inflammatory chemokines are: CCL-3, -4 and -5, which activate CCR5; CCL-2, -7, -8, which activate CCR2; CXCL-1, -2, -3, -5, -6, -7 and -8, which activate CXCR2; and, CXCL-9, -10 and -11, which activate CXCR3. What is apparent from this is that multiple chemokines can activate a given receptor. Conversely, chemokines can bind to more than one receptor, for example CCL5 can also bind to CCR1 and CCR3 [10,11]. The complex array of chemokine/chemokine receptor interactions is illustrated in Figure 1. The property of chemokines to bind to multiple chemokine receptors, together with the diverse cellular expression of chemokine receptors, have led to the suggestion of pharmacological redundancy within the chemokine system, such that targeting a single chemokine receptor may not be sufficient for therapeutic efficacy [20]. Alternatively, it has been proposed that the pleiotropic nature of chemokines may actually represent a mechanism for specific spatial and temporal control and activation [21]. Future Med. Chem. (2014) 6(1)

Chemokines regulate the movement, trafficking and positioning of cells of the immune system and, as such, are chemical mediators of the immune response. Not surprisingly, in the case of autoimmune and inflammatory disease, the same chemokines recruit cells to sites of inflammation and, therefore, become mediators of the inflammatory response. Different cell types are recruited by different chemokines. Neutrophils, typically the first cells to be recruited to an inflammatory site, express CXCR1 and CXCR2 and are recruited by CXCL1, CXCL2 and CXCL8. The switch from a neutrophil-mediated acute inflammatory response to a chronic inflammatory response is mediated by monocytes and macrophages that express CCR1 and CCR2 and migrate in response to CCL3, CCL5, CCL7, CCL14 and CCL2, CCL7, CCL8, CCL13, respectively. T cells that express CXCR3 and CCR5 are recruited by CXCL9, CXCL10 and CXCL11, ligands for CXCR3; and CCL3, CCL4 and CCL5, ligands for CCR5 [22]. In allergic disease, such as asthma, eosinophils are recruited by CCL11 acting via CCR3 [23]. Although this is not a comprehensive list, it can be seen that chemokines and their receptors play a major role in the inflammatory response and are potential therapeutic targets in these diseases [10,23]. The homeostatic chemokines have also been shown to be mediators of the inflammatory response. CXCL12 and its receptor, CXCR4, have been implicated in cellular recruitment in rheumatoid arthritis [24–27] and asthma [28]. CCR7 and CXCR5 are responsible for secondary lymphoid organ formation by recruitment and positioning of T and B cells, respectively [29,30]. The appearance of ectopic or tertiary lymphoid structures, lymphocyte clusters with lymphoidlike organization, have been found at sites of inflammation in autoimmune disease and both secondary and tertiary lymphoid organs have been postulated to be involved in autoantibody production. Both receptors have been implicated in the pathology of autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis [31]. Interestingly, chemokines and their receptors play a role in metabolic-related disorders [32,33]. The initiating step in the formation of an athero sclerotic plaque is the recruitment of monocytes in response to chemokines produced by endothelial and smooth muscle cells, in response to oxidized LDL. CCR2 is the predominant chemokine receptor involved in trafficking and recruitment of monocytes to sites of inflammation, and studies using both and CCR2-/- and CCL2-/- (the future science group

Chemokine receptor modeling: an interdisciplinary approach to drug design

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Figure 1. Chemokine receptors and their chemokine ligands. The systematic and descriptive names are given for the chemokines. The chemokine receptors are subdivided into the four classes, CC, CXC, C and CX3C for the typical chemokine receptors on the left. Atypical chemokine receptors are on the right. DARC: Duffy antigen receptor for chemokines.

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Review | Fricker & Metz Key Term Molecular modeling:

Molecular modeling describes, in broad terms, the utilization of theoretical methods and computational techniques to investigate the properties of molecules. Structure-based design is a component of molecular modeling that most commonly describes the binding of molecules (proteins and small molecules) with force field and molecular dynamics or Monte Carlo simulation methods in three dimensions to a target, such as a protein. The structure of the target can be obtained by applying spectroscopic methods, such as NMR and x-ray crystallography or using comparative homology modeling techniques. Computational tools can be used to generate binding hypotheses and the suggested compounds once synthesized by medicinal chemistry can help to define binding models to explain structure activity data.

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ligand for CCR2) gene knockout in mouse models of atherosclerosis have provided strong evidence for the role of CCR2 in atherosclerosis. In addition, both CX3CR1 and CCR5 and their ligands CX 3CL1 and CCL5, respectively, have been demonstrated to play a part in the development of the atherosclerotic plaque [34,35]. These three chemokine receptors appear to play distinct as well as overlapping roles in monocyte recruitment and adhesion and atherosclerotic plaque formation. Low-grade inflammation and macrophage infiltration into adipose tissue also play a role in the development of insulin resistance, Type 2 diabetes and related obesity [32,33]. Ligands for CCR2 (CCL2) and CCR5 (CCL5) are found in adipose tissue and animal studies support a role for CCR2- and CCR5-mediated monocyte recruitment to adipose tissue in obese fat-fed mice. A CCR2 antagonist, CCX140-B, has recently completed a Phase II clinical trial in Type 2 diabetic patients [33]. Cancer cells can co-opt chemokine receptors for their own use. The first indication that chemokines and their receptors could be involved in cancer was the discovery that the receptor CXCR4 could direct the metastasis of breast cancer cells [36]. Since then, CXCR4 has been implicated not only in cancer metastasis but also in cancer survival at the metastatic site, where there is high expression of CXCL12, such as lymph nodes, lungs, liver or bone marrow. Other chemokine receptors have also been found to play a role in organ specific metastases of various malignancies including CCR5, CCR7, CXCR3 and CXCR5 [37,38]. In addition, chemokine receptor expression is important on supporting stromal cells. CXCL12 can also recruit CXCR4-expressing endothelial precursor cells and angiogenic-supporting cells, therefore promoting vasculogenesis and supporting tumor growth [38–40]. The ligands for CXCR1 and CXCR2 can also promote angiogenesis [41]. Chronic inflammation predisposes to some forms of cancer [42]. Triggers for cancer-related chronic inflammation can include infections such as Helicobacter pylori, which is associated with gastric cancer. Cancer-related inflammation is associated with chemokinemediated accumulation of inflammatory cells, such as tumor-associated macrophages, which are recruited by CCL2 [43]. As in cancer, HIV, the causative agent of AIDS, has exploited chemokine receptors on host cells to facilitate infection. The gp120 subunit of the HIV envelope glycoprotein binds Future Med. Chem. (2014) 6(1)

sequentially to proteins on the host cell, initially to CD4 and then to a chemokine co-receptor [3]. Although several chemokine receptors have been found to mediate HIV infection in vitro, CCR5 and CXCR4 are the two clinically relevant chemokine receptors [2]. There is no complete cure for AIDS as yet, although the infection can be successfully managed by a combination of nucleoside analog reverse transcriptase inhibitors, with either a non-nucleoside reverse transcriptase inhibitor or a protease inhibitor [44]. This regime is known as highly active antiretroviral therapy (HAART). However HIV has a highmutation rate and mutations in these targeted proteins lead to drug resistance, so there is frequently a need to change the treatment regimen [45]. Furthermore, the virus cannot be totally eradicated due to a latent reservoir of provirus that persists in reservoirs, such as CD4+ T cells, which can reactivate upon cessation of HAART [46,47]. With the success of HAART, HIV has now become a chronic treatable disease in the Western world, however, there is still a need for multiple agents with different mechanisms of action to overcome the above challenges. Chemokine receptors therefore appear as attractive therapeutic targets. Yet, in spite of this, there are only two chemokine receptor antagonists approved for clinical use: maraviroc, a CCR5 antagonist, and plerixafor, a CXCR4 antagonist, suggesting that chemokine receptor drug discovery is challenging [20,21]. One of the features of the successful discovery of both these drugs has been our understanding of how they interact with their respective receptor targets. In this article, we will review the role that molecular modeling has played in this understanding and how it can facilitate drug design. In particular, we will describe how our efforts in developing different CXCR4 small-molecule antagonists, and our studies on how these molecules interact with this chemokine receptor at a molecular level, provided the foundation for using molecular modeling for the prospective design of novel CCR5 antagonists. Molecular modeling approach to GPCR drug design The therapeutic and commercial significance of GPCRs for the pharmaceutical industry is reflected in that approximately half of the drugs currently on the market target GPCRs; it is, therefore, not surprising that approximately 30% of the drug-discovery efforts are dedicated toward this class of receptors. However, future science group

Chemokine receptor modeling: an interdisciplinary approach to drug design despite considerable successes, GPCRs should not be considered as the low-hanging fruit of medicinal chemistry and drug discovery. The identification of novel GPCR-targeted drugs faces many challenges, including selectivity. Recent advances in the molecular modeling of drug–GPCR interactions can provide new insights and guidance for drug discovery and medicinal chemistry and there is an increasing number of examples of drug-design efforts from lead identification by means of virtual screening to the final stage, lead optimization through the interplay of molecular biology, medicinal and computational chemistry [48–51]. Until recently, molecular modeling of GPCRs was dependent upon the published crystal structure of rhodopsin, the archetypal 7TM protein. This process has now been facilitated by the elucidation of several high-resolution x-ray crystallographic structures of GPCRs, including those of the chemokine receptors CXCR4 and CCR5, enabling enhanced in silico modeling and ligand docking. These structures, together with biochemical and biophysical techniques, such as site-directed mutagenesis, chimeric receptors, surface plasmon resonance, NMR, EPR and hydrogen-deuterium exchange mass spectrometry, allow us to explore drug–receptor interactions at the molecular level and to use this information for drug design. As introduced above, the assistance of these projects by computational efforts has significantly improved with the determination of the first crystal structure of a GPCR, bovine rhodopsin [52]. Although the sequence similarity of bovine rhodopsin to other class A GPCRs is less than 25% (e.g., CCR5 and CXCR4) these receptors share a common fold, the 7TM region. This similar tertiary structure is maintained through structural mimicry. This is supported by several observations obtained using several different methodologies, such as the substituted-cysteine accessibility method, which provided good agreement between accessible residues found in the dopamine D2 receptor and the rhodopsin structure; cysteine crosslinking; double revertant mutants; engineered His2Zn2+-binding sites; and spin labeling and ligand receptor interactions obtained by single-point mutations [53,54]. However, local differences in the 7TM region between the structural template and any of the class A GPCRs under investigation have to be accounted for. For example, the kink angle of the extracellular part of TM2 in chemokine receptors is different to that in future science group

| Review

rhodopsin, which is caused by a TXP motif in the former compared with a GGP motif in the latter. This causes TM2 to reorient relative to the other TM helices [55]. Other structural parts of class A GPCRs, such as the three extracellular loops (ECL), the three cytosolic loops, the N-terminal domain and the cytosolic C-terminal domain are less structurally conserved [49]. Using only the 7TM region of bovine rhodopsin as a structural template for comparative homology modeling, and obtaining the structurally variable regions by means of different loopmodeling techniques, has provided receptor models which have proved useful for virtual screening. Rather surprisingly, despite the fact that the available structure of bovine rhodopsin represents the inactive form, virtual screening campaigns with homology models based on this structural template have yielded small-molecule agonists and antagonists [51,56,57]. In the last 5 years, several crystal structures of other class A GPCRs have been determined, including the adrenergic receptors b2 AR, b1AR, adenosine A2A, D3 dopamine, CXCR4, CCR5, histamine H1, sphingosine-1 phosphate, M2 and M3 muscarinic, and the µ opioid receptors. Depending on the GPCR under investigation, this enables the creation of more reliable in silico homology models by better describing the 7TM domain structure as well as comparatively modeling additional structural domains [51,58,59]. With the availability of more GPCR crystal structures of targets, which importantly are also targets for drug discovery, such as b2 AR, A2A, D3, CXCR4 and CCR5 receptors, it was possible to conduct docking competition experiments that revealed that computer-aided techniques are well suited for this class of targets [60]. Since it is realistically not possible to obtain crystal structures for every GPCR, discovery projects would greatly benefit from current advances in structural biology of GPCRs in combination with computer-aided techniques [59]. Besides structural biology, biochemistry plays a very important part in identifying the antagonist, agonist and allosteric binding pockets of GPCRs. Mutagenesis helps to identify residues crucial for small-molecule binding. One commonality across the whole family of class A GPCRs is that small molecules bind on the extracellular side of the 7TM core, beneath the ECL2. These results are important for binding-site definitions for virtual screening. Lead optimization efforts benefit from the readily www.future-science.com

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Review | Fricker & Metz Key Term Site-directed mutagenesis: Technique that enables the selected and specific changes or mutation in the DNA sequence of a gene. This will result in the coding of an alternative amino acid in a protein. The most commonly used approach typically utilizes polymerase chain reaction-based methods using primers designed containing the specific mutation. The technique of site-directed mutagenesis has been used to characterize gene and protein structure–function relationships, protein–protein interactions, enzyme active sites, and binding domains of proteins.

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available experimental data, which can be used to refine small-molecule binding (vide infra). While progress in computational and spectroscopic tools, and utilization of a multidisciplinary approach to medicinal chemistry and the drug-discovery process all serve the purpose of reducing timelines and costs for drug discovery, the major bottleneck for new therapies targeting GPCRs in general, and chemokine receptors more specifically, remains the proof of concept linking disease pathology to the specific target. This ultimately depends on our knowledge of the role of chemokine biology in human disease. Chemokine receptor models & their role in drug design The availability of GPCR crystal structures provides good starting points for structurebased computational support [51,61]. Furthermore, the use of computational techniques can be optimized if biological data of the target are available. Biochemistry is, therefore, another key player for identifying small-molecule binding sites. In this review, we will present examples of binding site studies and prospective lead identifications for a GPCR subfamily, the chemokine receptors. Examples how biochemical data can be used for lead optimization are presented later. Chemokine receptors as typical GPCRs can exist in multiple conformational states from an activated ligand to inactive [62]. In addition, they can be post-translationaly modified, notably by tyrosine sulfation or N-glycosylation at the N terminus [63]. This is significant for cognate ligand interaction as it is generally accepted that chemokines bind to their receptors in a two-stage process: first, by binding the core domain of the chemokine protein to the N-terminal domain of the receptor; followed by the second step, in which the flexible N-terminus of the chemokine interacts with the extracellular domain and TM regions of the receptor [9]. Different conformations of both CXCR4 and CCR5 have been demonstrated based on antibody recognition [62,64]. It has been proposed that HIV may exploit these different conformations for viral entry in the presence of CCR5 inhibitors. This conformational complexity should be borne in mind when considering that much of the chemokine receptor homology modeling until recently was based upon the crystal structure of the inactive state of rhodopsin. In spite of this apparent limitation, Future Med. Chem. (2014) 6(1)

molecular modeling of chemokine receptor/ inhibitor interactions has proved to be a useful tool to guide medicinal chemistry and, given the relative recent publication of the CXCR4 crystal structure in 2010 [65], it is not surprising that the majority of work has been undertaken with other structural templates, such as bovine rhodopsin or b2 AR. Several studies confirmed the suitability of these crystal structures as template (vide supra) and this has been confirmed by mostly virtual screening results and a few lead optimization examples [51,56,57,66–69]. The CXCR4 crystal structure and a homology model were used for screening for prospective small-molecule antagonists. Not surprisingly, crystal structure based docking performed better in identifying several specific potent compounds with IC50 values as low as 306 nM, while homo logy-based docking yielded only one nonspecific hit structurally related to known CXCR4 antagonists. The results are in support of structurebased efforts to discover lead compounds for chemokine receptors [70]. A new allosteric binding site in CXCR2 was identified by using different receptor orthologs, chimeric proteins, site-directed mutagenesis and in silico modeling. The homology model was created using the CXCR4 crystal structure as template. This new binding site is comparable to small-molecule binding sites found in other chemokine receptors, such as CCR5 and CXCR4 [71]. Targeted photo-crosslinking between CCR5 and maraviroc was used to study ligand–receptor interactions. This method introduced minimal modifications to the small molecule and receptor and, therefore, could complement existing biophysical methods. It is also of interest that a potential second binding site of maraviroc on the extracellular surface of CCR5 was determined. This could be further investigated by mutagenesis and computational studies. In general, this method seems promising in identifying the binding site of GPCR allosteric modulators [54,55]. Surface plasmon resonance spectroscopy was applied to identify novel fragment-like antagonists for CCR5. This biosensor assay protocol can be applied for screening other GPCRs. It has the advantage of directly measuring small molecule–receptor interactions and can complement other approaches that rely on functional responses [72]. The understanding of how ligands interact with GPCRs can also extend to atypical chemokine receptors. Both DARC and D6 can bind future science group

Chemokine receptor modeling: an interdisciplinary approach to drug design several chemokines, for example DARC can bind up to 20 CC and CXC chemokines [15]. It is, therefore conceivable that an understanding of the promiscuous binding mode of DARC and D6 may provide insights into the design of novel chemokine receptor inhibitors [73]. A challenge with these atypical receptors is that they do not signal in a conventional manner hence assessing biological activity of inhibitors is challenging. However ligand binding and b-arrestin recruitment have been successfully used to evaluate novel CXCR7 antagonists [74–76]. This atypical receptor binds CXCL12 and CXCL11 and has been implicated in tumor survival and development [74], as well as been hypothesized to regulate the CXCR4mediated response to CXCL12 [77]. Inhibitors of CXCR7 have been designed using a novel homology modeling method based on using five GPCR crystal structures, including CXCR4 [76]. While these new methods certainly hold promise to increase understanding of how agonists and antagonists bind to GPCRs, more case studies, especially prospective studies, will allow us to fully evaluate the potential of these approaches. The following sections will provide detailed examples of the interplay between mutagenesis and molecular modeling. This approach was used on CXCR4 for calibrating small-molecule interactions at molecular level, and it was prospectively applied for CCR5 for defining binding-site interactions for compound design and optimization. Modeling CXCR4 inhibitor interactions CXCR4 inhibition as a target for therapeutic intervention in disease It had been known that HIV entry was mediated not only by gp120 binding to CD4, but also required involvement of one or more co-receptors.

| Review

In 1996, two co-receptors were identified, the chemokine receptors CCR5 and CXCR4. HIV infection is a complex multistep process in which the viral envelope proteins, gp120 and gp41, interact with host-cell membrane proteins. Initially the gp120 protein binds to CD4 on the host cell. This is followed by a conformational change, which allows gp120 to bind to a coreceptor, CCR5 or CXCR4. This is followed by a further conformational change resulting in insertion of gp41 into the cell membrane and promotion of viral fusion and cell entry. The virus that uses CCR5 as a co-receptor is characterized as CCR5-using or a R5 virus, and a virus that uses CXCR4 is referred to as CXCR4-using or a X4 virus. CCR5 is the principal co-receptor for viral transmission. CXCR4 usage emerges in the later stages of the disease process and is associated with a rapid decrease in CD4 T-cell count and accelerated disease progression [2]. Interestingly, the first non-peptidic smallmolecule inhibitor of either co-receptor was identified through a drug-discovery program for antiviral, HIV-specific, drugs. It was initially identified as a potent inhibitor of HIV infection with activity against T-tropic strains of HIV with EC50 values in the range of 2.5–12.5 nM. It was only with the later discovery of the viral use of CXCR4 and CCR5 as co-receptors for CD4 that the mechanism of action was elucidated. Therefore, plerixafor, formerly known as AMD3100, became the first small-molecule CXCR4 antagonist [78]. Molecular pharmacology studies confirmed that plerixafor could inhibit ligand binding to CXCR4, inhibit CXCL12 (SDF-1) mediated G protein binding and chemotaxis (Table 1) [79,80]. A Phase I/II clinical trial in HIV-infected patients both validated CXCR4 as a therapeutic target for HIV and demonstrated the efficacy of plerixafor to reduce the X4 viral load in HIV-1 infected subjects [81].

Table 1. Comparison of the properties of three CXCR4 inhibitors. CXCR4 inhibitor

Ligand Calcium flux‡ GTP binding† (nM) binding‡ (nM) (nM)

Plerixafor 651 AMD3465 41.7 AMD11070 12.5

572 12.1 9.0

27.3 10.4 39.8

Chemotaxis‡ HIV Oral (nM) activity § bioavailability (nM) (%) 51 8.7 19.0

7.4 6.1 10.1

22/80 ¶

Inhibition of 125I-SDF-1/CXCL12 ligand binding to CXCR4 on CCRF-CEM cells. Result is the IC50 , concentration (nM) giving 50% inhibition of ligand binding. ‡ Inhibition of SDF-1/CXCL12-stimulated responses in CCRF-CEM cells. Results are expressed as IC50 , concentration giving 50% inhibition. § Inhibition of HIV infectivity of HIV-1 NL4.3 virus strain in peripheral blood mononucleated cells. Result is expressed as IC50 , concentration giving 50% inhibition. ¶ Percent of oral bioavailability in two species: rats/dogs. †



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Review | Fricker & Metz Key Term Mobilization of hematopoietic stem cell:

Cells of the immune system are derived from hematopoietic stem cells (HSCs) by a process known as hematopoiesis. In adults this process takes place in the bone marrow. Leukemia and lymphoma are diseases of the cells of the hematopoietic system. In many cases the only treatment option is a HSC transplant. One method of obtaining HSCs is to mobilize them from the bone marrow into the peripheral blood and then collect them by a process known as apheresis. The collected cells are then frozen and stored until the time of transplant. Techniques for mobilization include the use of cytotoxic chemotherapeutic agents and/or cytokine growth factors, such as G-CSF. HSCs express the chemokine receptor CXCR4, which plays a key role in the retention of HSCs in the bone marrow. The CXCR4 antagonist plerixafor is an approved drug for HSC mobilization in combination with G-CSF for autologous transplant for patients with multiple myeloma and non-Hodgkin’s lymphoma.

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In the case of HIV, the virus has usurped the function of a naturally occurring protein on the host cell. However, CXCR4 plays a fundamental role in normal hematopoiesis and embryonic development. This was first demonstrated by knockout studies in mice in which either Sdf-1/ Cxcl12 or CxcR4 gene knockouts die in utero. The principal defects observed were impairments in myelopoiesis and lymphopoiesis, cerebellar development, cardiac development and vascularization of the GI tract [82]. The CXCL12/ CXCR4 chemokine/chemokine receptor pair is an important component of the mechanism of retention of hematopoietic stem cells (HSC) in the bone marrow [83–85]. Inhibition of CXCR4 by plerixafor results in mobilization of HSCs from the bone marrow into the circulation. HSC transplantation is a significant component of the treatment options for hematological malignancies, such as leukemias and lymphomas. Direct aspiration and collection from the bone marrow was the most common method to obtain HSCs, however, HSC mobilization is now often a preferred, less painful, method for collecting HSCs for transplant. There are two types of transplant, autologous and allogeneic. In an autologous transplant the recipient receives their own HSC, which have been previously harvested and stored; whereas for allogeneic transplant the recipient receives cells from a healthy human lymphocyte antigenmatched donor, frequently a sibling. Allogeneic transplants are primarily used to treat leukemias, such as acute lymphoblastic leukemia, and acute and chronic myeloid leukemia, and is often the only curative treatment option. Auto logous transplants form part of the treatment of non-Hodgkin’s lymphoma (NHL), Hodgkin’s disease, and multiple myeloma (MM) [86]. In this instance HSCs are first collected from the patient who is then treated with myeloablative high-dose chemotherapy and/or total body irradiation, which not only destroys the diseased cells but also the hematopoietic cells. The bone marrow is then reconstituted by transplanting the previously collected HSCs. Commonly used methods for HSC mobilization are treatment with cytokines such as G-CSF either with or without chemotherapeutic agents, however, up to 30% of patients may fail to mobilize enough cells for a transplant [86]. Treatment with plerixafor and G-CSF was shown to significantly increase HSC mobilization compared with G-CSF alone, both in healthy volunteers and in patients with NHL and MM. Plerixafor was evaluated in two Future Med. Chem. (2014) 6(1)

successful randomized Phase III clinical trials and was subsequently approved by the US FDA in 2008 in combination with G-CSF, to mobilize HSC to the peripheral blood for collection and subsequent autologous transplantation in patients with NHL and MM. This was followed by approval in Europe by the European Medicines Agency in 2009 [87–89]. CXCL12 is regarded as being a homeostatic chemokine because it is constitutively expressed and is integral to developmental processes, including the development of the immune system. However, CXCL12 and CXCR4 have been found to be involved in leukocyte recruitment in autoimmune and inflammatory disease. CXCR4 and CXCL12 expression is elevated on CD4+ T cells in the synovium of patients with rheumatoid arthritis and contributes to T-cell recruitment [22]. CXCR4 has also been implicated in leukocyte recruitment in other inflammatory diseases, such as uveitis and asthma. Treatment with the CXCR4 inhibitor, plerixafor, attenuated disease in rodent models of rheumatoid arthritis and asthma [26–28]. There are a number of similarities between the biology of tumor progression and inflammatory disease, including increased cytokine and chemokine expression, and cell migration [90]. CXCR4 expression has been demonstrated on hematological cancers, and on a variety of solid tumors of different tissue origins [39,91]. CXCR4 has been proposed to act as a proliferative and survival factor, a mediator of tumor vasculogenesis, and to direct and promote metastasis. Positive outcomes have been observed using CXCR4 blockade by antibodies in mouse cancer models using human tumor xenografts. Treatment with anti-CXCR4 antibodies has been shown to be curative in a model of NHL [92], and to inhibit metastasis in a model of breast cancer [36]. Plerixafor has been shown to be able to mobilize leukemia cells and treatment with plerixafor in combination with cytotoxic therapy was shown to decrease leukemia burden in a model of acute promyelocytic leukemia. Similarly, treatment with the CXCR4 inhibitor AMD3465 increased the sensitivity of FLT3 mutated acute myeloid leukemia cells to the kinase inhibitor sorafenib. Preliminary clinical studies hint at the possible role of CXCR4 inhibition to sensitize leukemia cells to chemotherapy. Defining

the plerixafor binding site Plerixafor is a bicyclam with two 1,4,8,11-tetraazacyclotetradecane (cyclam) rings linked by a 1,4-phenylenebis(methylene) moiety (Figure 2) future science group

| Review

Chemokine receptor modeling: an interdisciplinary approach to drug design [78,93,94].

It has a low molecular weight (502 for the free base) and at physiological pH it is partially protonated giving it two positive charges on each cyclam ring, with an overall charge of +4, and is subsequently water soluble. The single 1,4,8,11-tetraazacyclotetradecane ring has an overall positive charge of +2 at physiological pH. Structural studies have demonstrated that the protonated cyclam ring has the ability to form a direct, hydrogen-bonded stabilized complex with carboxylic acid groups. The cyclam rings can also chelate metal ions, and transition metal complexes of plerixafor have an enhanced binding affinity for CXCR4. It was hypothesized that this may allow plerixafor to interact with metal chelating amino acid residues such as histidine. The hypothesis that plerixafor could interact with CXCR4 in the aforementioned fashion was supported by observations that mutations of acidic amino acids in the ECL2 and TMIV of CXCR4 conferred resistance to plerixafor. Therefore, the potential consequence of the localized positive charges of plerixafor is the opportunity for charge–charge interactions with the target receptor CXCR4 [95]. This makes plerixafor somewhat unique as a nonpeptide GPCR antagonist. The majority of nonpeptide antagonists of other GPCRs are more hydro phobic (this concept will be explored further when considering CCR5 antagonists). The binding mode of plerixafor to CXCR4 was probed by site-directed mutagenesis of selected aspartate and histidine residues within TM regions III, IV, V and VII and ECL2, and the effect of these mutations on the binding of the either radiolabeled Met-SDF-1a or the CXCR4-specific antibody 12G5 to the receptor [95]. Aspartates were replaced with asparagine, and histidines with alanine. Comparative studies were performed with a series of cyclam analogs, including the single ring cyclam. It should be noted that the binding affinity (K i) of cyclam to CXCR4 is several orders of magnitude less than plerixafor (13 µM compared with 74 nM for plerixafor using 125I-Met-SDF-1a). Cyclam had no effect on 12G5 binding to CXCR4, whereas plerixafor inhibited 12G5 binding with a K i of 550 nM. The 12G5 antibody is CXCR4conformation dependent and recognizes epitopes on the first- and second-extracellular loop, whereas CXCR4 has been postulated to interact with the TM regions of the receptor [9,64]. As the binding of the two ligands, Met-SDF-1 and 12G5 antibody, were affected differently by the bicyclam plerixafor and the monocyclam this future science group

H2

H2 N+ N+

H2 N+

N+ H2

H

N

N H2 +

N

N

H2+ N

N N

HN

NH

N+ H2

Plerifaxor

AMD3465

O

N

N N H+ N

NH3+

N+ H

Br

N

NH AMD11070

O-

O

Schering-C

O N+

O

F

N+

O

N

F

N+ H

N H

N N

F

N O

Vicriviroc

TAK779

F F

O N

O -

O

O

NH N

NH NH+

O

OH

N

O

Aplaviroc

N N

Maraviroc

Figure 2. Structures of chemokine receptor inhibitors: CXCR4 inhibitors plerixafor (AMD3100), AMD3465, AMD11070; and, CCR5 inhibitors SCH-C, TAK779, aplaviroc, vicriviroc and maraviroc.

further indicates that the interaction of these ligands with CXCR4 are not identical. The mutation D171N in TMIV abrogated inhibition of Met-SDF-1a binding by both plerixafor and cyclam. Interestingly, plerixafor inhibition was also abrogated by the D262N mutation in TMVI. None of the histidine mutations had a significant effect on plerixafor inhibition. Based on these observations, a binding mode was proposed in which the binding for plerixafor to CXCR4 was defined by Asp171 in TMIV and Asp262 in TMVI, where each of the cyclam moieties in plerixafor binds to one of the aspartates via interaction of the aspartate carboxylate with a protonated nitrogen of a cyclam ring (Figure 3A). www.future-science.com

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Review | Fricker & Metz

D171

W94 D97

D171

W94

D171 W94

Y116

Y116 E288

Y45

D262

E288 D262

Y45

Y45

D262 E288

H281

D171

D171

D171

W94 D262

Y116

Y116

E288

E288 D262

Y45

Y45

D262

W94 D97

W94

Y45

E288 H281 I W94

D171

D171

D171 D97

Y116 W94 Y45 D262

Y116 D262

Y45

D262

E288

W94

E288 Y45

E288

Figure 3. Predicted binding modes. (A–C) Plerixafor, (D–F) AMD3465 and (G–I) AMD11070. Reproduced with permission from [97] .

The unique polar binding interaction of plerixafor with CXCR4 among chemokine receptors is presumably responsible for the selectivity of plerixafor for CXCR4 over other chemokine receptors; plerixafor does not significantly bind to or affect the function of other chemokine receptors, including the closely related CXCR3. The plerixafor binding site was further elaborated in an elegant study in which the binding site for plerixafor was transferred onto CXCR3 [96]. Additional mutagenesis studies identified Glu288 on TMVII as another possible acidic amino acid capable of interacting with a cyclam ring of plerixafor. This amino acid triad, AspIV:20, AspVI:23 and GluVII:06 (defined by their relative positioning within the TM regions) is unique to CXCR4. CXCR3 has two equivalent residues; AspIV:20, AspVI:23, 100

Future Med. Chem. (2014) 6(1)

but has a serine (Ser340) at the position VII:06. In addition, CXCR3 has a lysine at position VII:02 (Lys300) which could potentially form a salt bridge with AspIV:20 (Asp186 on CXCR3), therefore neutralizing one of the two aspartate residues. In order to reconstruct the CXCR4 binding site, two mutations were introduced into CXCR3; K300A and S304E. Using phosphatidyl–inositol turnover as a functional readout of receptor activity it was demonstrated that this reconstructed CXCR3 responded to the CXCR3 ligands IP-10 and ITAC, and that this response could now be inhibited by plerixafor, in contrast to the lack of effect on the wild-type CXCR3. These data now indicated that plerixafor interacted with CXCR4 via three acidic anchor points, two at one end of the binding pocket, and a third at the opposite end. future science group

Chemokine receptor modeling: an interdisciplinary approach to drug design These three interactions were independently confirmed using site-directed mutagenesis together with inhibition of 125I-CXCL12 binding to CXCR4 (Table 2). However, in the same study, additional interactions were identified suggesting two alternative binding modes, that further extend into the TMI, TMII and TMVII regions [97]. In both binding modes one of the double protonated cyclam rings potentially forms a cationic p–interaction with the aromatic side chain of Trp94 in TMII and a hydrogen-bond interaction with Tyr45 in TMII. In one of the proposed alternative binding modes, the second cyclam ring utilizes Asp171 as an interaction site (Figure 3B). In another proposed binding mode, the second cyclam ring forms an ionic hydrogenbond interaction with Asp262 and an additional p-stacking interaction is proposed between the phenyl spacer and Tyr116 in TMIII (Figure 3C). From a practical pharmacology perspective, the importance of these interactions on plerixafor inhibition of HIV infection was investigated. HIV entry was abrogated by the D262N mutation and the D171N/D262N double mutation, demonstrating that these sites on CXCR4 also have an influence on HIV entry, possibly via the interaction of the highly basic V3 loop of gp120 of some X4 virus strains with CXCR4 [98]. Evolution

of an orally bioavailable CXCR4 antagonist Plerixafor is not orally bioavailable and has to be administered either intravenously or subcutaneously. Although this is acceptable for HSC mobilization an orally bioavailable drug is to be preferred for other applications such as anti-HIV therapy. The strategy that was adopted to confer oral bioavailability was the redesign of the azamacrocyclic pharmacophore, while maintaining the critical interactions with the basic amino acid residues identified by the mutagenesis and modeling studies. Structure–activity studies indicated that there was structural redundancy in the cyclam structure and that all four nitrogens were not required for anti-HIV activity [93]. Therefore, this facilitated the reduction of the number of basic amine groups and, hence, the overall positive charge at physiological pH. The simplest replacement for a cyclam ring was reasoned to be a diamine-segment, representing the first two amino groups of the macroc yclic ring. However, whereas the introduction of a benzylamine group substantially lowered antiHIV activity, the introduction of a pyridyl group restored anti-HIV activity. In addition, it was future science group

| Review

discovered that the positional specificity of the pyridine N was important and that 3-pyridyl and 4-pyridyl analogs were inactive, whereas the 2-pyridyl had potent activity. This molecule, AMD3465 (Figure 2), which is an asymmetrical molecule with one monocyclam replaced by an aminomethylpyridine moiety, retained potent CXCR4 inhibition demonstrating that CXCR4 inhibition could still be achieved with a molecule containing a single cyclam ring [99–101]. The binding site of AMD3465 was probed by site-directed mutagenesis in two separate studies, using sets of amino acid substitutions that were identical to both and those that were exclusive to each study, therefore, providing an opportunity to extensively map the AMD3465 binding site. Using a combination of ligand (125I-12G5 antibody) binding and receptor function assays demonstrated the dependence on the tripartite binding site of Asp171, Asp262 and Glu288 [97,102]. This binding motif was confirmed using a competition binding assay with 125I-CXCL12 (Table 2). Importantly, additional amino acid interactions were identified with His281 on TMVII being the most significant. The importance of this residue was confirmed by competition binding assays in two separate studies. Interestingly, it was found that the functional assays, such as receptormediated phosphatidyl-inositol turnover or calcium flux, were less sensitive to mutations in the receptor and, hence, presented a less complex mutational analysis. Again, His281 was identified as a key interaction point for Table 2. Effects of CXCR4 single-site mutations on 125I-SDF-1a/ CXCL12 competitive binding of CXCR4 inhibitors. Location

Mutant

Plerixafor†

AMD3465†

AMD11070†

TMI TMII TMII TMIII TMIII TMIV ECLII ECLII ECLII TMV TMVI TMVI TMVII TMVII

Y45A W94A D97N Y116A Y121A D171N N176A R183A V196A H203A Y255A D262N H281A E288A

10.0 14.0 3.5 9.6 0.5 11.4 0.9 0.9 1.4 0.6 2.8 17.8 0.2 35.0

21.6 89.5 3.6 15.7 0.3 27.2 1.2 0.9 2.5 0.6 4.5 172.0 131.4 68.7

39.8 107.5 289.0 3.5 1.7 1248.6 0.8 0.14 2.5 0.9 1.2 12.5 2.2 460.3

Fold difference in Ki with respect to wild-type. ECL: Extracellular loop; TM: Transmembrane region. †


101

Review | Fricker & Metz AMD3465 using these functional readouts. Structure–activity studies with analogs of AMD3465 suggested that the cyclam moiety bound within the binding pocket defined by TM-III, -IV and -V. The identification of the interaction with His281 suggests that AMD3465 may occupy the CXCR4 receptor in a different fashion to plerixafor, and yet, similar to plerixafor, it is possible to dock the monocyclam into the binding site of CXCR4 in several ways (F igure 3D–F) . We hypothesized a potential aromatic interaction between His281 and the pyridine ring of AMD3465. As His281 is located at the interface of TMVII and ECL3 and is approximately two helical turns above Glu288, an ionic interaction with the protonated dibenzyl-amine is also possible. The double-protonated cyclam ring of AMD3465 can then interact with either Trp94 (F igure 3D) , Asp171 (Figure 3E), or Asp262 (Figure 3F) in the TM-III, -IV and -V binding pocket [97]. The above studies demonstrated that CXCR4 inhibition could be retained with only one cyclam ring, with partial redesign of the pharmacophore maintaining the crucial interactions responsible for the binding of the parent molecule plerixafor to the receptor. Although the overall positive charge had been reduced on AMD3465 to +2 to +3 compared with +4 for plerixafor, AMD3465 was still not orally bioavailable. The observation that not all four nitrogens were required for HIV activity suggested that further modifications could be made to the remaining cyclam ring. This led to further redesign to give an orally bioavailable potent CXCR4 inhibitor with antiviral activity, AMD11070 [103]. A MD11070 (N1-[1H-benzimidazol-2- ylmethyl]-N1-([S]-5,6,7,8-tetrahydroquinolin8-yl)-butane-1,4-diamine) is a nonmacrocyclic molecule (Figure 2). It has three major components radiating from a central nitrogen atom; a 4-amino butyl chain, benzimidazol-2-ylmethyl- group, and a 5,6,7,8-tetrahydroquinolin-8-yl- group. Mutagenesis studies again indicated the importance of the three amino acids, Asp171, Asp262 and Glu288, for AMD11070 interaction with CXCR4. However, these studies also identified three other amino acids, Asp97 and two aromatic amino acids Trp84 in TMII and Tyr45 in TMI, suggesting unique interactions for this molecule with CXCR4 (Table 2) [97]. Interestingly, Asp97 is required for HIV gp120 interaction with CXCR4. These data, therefore, suggest that AMD11070 when 102


protonated could interact with the negatively charged amino acids, Asp97, Asp171, Asp262 and Glu288. However, as with both plerixafor and AMD3465 no single binding mode can explain all the possible amino acid interactions identified from the mutagenesis studies, therefore, alternative binding modes have been proposed to accommodate this. In Figure 3G , the protonated primary amine of AMD11070 is shown forming an ionic hydrogen bond with Asp97 and cationic p-interaction with Trp94, while the benzimidazole can form a hydrogen bond interaction with Tyr45. In this binding mode, there are no direct interactions with the negatively charged amino acid triad of Asp171, Asp262 and Glu288 as proposed for plerixafor (F igure 3A) . In the alternative binding mode shown in F igure 3H , the primary amine of AMD11070 is now shown interacting with Asp171, while the benzimidazole forms aromatic interactions with Tyr45 and Trp94 in a binding pocket defined by TMI, TMII and TMVII. In contrast to this, the third binding mode is defined by ionic interactions between the primary amine and Asp262 and the tertiary amine and Glu288 (Figure 3I). Biochemistry and computational chemistry, therefore, supported medicinal chemistry in the evolution of small-molecule CXCR4 inhibitors from plerixafor, through AMD3465 to AMD11070 facilitating an understanding of their interactions with CXCR4. For each compound, the mutagenesis data could be used to develop binding hypotheses in which the retention of the necessary interactions with charged amino acid residues on CXCR4 were confirmed. Historically, many GPCR-targeted drugs act as orthosteric inhibitors, however, recently attention has been given to the many opportunities for allosteric interaction. Several smallmolecule antagonists of chemokine receptors, such as the CXCR1/2 inhibitors repertaxin and Sch527123 [104,105], and the CCR5 inhibitors TAK-779, SCH-C and aplaviroc [106–108], are allosteric inhibitors of their respective chemokine receptor. As the relative size of the natural chemokine ligands is 8–10 kDa, it is not surprising that these small-molecular-weight (350–500 Da) ligands are not competitive, orthosteric inhibitors, but exert their effect by interaction at an alternative site to that of the cognate ligand. CXCL12 is thought to bind to CXCR4 in a two-stage process, with initial binding of the core domain of CXCL12 future science group

Chemokine receptor modeling: an interdisciplinary approach to drug design to the N-terminus of CXCR4 with subsequent presentation of the unstructured N-terminus of the CXCL12 ligand to the extracellular loops and the TM region of CXCR4. The latter interaction is responsible for triggering chemokine receptor activation and signaling [9,109]. Plerixafor can displace the N-terminus of CXCL12 from CXCR4 but does not interfere with the binding of the chemokine core domain to CXCR4 [110]. Key amino acids involved in receptor signaling have been identified in ECL-2 and -3, and Asp97 as well as Glu288 have been identified as important TM interaction sites. AMD11070, therefore, can potentially interact with residues involved in CXCL12 interactions, but can also occupy unique binding sites, suggesting an allosteric, mixed-inhibition model [97,111]. Mechanistic studies with AMD11070 provided further support that it is an allosteric inhibitor of CXCR4 [111]. By comparing the dose–response of CXCL12 in the calcium flux assay, in the presence of increasing concentrations of inhibitor, it was found that the dose–response curves were depressed with increasing AMD11070 concentration, and the inhibitory effect of AMD11070 could not be overcome by high ligand concentrations. A comparison of the effect of the inhibition of CXCR4-mediated calcium flux stimulated by different CXCL12 isoforms provides further support for an allosteric mechanism. AMD11070 inhibits signaling via both ligands but with different K i values, which is indicative of an allosteric interaction. Therefore, both molecular modeling and biochemical studies support an allosteric mechanism for AMD11070. Modeling CCR5 inhibitor interactions CCR5 inhibition for therapeutic intervention in disease CCR5 differs from CXCR4, the other HIV co-receptor, in a number of fundamental ways. CCR5 has several ligands, including MIP-1a/ CCL3, MIP-1b/CCL4, RANTES/CCL5 and MCP-2/CCL8. The chemokine ligands for CCR5 are inflammatory chemokines (induced as components of the inflammatory response) unlike CXCL12, which is a homeostatic chemokine. CCR5 is expressed on multiple cell types, primarily T cells including effector/memory T cells, T helper 1 cells; natural killer and natural killer T cells; and antigen presenting cells, such as immature dendritic cells, and monocytes, macrophages and microglia [112–114]. The expression of CCR5 frequently follows a temporal future science group

| Review

pattern. For example, CCR5 is present on immature dendritic cells, which can take up microbes for antigen presentation. Upon maturation, the dendritic cells lose expression of CCR5 but increase expression of CCR7, which promotes homing to lymphoid tissue. The expression of CCR5 on cells involved in both the innate and adaptive immune response is compatible with a role in the normal immune response to infection. CCR5 has been associated with several diseases besides HIV. CCR5 and its ligands have been shown to have a role in several aspects of tumor biology. CCR5 mediates T-cell recruitment to tumors. However, whether this is beneficial or detrimental is unclear. Recruitment of CD8 T cells may be components of tumor surveillance and suppression. Alternatively, recruitment of immune suppressive T regulatory cells may be employed by the tumor to evade the immune response and facilitate tumor growth [115]. In this context, CCR5 and its ligands have been suggested to be prognostic indicators of poor outcome in several tumors. CCR5 has also been shown to mediate tumor/stroma interactions and has been implicated in tumor metastasis [116]. CCR5 and its ligands are involved in the inflammatory response directing monocytic cells and lymphocytes to sites of inflammation, and CCR5 has been associated with rheumatoid arthritis [22], Crohn’s disease [117] and multiple sclerosis [114]. However, CCR5 inhibitors have not been efficacious in inflammatory diseases with maraviroc and other CCR5 inhibitors failing to demonstrate efficacy in clinical trials for rheumatoid arthritis [118,119]. In contrast to this, the pursuit of CCR5 inhibitors for HIV has proven to be more fruitful. CCR5 is a focus for the pharmaceutical industry as a target for HIV, and much of the drug-discovery efforts around this receptor have focused on this indication [3,4]. Several inhibitors entered clinical trials culminating in the approval of maraviroc by the FDA for use in treatment experienced and treatment-naive patients with CCR5-using virus [120–122]. Several other CCR5 inhibitors have entered clinical trials against HIV, but with less successful outcomes related either to toxicity issues or lack of efficacy. A dual CCR2b/CCR5 inhibitor, TAK-779, failed because of poor pharmacological and/or toxicological properties as well as lack of oral bioavailability [123]. GlaxoSmithKline halted the development of its CCR5 inhibitor aplaviroc after patients in Phase II and III www.future-science.com

103

Review | Fricker & Metz trials experienced liver toxicity [124]. A Schering–Plough compound, SCH-C, did not progress beyond Phase I safety studies because of the observed prolongation of the QT interval, which is associated with acute ventricular arrhythmia and sudden cardiac death. A follow-on compound, vicriviroc, progressed further, but Phase II studies involving treatmentnaive patients were stopped because of increased likelihood of viral load rebound compared with a group of patients using standard therapy [125]. Following promising Phase II data in treatmentexperienced HIV-positive patients Phase III clinical trials were conducted. However, the results of these trials were disappointing with no efficacy gains when vicriviroc was added to optimized background therapy [126]. Merck, which took over development of the drug, stopped development of vicriviroc in 2010. Defining

the binding site for CCR5 inhibitors Several groups have worked to explore the binding sites of CCR5 inhibitors. The common features of these efforts have been a combination of site-directed mutagenesis of the CCR5 receptor, a biological readout, and molecular modeling based on the creation of a homology model most commonly based on the crystal structure of rhodopsin, the only available crystal structure at the time these studies were performed. The variables in these studies are the different inhibitors that have been studied and the biological readouts. The biological readouts used include ligand binding, HIV infectivity, single-cycle HIV entry and cell fusion. All of the readouts directly probe the interaction of the HIV gp120 interaction with CCR5, with the exception of ligand binding, which probes the interaction of inhibitors with the physiological ligand binding site. The HIV–inhibitor interaction is pharmacologically more relevant, but empirically ligand-binding studies have provided similar conclusions [106,107,127–131]. The common conclusion that unites all of these studies is that the inhibitors studied bind to CCR5 in two hydrophobic binding pockets defined by the extracellular face of the TM regions TMI–VII, below the extracellular loops ECL1 and ECL2. In contrast, the HIV gp120 primarily makes contact through ECL2 and the receptor N terminus. This suggests that in general the CCR5 inhibitors act as allosteric inhibitors, which is supported by receptor pharmacology studies [108]. 104


This small-molecule binding site in CCR5 is rather hydrophobic as exemplified by the lipophilic compounds with all the safety issues associated with lipophilicity (see above). In our own work, we built upon the foundation provided by the many publications on CCR5 inhibitors. We first explored the use of a cell–cell fusion assay, in which one cell expressed HIV-1 JRFL viral envelope protein and the Tat transcription factor (CHO-Tat10), and the partner cell line, which expressed both the primary receptor for the viral envelope protein, CD4 and the CCR5 co-receptor together with the LacZ gene under the control of the HIV-1 LTR promoter. Fusion of the two cells, therefore, represented the initial stage in viral infectivity, with a reporter readout, the expression of the enzyme b-galactosidase, which could be assayed with a chemiluminescent substrate. We validated the fusion assay and demonstrated that the results obtained using this assay agreed with those provided by alternative readouts, including ligand binding and viral inhibition, and, therefore, that the fusion-inhibition assay was a good model of the infection process. The advantages of this assay were a biologically relevant assay, the ability to use transient transfection of CCR5 and CCR5 mutant receptor and a simple readout allowing high-throughput testing of inhibitors [128]. We then proceeded to investigate the effect of fusion inhibition upon introduction of rationally selected single-point mutations. A mutagenesis/ modeling approach was applied, in order to map the binding site of known small-molecule allosteric CCR5 inhibitors, we expanded the number of single-site mutations over those previously reported so as to cover the whole of the potential binding site, including the TM regions, extracellular loops and the N-terminus. A ‘mutant fingerprint’ describing the dependency of CCR5 inhibitors on a set of single-site mutations was used to generate small-molecule interaction hypotheses (Table 3) . We evaluated several known reported inhibitors, including SCH-C, TAK-779, vicriviroc, maraviroc and aplaviroc (Figure 2). The latter was of particular significance as it allowed us to identify polar residues as potential interaction sites. These data confirmed the presence of two hydrophobic binding regions for the CCR5 inhibitors defined by TMI, TMII, TMIII and TMIII, TMIV, TMV, TMVI, TMVII, respectively (Figure 4). Although all the compounds share a common binding site, their mutant fingerprints were different suggesting the future science group

Chemokine receptor modeling: an interdisciplinary approach to drug design possibility of different ligand-binding modes. One common residue that was identified as being essential for almost all compound interactions was Glu283, which was determined to be at the center of the binding site. This glutamate is highly conserved in many chemokine receptors and appears to serve as a common anchor point for nonpeptide chemokine receptor inhibitors [132]. Upon mutation to alanine, the inhibition of the viral envelope/CCR5 interaction was significantly attenuated. An explanation provided for this observation was that the CCR5 inhibitors interact via their positively charged nitrogen with Glu283 forming a strong directional ionic hydrogen bond interaction. The significant exception to this was the dual CCR5/CCR2b inhibitor TAK-779. The dependencies of the other mutations were found to be somewhat less due to the nature of their interactions being mainly hydrophobic and therefore weaker. Of particular interest to future inhibitor design was the identification of the potential for ionic interactions of the carboxylic acid group of aplaviroc with lysine residues either in the N-terminus (Lys26) or extracellular loop 2 (Lys191) [128]. These latter ionic hydrogen-bond interactions are weaker than the Glu283 interaction as the lysines reside in the inherently more flexible loop regions (Figure 4). It remains to be seen if the differences in mutant fingerprints result in different HIV resistance profiles.

| Review

Table 3. The effect of single-site mutations on the CCR5-mediated cell–cell fusion inhibition by CCR5 inhibitors. Location Mutant† SCH-C† TAK-779† Aplaviroc† Vicriviroc† Maraviroc† N-terminal K26A L33A TM1 Y37A TM1 Y37F F79A TM2 W86A TM2 T105A TM3 Y108A TM3 Y108F F109A TM3 F113A TM3 K191A ECL2 I198A TM5 Y251A TM5 Y251F E283A TM7 T284A TM7

1.5 28 208 0.3 27 1367 17 60 9.2 1.9 1.3 8.9 75 0.3 0.2 2077 0.9

1.3 32 273 16 8.3 378 0.4 146 111 7.0 0.7 3.7 0.6 4.4 0.1 5.5 39

6.6 1.7 0.6 0.4 21 477 8.9 16 22 2620 8.8 6.3 110 0.2 1.9 1273 4.2

2.0 22 395 0.5 192 1205 2.9 51 46 27 9.8 2.0 83 3.3 0.4 12109 0.9

0.5 0.2 1.7 2.5 7.1 83 1.1 207 51 0.6 0.2 3.4 256 69 28 11118 5.7

Fold difference of mutant IC50 /wild type IC50. ECL: Extracellular loop; TM: Transmembrane region. Reproduced with permission from [128] © Elsevier (2011). †

design of CCR5 inhibitors. Two case studies are described that highlight the value of such experimental data in which the ionic interactions with either Lys26 and/or Lys191 are utilized to design compounds to overcome potential cardiotoxicity in one case, and to optimize antiviral activity in a second case [133]. Inhibition of the hERG ion channels is used as an in vitro predictor of QT prolongation in vivo and hence for cardiovascular toxicity. This is a frequently occurring problem in drug design and several strategies have been suggested to remove hERG inhibition from organic

Small-molecule

compound design using CCR5 binding-site hypotheses The information obtained through determining the mutant fingerprints of known CCR5 smallmolecule antagonists was used for the prospective

K26

E283

K191 Y251

L33 Y37

I198 W867

Y108

Figure 4. Proposed model of the CCR5 binding site shared by small-molecule antagonists. (A) For clarity, only the transmembrane region at the extracellular end is depicted together with other important pharmacophore elements; (B) representation without secondary structure; red and magenta spheres: polar binding site regions; cyan: hydrophobic regions. Reproduced with permission from [133] © American Chemical Society (2011).



105

Review | Fricker & Metz K26

Y251

E283

K191

E283

Y251

L33 L33 Y37

W86

Y108

Y37

Y108

W86

Figure 5. Proposed binding modes of compound 7 from the thiophene-3-yl-methyl urea series. hERG inhibition was abrogated by targeting binding to lysine groups on CCR5. (A) The carboxylic acid group interacts with Lys191; (B) Carboxylic acid interacts with Lys26. With exception of Lys26, which is used for binding-mode prediction, only residues are shown that have an impact on inhibition upon mutagenesis results. Reproduced with permission from [133] © American Chemical Society (2011).

molecules. One strategy is to reduce the lipophilicity by the use of zwitterions. A series of compounds based upon thiophene-3-yl-methyl urea was found to be potent CCR5 antagonists and inhibitors of R5 HIV-1 replication. However, members of this series were found to be inhibitors of hERG (1, 2 & 3; Table 4). The hERG activity in this series could be abrogated by attaching a carboxylic group via a linker therefore enabling interaction with Lys191 (5 & 7; Figure 5; Table 5) and alleviating the interaction with hERG (1, 2 & 3 vs 4, 7 & 9; Table 4). In

agreement with the predicted binding models, compounds were proposed that indicated that the linker needed to have a certain length in order to provide anti-virally active compounds (4, 5 & 7; Table 4). Based on the proposed binding mode the flexible linkers were successfully replaced by a benzyl linker. The proposed compound 9 was duly synthesized and shown to maintain potency in the fusion assay with wild type CCR5 receptor, and more importantly retain antiviral potency, while removing hERG inhibition (Table 4).

Table 4. Cell fusion inhibition, anti-HIV-1 (BaL) activity, cellular cytotoxicity and hERG inhibition of compounds of the thiophen-3-yl-methyl-urea series. S R1

H N

Cl

N O

H N

N

N O

R2

Compound

R1

R2

LogD RANTES binding Cell fusion IC50 (nM) IC50 (nM)

HIV-1 PBMC IC50 (nM)

PBMC CC50 (µM)

hERG IC50 (µM)

1

CH3

CH3

0.59

3.6

0.3

16.0

>40.6

2.8

2

CH3O

CH3

0.73

4.0

0.3

15.0

>35.2

4.0

3

CH3O

Cl

1.29

2.2

0.08

1.9

>37.8

1.9

4

HO2C(CH2)2

CH3

-0.92

nd

26.1

nd

nd

>40

5

HO2C(CH2) 3

CH3

-0.51

nd

2.3

690.0

>35.5

nd

6

H3CNHOC(CH2) 3

CH3

-0.11

nd

1.0

413.0

>34.6

>50

7

HO2C(CH2) 4

CH3

-0.06

nd

0.5

125.0

>34.6

>40

8

H3CO2C(CH2) 4

CH3

1.22

nd

0.3

nd

nd

nd

9

HO2C-4-PhCH2

CH3

0.48

12.6

0.48

6.9

>32.2

>50

nd: Not done; PBMC: Peripheral blood mononucleated cell; RANTES: Regulated upon activation normal T cell expressed and presumably secreted. Reproduced with permission from [133] © American Chemical Society (2011).

106



Chemokine receptor modeling: an interdisciplinary approach to drug design In a second case study, the binding hypotheses were used to guide medicinal chemistry in moving from a promising hit compound to a lead candidate in a rapid time frame. Medicinal chemistry had identified a 4-phenylimidazolidine-2-one cyclic urea series with modest anti-viral activity (Table 6). The mutant fingerprint of the parent molecule (10) confirmed binding interactions within the hydrophobic regions of CCR5 and with Glu283 (Table 7). After this experimental confirmation that the initial hit compound bound to the same site as other small-molecule antagonists structural modifications were made that allowed the interaction with either of the lysines in the extracellular domain (Figure 6C & 6D). Moreover, in order to further explore structure–activity relationships substitutions of the phenyl ring with a carboxylate, an ester or an amide were proposed. The suggested compounds should have a weaker interactions with the lysines, which was experimentally confirmed by determining their mutant fingerprint (Table 7). A comparison of the predicted binding modes of the parent compound 10 and the proposed lead compound 13 are shown in Figure 6. The value of this example cannot be overstated, as this is, to our knowledge, the first published example for using mutagenesis data combined with molecular modeling prospectively for lead optimization.

| Review

Table 5. The effect of single-site mutations on the CCR5-mediated cell–cell fusion inhibition by compounds of the thiophen-3-ylmethyl-urea series. Location

Mutant

Compound 3

4

5

6†

7†

8†

†

†

†

N-ter TM1 TM1 TM2 TM3 ECL2

K26A L33A Y37A W86A Y108A K191A

nd 57 712 958 73 nd

1 35 46 47 12 5

2 17 383 275 11 12

1 25 211 301 21 1

5 55 737 496 64 10

1 22 392 330 36 2

TM5 TM7

Y251A E283A

50 3846

8 47

22 411

26 2252

69 5833

102 6329

Fold difference of mutant IC50 /wild type IC50. ECL: Extracellular loop; nd: Not done; TM: Transmembrane region. Reproduced with permission from [133] © American Chemical Society (2011). †

molecular interactions of inhibitors with this chemokine receptor. A small-molecule isothiourea IT1t was found to occupy a binding pocket defined by TMI, II III and VII located closer to the extracellular loop compared with other GPCR structures. This new binding pocket has already been used to discover new CXCR4 inhibitors [70]. The symmetrical isothiourea contains two protonated nitrogens one of which can form a salt bridge with Asp97 with the protonated imidazothiazole making a salt bridge with Glu288. Both interactions were predicted by mutational analysis. A larger 16mer cyclic peptide inhibitor CVX15 was also co-crystallized with CXCR4 and was found to occupy both predicted binding pockets. Once again, acidic amino acid interactions predicted by mutational analysis were confirmed by the crystal structure [65]. The publication of the crystal

New

insights provided by the chemokine receptor crystal structures The recent elucidation of the crystal structure of CXCR4 has provided further insights into the

Table 6. Cell fusion inhibition, anti-HIV-1 (BaL) activity, cellular cytotoxicity and hERG inhibition of compounds of the 4-phenylimidazolidine-2-one cyclic urea series.

N

X

N O

R N

Compound

X

R

LogD

125

I RANTES binding IC50 (nM)

Cell fusion IC50 (nM)

HIV-1 PBMC IC50 (nM)

PBMC CC50 (µM)

hERG IC50 (µM)

10 11

CH2 CH2

Br O-Ph-4-CO2CH3

6.83 7.78

63 (n = 2) 30

700 (n = 36) 65

nd nd

nd nd

2.7 15.8

12

CH2

O-Ph-4-CONH2

6.18

18

29

40 (n = 3)

14.4

2.8

13

CH2

O-Ph-4-CO2H

5.68

39

13 (n = 2)

34 (n = 4)

>30.6

4.0

14

O

O-Ph-4-CO2H

3.90

25

16 (n = 6)

190 (n = 2)

>32.5

>40

nd: Not done; PBMC: Peripheral blood mononucleated cell; RANTES: Regulated upon activation normal T cell expressed and presumably secreted. Reproduced with permission from [133] © American Chemical Society (2011).



107

Review | Fricker & Metz Table 7. The effect of single-site mutations on the CCR5-mediated cell– cell fusion inhibition by compounds of the 4-phenylimidazolidine-2-one cyclic urea series. Location Mutant

Compound 10

N-terminal TM2 TM3 ECL2 TM5 TM7

K26A W86A Y108A K191A I198M E283A

†

nd 18 16 nd 22 22

11†

12†

13†

14†

4 212 5 3 9 212

1 458 13 3 15 359

16 920 8 6 14 161

14 346 7 12 33 346

Fold difference of mutant IC50 /wild type IC50. ECL: Extracellular loop; nd: Not done; TM: Transmembrane region. Reproduced with permission from [133] © American Chemical Society (2011). †

structure of CXCR4 holds significant promise of using molecular modeling for in silico ligand/ inhibitor design, both for smaller cyclic peptides and small molecules, for chemokine receptors [68,134,135]. The crystal structure of CCR5 bound with the inverse agonist maraviroc was recently published in September 2013. This structure

confirms the suggested small-molecule binding site derived from mutagenesis data. Furthermore, a comparison of the CXCR4 and CCR5 crystal structures demonstrates that the 7TM bundle of both chemokine receptors are structurally conserved with a root-mean-square deviation of 1.8 Å [136]. Future perspective The realization of the importance of chemokines and their receptors as components of the immune response has resulted in an increasing expansion in this area of research, since the discovery of IL-8 (CXCL8) and MCP-1 (CCL2) in the late 1980s. They are mediators of the inflammatory response and play key roles in the pathogenesis of autoimmune diseases. The discovery that two chemokine receptors, CXCR4 and CCR5, acted as co-receptors for HIV infection of host cells catalyzed drugdiscovery efforts aimed at chemokine receptors. There are now two approved drugs targeting chemokine receptors, maraviroc for CCR5, and plerixafor, a CXCR4 inhibitor. There is promise for more drugs with vercirnon (also known as Traficet-EN™, CCX282 or GSK1605786),

E283

I198 W86

E283

I198

W86 Y108

Y108

K191

K191 K26

K26

E283 W86

I198

E283

I198

W86

Figure 6. Binding modes of 4-phenylimidazolidine-2-one CCR5 inhibitors 10 and 13. (A) The 4-bromobenzyl group interacts with Trp86 and the cyclic urea with Ile198; (B) the cyclic urea interacts with Trp86 and the 4-bromobenzyl group with Ile198. (C) The carboxylic acid group interacts with Lys191; (D) carboxylic acid interacts with Lys26. With exception of Lys26, which is used for binding mode prediction, only residues are shown that have an impact on inhibition based upon mutagenesis results. Reproduced with permission from [133] © American Chemical Society (2011).

108



Chemokine receptor modeling: an interdisciplinary approach to drug design a first-generation orally administered CCR9 inhibitor currently in a pivotal Phase III trial (SHIELD-1) for moderate-to-severe Crohn’s disease with data expected in late 2013 [137,138]. (While this review was in preparation GlaxoSmithKline announced top-line results for the SHIELD-1 trial. The trial did not achieve the primary endpoint of improvement in clinical response and the key secondary endpoint of clinical remission. GlaxoSmithKline has returned all the rights to vercirnon to Chemocentryx, which still regard the CCR9 inhibitor as a valuable asset). Chemokine receptors are, therefore, promising drug targets, but progress has been slow in finding drugs acting on these targets. This is, in part, inherent in the apparent pharmacological redundancy associated with chemokine receptors, and biological hurdles, such as ortholog differences between mouse, the common animal model for inf lammatory disease, and humans [5,20]. However, a growing knowledge of the biology of chemokines and a complementary understanding of the pharmacology of chemokine receptor inhibition will assist in overcoming these challenges [21]. Furthermore, new additions to the drugdiscovery tool box will facilitate chemokine receptor drug discovery. One major tool available to medicinal chemistry is molecular modeling of drug–receptor interactions. Until the publication of the crystal structure of rhodopsin in 2000, there was no high-quality 3D structure of a GPCR available. Recently, rapid progress has been made in this area including the elucidation of the structure of CXCR4 [65] and CCR5 [136], which represents a major step forward by introducing structural-based design into the chemokine receptor field. In addition, these structures allow homology modeling of chemokine receptors, which will lead to the wider application of in silico modeling of inhibitor–receptor interactions. This modeling process can be used not only to explore interactions of known inhibitors, but can be used for virtual screening and design of new inhibitors and ligands [68]. We have reviewed our experiences of molecular modeling and chemokine receptor drug design. We have combined an empirical approach with computational chemistry by using the tools of molecular biology to generate targeted single-site mutations in CXCR4 and CCR5, biological readouts of receptor inhibition, and molecular modeling to build a picture future science group

| Review

of the binding sites for inhibitors of CXCR4 and CCR5 [97,128]. In the case of CXCR4, the molecular models developed for CXCR4 inhibitors helped to support the evolution of a new orally bioavailable inhibitor by retaining the key interactions with the receptor, but in a novel structure with improved drug-like properties. In the case of CCR5, we probed the binding site of a number of structurally diverse inhibitors, building upon information in the literature, and utilized this information to design novel CCR5 inhibitors, and to mitigate hERG interactions [133]. CXCR4 and CCR5 are just two examples of where in silico modeling has facilitated drug design. The crystal structure of CXCR4 has greatly facilitated chemokine receptor modeling. Molecular models based on CXCR4 have enabled structure-based virtual screening for new small-molecule ligands not just for CXCR4 but also other chemokine receptors, such as CCR2, CCR3 and CCR4. It is anticipated that similar efforts will be undertaken utilizing the recently published crystal structure of CCR5. The b2 AR receptor structure has also been used a template for modeling of the chemokine receptors CCR1, CCR2 and CCR5. However, there are still challenges, as the CXCR4 model as well as the CCR5 model are based upon a conformationaly restricted structure of the inactivated receptor, in which the receptor was stabilized by introducing thermostabilizing amino acid modifications and insertion of stabilizing peptides into the third intracellular cytoplasmic loop between TMV and VI (T4 of lysozyme for CXCR4 and Met1-Glu54 of rubredoxin for CCR5). GPCRs undergo dynamic structural changes so this model only provides one picture of how inhibitors may interact with the receptor target. An alternative approach has been used to obtain a 3D image of CXCR1 using NMR spectroscopy of the receptor in liquid crystalline phospholipid bilayers therefore, providing a more physiological environment [139]. With the ever-increasing challenges facing modern drug discovery the interdisciplinary combination of empirical molecular biology, structural biology, computational chemistry and medicinal chemistry provides a modern approach to drug discovery. As the examples of CXCR4 and CCR5 reviewed above demonstrate this approach can successfully facilitate drug discovery for novel chemokine receptor inhibitors. www.future-science.com

109

Review | Fricker & Metz Financial & competing interests disclosure S Fricker is an employee of Genzyme (a Sanofi company), M Metz is an employee of Sanofi. The authors have no other relevant affiliations or financial involvement with any

organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary The physiology & pathophysiology of chemokine receptors Chemokines and their receptors are integral components of the immune response, regulating lymphocyte development, homing and trafficking and play a key role in the pathophysiology of many diseases including HIV, multiple inflammatory diseases and cancer. Chemokine receptors are members of the G protein-coupled receptor (GPCR) family and are attractive targets for drug discovery. Molecular modeling approach to GPCR drug design

Recent advances in the molecular modeling of drug–GPCR interactions can provide new insights and guidance for drug discovery and medicinal chemistry. This was facilitated with the publication of the crystal structure of the archetypal class A seven transmembrane protein rhodopsin. This has been further enhanced by the recent elucidation of several high-resolution x-ray crystallographic structures of GPCRs, including that of the chemokine receptors CXCR4 and CCR5. This crystal structure information has enabled enhanced in silico modeling and ligand docking of both cognate ligands and drugs. Chemokine receptors & drug design

CXCR4 and CCR5 were identified as co-receptors for HIV infection and inhibitors of both receptors have been pursued as therapies for HIV. However, both receptors have been implicated as mediators of autoimmune and inflammatory diseases, and to play roles in cancer biology. Inhibitors of these two receptors, therefore, have multiple pharmacological applications. CXCR4 as a target for therapeutic intervention in disease

CXCR4 plays a key role in the homing and retention of hematopoietic stem cells (HSC) in the bone marrow. Pharmacological inhibition of CXCR4 leads to mobilization of HSC from the bone marrow. HSC transplant is a major therapeutic option for hematological malignancies. The CXCR4 antagonist plerixafor has been approved for HSC mobilization for autologous transplant for patients with multiple myeloma and non-Hodgkin’s lymphoma. Defining the plerixafor binding site

The plerixafor binding mode was probed by a combination of site-directed mutagenesis of the CXCR4 receptor and homology modeling based upon the published crystal structure of rhodopsin. The binding site was defined by the ionic interaction of charged nitrogen atoms on plerixafor with the carboxylate groups on amino acids Asp171, Asp262 and Glu288. This approach was further used to define the inhibitor binding site in support of the evolution of an orally bioavailable CXCR4 antagonist. Defining the binding site for CCR5 inhibitors

The inhibitor binding site for previously identified CCR5 inhibitors was probed and shown to be composed of two hydrophobic binding regions defined by TMI, TMII, TMIII and TMIII, TMIV, TMV, TMVI, TMVII, respectively. In addition, three potential compound-specific ionic interactions were identified with Glu283 in TMVII, Lys26 on the N-terminus and Lys191 on ECL2. Small-molecule compound design using CCR5 binding site hypotheses

The model of the CCR5 inhibitor binding site was used to as a component of a medicinal chemistry program to prospectively design novel CCR5 inhibitors. The use of the combination of medicinal chemistry, site-directed mutagenesis and molecular modeling for lead optimization identified novel CCR5 inhibitors with reduced hERG activity and potent CCR5 inhibition with antiviral activity.

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