Helix mimetics: Recent developments.

Progress in Biophysics and Molecular Biology xxx (2015) 1e8

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Helix mimetics: Recent developments Andrew J. Wilson a, b, * a b

School of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2015 Received in revised form 21 May 2015 Accepted 22 May 2015 Available online xxx

The development of proteineprotein interaction (PPIs) inhibitors represents a challenging goal in chemical biology and drug discovery. PPIs are problematic targets because they involve large surfaces with less well defined features and recognition motifs that are less amenable to conventional experimental and computational ligand discovery methodologies. a-Helix mediated PPIs represent a sub group with a clearly defined interface and thus may be more amenable to the development of generic ligand discovery methods. Indeed, this is borne out in numerous studies using peptides covalently constrained into a helical conformation resulting in improvement of myriad biophysical and cellular properties. It is however desirable to have small molecule alternatives: a helix mimetic (proteomimetic) is a generic small molecule scaffold that projects functional groups in a similar spatial orientation so as to mimic the presentation of key amino acid side chains from the helix that mediates the PPI. The first true example of a helix mimetic was described over a decade ago however this approach has not yet been elaborated to the extent that it receives similar levels of attention to constrained peptides. This review explores recent significant developments in the area of small molecule a-helix mimetics and provides a critical overview of success stories, potential limitations of the approach, and areas for future development. © 2015 Published by Elsevier Ltd.

Keywords: Proteineprotein interactions Inhibitors a-Helix a-Helix mimetic Chemical biology

1. Introduction The development of inhibitors of proteineprotein interactions (PPIs) represents a central challenge in chemical biology and medicinal chemistry that must be addressed in order to deliver better temporal understanding of signaling pathways and starting points for molecular therapeutics development (Surade and Blundell, 2012; Morelli and Hupp, 2012). a-Helix mediated PPIs (Edwards and Wilson, 2011; Bergey et al., 2013) e involving the binding of an a-helical sequence from one protein to a cleft or the surface of its partner (Fig. 1a) e have emerged as a target class amenable to small molecule orthosteric inhibition (Azzarito et al., 2013) resulting in a number of clinical candidates (Vu et al., 2013). Similarly, classical a-Helix mediated PPIs such as p53/hDM2(X) (Kussie et al., 1996; Hoe et al., 2014) and Bcl-2/BH3 family interactions (Sattler et al., 1997; Czabotar et al., 2014) have served as excellent models for the elaboration of novel chemical

* Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK. E-mail address: [email protected].

intervention methodologies e.g. constrained peptides (Walensky and Bird, 2014) and foldamers (Smith et al., 2013; Boersma et al., 2011; Lee et al., 2009). One such approach (Cummings and Hamilton, 2010; Moon and Lim, 2015) involves the use of ligands termed in various publications as proteomimetics, (Orner et al., 2001) a-helix mimetics, (Azzarito et al., 2012) pharmacological chaperones (Oh et al., 2014) and topographical mimics; (Lao et al., 2014a) these all utilize a common scaffold to project essential binding side chains in a 3D spatial orientation so as to recapitulate the helical pharmacophore (Fig. 1b). The concept, first introduced by Rees and co-workers (Nolan et al., 1992) and truly exemplified by the Hamilton group (Orner et al., 2001; Yin et al., 2005a, 2005b) has resulted in a number of successful studies with small molecule helix mimetics shown to act selectively on their target(s) (Azzarito et al., 2015) as well as functioning in cells (Barnard et al., 2015) and in animal models (Lao et al., 2014b). To have true credibility and utility, there needs to be widespread use of the helix mimetic approach to identify inhibitors (a) that inhibit multiple PPIs with high potency and selectivity in animal models to serve as drug development candidates and (b) to serve as probes that can be used to validate potential drug targets and interrogate cellular biology to reveal new understanding of intracellular biological signalling networks.

http://dx.doi.org/10.1016/j.pbiomolbio.2015.05.001 0079-6107/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Wilson, A.J., Helix mimetics: Recent developments, Progress in Biophysics and Molecular Biology (2015), http://dx.doi.org/10.1016/j.pbiomolbio.2015.05.001

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A.J. Wilson / Progress in Biophysics and Molecular Biology xxx (2015) 1e8

Fig. 1. The helix mimetic approach (a) representative example of a helix mediated interaction; p53/hDM2 (PDB ID: 1YCR) (b) schematic depicting a helix mimetic and how it should reproduce vectoral presentation of side chains from one of the partners found at the PPI interface.

2. Helix mimetics: criteria for successes A diverse array of scaffolds has been reported in the literature which, are proposed to mimic the structure of an a-helix: to inhibit a helix mediated PPI, the scaffold needs to be appended with functionality that matches (closely) the side chains found on the helix which it mimics. The number of scaffolds reported, dictate that the majority discussed in this article functionally mimic an ahelix; that is to say they have been shown in a biophysical and/or cellular context to act as inhibitors of a PPI. As outlined above, helix mimetics have now been shown to represent a viable methodology for the identification of cell-permeable PPI inhibitors, however to date there have been no published crystal structures demonstrating a helix mimetic exhibits its designed molecular mode-of-action. Indeed, the solitary published crystal structure (for a KSHV protease dimerisation inhibitor), revealed an allosteric rather than direct orthosteric mode of inhibition (Lee et al., 2011). Furthermore whereas peptide constraining methodology (Walensky and Bird, 2014) has led to proof-of-concept that such reagents have therapeutic potential (Chang et al., 2013) and has been used to reveal structural and mechanistic understanding of biological processes, (Gavathiotis et al., 2008) this is not the case for helix mimetics. A critic might reasonably suggest that it is still necessary to show this and that it may be unlikely for such simple or “baroque” ligands possessing only limited similarity to the entire binding region upon which they are based. In this regard, it is perhaps instructive to consider what makes a good/useful chemical probe: criteria for chemical probe development (Edwards et al., 2011; Kodadek, 2010; Frye, 2010) suggest a probe should (a) demonstrate effectiveness in 2 assays, (b) have cellular IC50/EC50 (~1 mM) (c) show evidence of selectivity > 30 fold v relevant targets (d) show evidence of direct binding to target. Similarly, this class of ligand typically falls outside conventional small-molecule property space which may give cause for concern. It is worth noting that several helix mimetic scaffolds have been developed with a view to improving solubility, although the author is aware of only one published study (Lim et al., 2014) on helix mimetics where this property is measured (although for what it is worth, the vast majority of helix mimetics synthesized in the Wilson group labs seem to possess better solubility than Nutlin-3a). 3. Scaffolds amenable to library generation To address the criticism that a-helix mimetics are too simple to furnish highly potent and selective PPI inhibitors, it is either necessary to make the ligand more complex so as to mimic more of the pharmacophore upon which it is based, or to accept, as in many medicinal chemistry programs, that such small molecule starting

points for ligand development are inherently simple, but with sufficient exploration of the chemical space about the small molecule template, it is often possible to evolve potent and selective ligands. Thus, the ability to construct libraries of helix mimetics represents a means to achieve this goal and indeed, should be an attractive feature of the helix mimetic approach but relies on the availability of robust synthetic methods. Several solution based synthetic methods have been described for assembly of helix mimetics. Guy and co-workers (Lu et al., 2006) reported on the parallel assembly of 173 benzamide based compounds identified using structure-based computational design, leading to identification of low mM inhibitors of p53/hDM2. Boger and co-workers generated a library of 8000 aromatic oligobenzamide based helix mimetics (Shaginian et al., 2009) as mixtures using iterative solution based deprotection/unmasking followed by coupling cycles. This allowed identification of p53/hDM2 (Shaginian et al., 2009) and HIV gp41 assembly (Whitby et al., 2012) inhibitors. As helix mimetics typically comprise structurally similar repeat units, library assembly should more appealingly, be amenable to solid-phase synthetic methods. In 2010, the Wilson group reported on the first solid-phase synthesis of helix mimetics (Fig. 2a) (Campbell et al., 2010). The group focused on N-alkylated aromatic oligoamides (I in Fig. 2b). These were synthesized by N-terminal extension using Fmoc-protected monomers; a comparatively small library of ~30 inhibitor candidates was assembled using this approach and low mM p53/hDM2 inhibitors were identified. The same group improved upon this approach by employing microwave irradiation to facilitate synthesis of helix mimetics in ~4 h (Long et al., 2013), and also reported a microwave assisted synthesis of a related 3-O-alkylated aromatic oligoamide based helix mimetic scaffold (II in Fig. 2b) using a similar strategy (Murphy et al., 2013). Both methods allowed the incorporation of a diverse array of side chains covering the diversity of amino acid side-chain groups and beyond. In parallel Ahn's group reported on the assembly of the same 3O-alkylated aromatic oligoamide using a longer “reverse” sequence strategy (coupling of unfunctionalized monomers to the C-terminus followed by side chain addition) (Lee and Ahn, 2010). Finally, the Wilson group reported on a “hybrid” helix mimetic scaffold (IV Fig. 2b) comprising monomers from all of their previously described homo-oligomer scaffolds (I-III in Fig. 2b i.e. 2-O, 3-O or Nalkylated para-aminobenzoic acid building blocks) and a-amino acids using an Fmoc protected monomer extension approach (Azzarito et al., 2015). Arora and co-workers introduced a novel scaffold based upon an oligooxopiperazine backbone (Fig. 3a) (Tosovsk a and Arora, 2010).



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Fig. 2. Helix mimetic scaffolds amenable to conventional “peptide-based” solid-phase synthesis (SPS) methodology (a) example of an SPS synthesis developed for N-alkylated aromatic oligoamides (b) examples of aromatic oligoamide based helix mimetic scaffolds.

A synthetic route was developed that allowed assembly via peptide synthesis accompanied by the introduction of a bridging ethylene unit between successive nitrogens in the peptide backbone. Computational methods were used to inform the design and

Fig. 3. Different helix mimetic scaffolds amenable to library syntheses (a) oligooxopiperazine (b) pyrrolopyrimidine (c) triazineepiperazineetriazine.

synthesis of helix mimetics based on this scaffold that act as p53/ hDM2 and HIF-1a/p300 inhibitors (Lao et al., 2014a, 2014b). In all of the cases discussed above, the methodology has been harnessed to create small focused libraries intended for a specific target. To create screening libraries however, much larger libraries are needed and whilst the chemistries developed for aromatic oligoamide and oligooxopiperazine synthesis are in principle sufficiently robust to allow this, it has not been demonstrated. Lim and co-workers have developed two different scaffolds for which larger libraries have been accessed by solid-phase synthesis. In the first study, a pyrrolopyrimidine-based a-helix mimetic scaffold was introduced (Fig. 3b) (Lee et al., 2010). Using this approach a 900 member library was assembled and screened against the 53/hDM2 and p53/hDMX interactions to allow identification of submM inhibitors. In the second study a triazineepiperazineetriazine based helix mimetic scaffold was developed (Fig. 3c), which could be accessed using a SPPS strategy (Oh et al., 2014); using a peptoid encoding strategy a ~1500 member combinatorial library was assembled and screened in Mcl-1/BH3 and a-synuclein binding


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assays to identify inhibitors. A related phenyl-piperazine-triazine scaffold was also developed and a library screened to identify inhibitors that were selective for Mcl-1 over Bcl-xL (Moon et al., 2014).

4. Conformational considerations Helix mimetics tend to be depicted as rigid molecules presenting side-chains so as to reproduce the 3D projection of key side chains from the helix template upon which they are based. These are typically i, i þ 4 and i þ 7 side chains, although a number of scaffolds presenting different arrangements of helical side-chains and more than one face of a helix have also been described (Marimganti et al., 2009; Jung et al., 2013; Becerril and Hamilton, 2007). The reality is that these scaffolds adopt a range of conformations, some of which mimic the 3D side chain spatial projection of the helical template (Fig. 4a). The conformational landscape adopted by helix mimetics may therefore play a role in determining binding affinity towards target proteins e.g. due to the entropic cost of fixing rotatable bonds or the energy barrier between conformers. A number of mostly qualitative experimental studies have characterized the conformational space available to aromatic oligoamide based helix mimetics; (Prabhakaran et al., 2012; Plante et al., 2008) importantly, these have suggested that helix mimetics that differ in molecular structure have access to similar 3D conformational space resulting in similar inhibitory behavior (Fig. 4b) (Azzarito et al., 2012). Detailed experimental studies of helix mimetic conformational space however are difficult e in elegant studies by Burgess and co-workers, the conformational plasticity of peptidomimetic scaffolds has been exploited. Termed Exploring Key-Orientations on Secondary Structures (EKOS), the group have introduced a series of novel scaffolds (Fig. 4c) whose conformational landscape is quantitatively characterized in silico and the resultant ensemble matched against a broad range of secondary structure side-chain configurations

(Ko et al., 2012; Raghuraman et al., 2011; Ko et al., 2010). This means that in principle any one scaffold might mimic multiple different secondary structure side chain constellations. In follow-up studies the team illustrated that the same conformational plasticity and ability to mimic different side-chain orientations of multiple secondary structures is a common feature of many previously described scaffolds (Xin et al., 2013). Although, the team have not applied this approach to develop inhibitors of a-helix mediated PPIs, it has been exemplified for b-strands (Ko et al., 2012; Xin et al., 2014).

5. Tried and trusted targets? The vast majority of studies directed towards identification of functional helix mimetics have focused upon common helix mediated interactions, specifically p53/hDM2(X) and Bcl-2 family interactions. In contrast to constrained peptides, which have been tested on a broad range of targets (e.g. a recent review (Bird et al., 2013) highlighted examples of inhibition of ~25 distinct PPIs using all-hydrocarbon stapled peptides and this number has grown since), there are far fewer reports on PPI inhibition using helix mimetics. Although it is natural to develop new methodologies on tried and trusted model systems, this should change and several recent studies provide encouragement. Inhibitors of HIF-1a/p300 (Lao et al., 2014a, 2014b; Burslem et al., 2014), ER/co-activator, (Becerril and Hamilton, 2007; Ravindranathan et al., 2013) AKAP/ €fer et al., 2013) KSHV protease dimerization, (Lee et al., PKA, (Scha 2011) TS/DHFR (Martucci et al., 2013) and amyloid aggregation (hIAPP) (Hebda et al., 2009) have been described. The hIAPP aggregation inhibitors have an unusual mode of action in that they are proposed to sequester a helical intermediate en route to the bstrand conformer that assembles into b-sheet fibrils. Inhibitors of asynuclein aggregation have also been identified using a-helix mimetics as highlighted above, (Oh et al., 2014) although in this case, it was proposed that these ligands functioned by binding and

Fig. 4. Conformational properties of secondary structure mimetics (a) schematic depicting an a-helix mimetic as an ensemble of conformers only some of which mimic key a-helix side chains (b) schematic depicting how different helix mimetics can present similar orientations of side-chains due to covalent bond rotations (c) illustration of a “universal peptidomimetic” highlighting how different conformers can mimic different secondary structures.



stabilizing an intermediate tetramer. Finally, the ability of these ligands to have an unconventional molecular mode of action was further emphasized in a recent report on inhibitors of c-Myc function (Jung et al., 2015); rather than inhibiting c-Myc/Max dimerization, the helix mimetics were proposed to prevent interaction with DNA by binding to a helical region on the c-Myc/Max dimer that prevented complete formation of the “active” DNA binding heterodimer. 6. Selective inhibition of proteineprotein interactions A key challenge in any small-molecule inhibitor program is to identify ligands that are selective for their targets. For helix mimetics the majority of studies have focused upon identification of binders for a particular target so limited evidence of selectivity has been presented until recently. The first paper (Yin et al., 2005c) which reported on this described inhibitors that are selective for inhibition of p53/hDM2 using a terphenyl scaffold (Fig. 5a) e in this instance the inhibition of hDM2 was over 100-fold more favorable than Bcl-xL/Bcl-2. In the same paper a terphenyl scaffold bearing alternate side chains was shown to have the opposite preference, with Bcl-xL/Bcl-2 favoured over hDM2. Arora's group illustrated that it is possible to tune selectivity of oligooxopiperazine based helix mimetics (Fig. 5b) using the ROSETTA package to model optimal side chain groups (Lao et al., 2014a). Using this approach, a mimetic that bound hDM2 (Kd ¼ 0.3 mM), but not HIF-1a (Kd > 30 mM) was observed in addition to mimetics with opposing preference, binding HIF-1a (Kd ¼ 0.03 mM) but not hDM2 (Kd > 50 mM). Similarly the O-alkylated aromatic oligoamide scaffold was shown to be capable of selective recognition (Fig. 5c); the HIF-1a inhibitors discussed above were incapable of inhibiting the eIF4E/4G interaction (Burslem et al., 2014). One of the most recent studies by the Wilson group focused on a “hybrid” helix mimetic comprising p-aminobenzoic acid and amino acid building blocks (Fig. 5d); the initial low mM p53/hDM2 inhibitor identified in this study was tested against 4 other PPIs eIF4E/4G, HIF-1a/p300, Mcl1/NOXA-B and Bcl-xL/BAK, with only Mcl-1 exhibiting any inhibition (>4 fold selectivity in favor of hDM2) (Azzarito et al., 2015). Crucially this study also illustrated for the first time the role of side chain-spacing and stereochemistry in determining molecular recognition preference. A series of hybrids were synthesized with a drop-off in p53/hDM2 potency as the first two side-chain mimicking groups were positioned closer together. Regarding stereochemistry, the expected preference of hDM2 for one enantiomer of mimetic over the other was exemplified, but more surprisingly the protein selectivity was modulated; whilst one enantiomer was shown to exhibit hDM2 versus Mcl-1 selectivity, the other was shown to act as a dual inhibitor. In the studies by Lim and coworkers, distinct inhibitors of Mcl-1 and a-synuclein were obtained (Oh et al., 2014) from the same library of triazineepiperazineetriazine helix mimetics (Fig. 5e). The phenylpiperazine-triazine libraries were also shown to contain inhibitors with selectivity for Mcl-1 over Bcl-xL (Moon et al., 2014). Thus these studies illustrate that helix mimetics are indeed capable of exhibiting the selectivity profile characteristic of a chemical probe. Despite this, more stringent testing would be welcomeein most of these studies selectivity against only one further protein is assessed and it is perhaps instructive to consider that for a kinase inhibitor, it is standard to screen against many more kinases (Fedorov et al., 2007). 7. Cell based inhibitors and animal models A key objective in developing helix mimetics as chemical probes and small molecule inhibitors is to demonstrate they

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function in cells. In this regard very impressive progress has been made in recent years. The majority of studies have focused on inhibitors of hDM2 and Bcl-2 family interactions, however other targets have been addressed. Hamilton and co-workers demonstrated potent terphenyls act as pan Bcl-2 family PPI inhibitors (Kazi et al., 2011) using a battery of cellular readouts including coimmunoprecipitation, cytochrome c release, caspase and PARP induction. Compounds with IC50s in the low mM regime were identified that were proposed to function by inhibiting the interaction between pro- and antiapoptotic Bcl-2 family members, a conclusion supported by biophysical fluorescence anisotropy analyses. The O-alkylated aromatic oligoamides have been shown to be similarly potent inhibitors of the Bcl-2 family of PPIs using a broad array of experiments (Cao et al., 2013). Significantly, the compound used in these studies was shown to inhibit tumor growth in a lung cancer xenograft model. Furthermore, suitably functionalized O-alkylated aromatic oligoamide helix mimetics have been shown to inhibit the function of the androgen receptor; although no biophysical evidence was provided that these compounds inhibit the interaction between androgen receptor and its co-activators, potent sub mM activity was observed in cells and evidence of direct interaction with the target (through pull down using a biotin functionalized derivative) accompanied a panel of reporter and IP assays. Ultimately, the compound was also shown to act as an inhibitor of tumor growth in mice (Ravindranathan et al., 2013). In work by the Wilson group, N-alkylated aromatic oligoamide helix mimetics were shown to act as low mM inhibtiors of the p53/hDM2 interaction (Barnard et al., 2015) in fluorescence anisotropy assays and occupy the helix binding site on hDM2 by HSQC analyses. High content screening followed by Western analysis indicated induction of apoptosis, activation of downstream targets of p53 (p21), and, using biotin/FITC functionalized compounds, good uptake and direct interaction with hDM2 in several cell lines. The most potent compounds had comparable potency to Nutlin-3. Further analyses demonstrated interaction with Mcl-1 but not Bcl-xL indicating potential for a dual targeting approach (i.e. hDM2 and Mcl-1) as an anticancer strategy. Aside from providing direct evidence of selective binding biophysically and in cells, this work highlighted the importance of performing such studies on libraries of compounds as the biophysical properties were not always reproduced in a cellular context; the reason for this is unclear as similar compounds behaved differently. Schafmeister and co-workers introduced a very elegant spiroligomer scaffold (Fig. 5f) (Brown et al., 2012) that was also shown to inhibit the p53/hDM2 interaction in competitive fluorescence polarization assay; the affinity of the compound could be tuned by varying the stereochemistry of multiple residues within the backbone. In Huh7 cells where the p53/hDM2 negative feedback loop is inoperative, the compound had the unexpected effect of stabilizing hDM2, an effect proposed to arise as a consequence of stabilizing the protein to proteolysis and suggesting that helix mimetics can be used to develop new understanding of biological signaling. Finally, Arora and co-workers illustrated that oligooxopiperazine based helix mimetics can be used to inhibit the HIF1a/p300 interaction (Lao et al., 2014b). Mimetics were shown to act as single digit mM inhibitors in competition fluorescence polarization assays and map to helix 3 of the HIF-1a binding domain on p300 in 1He15N HSQC experiments. The compounds were shown to exert dose dependent effects on cell viability and affect HIF translational ability in a reporter assay. Furthermore, compounds were shown to modulate gene expression of multiple HIF target genes including VEGF a key player in angiogenesis and the development of new blood vessesl under hypoxic conditions. The compounds were also shown to reduce tumor growth rate in a mouse tumor xenograft model.


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Fig. 5. Structures and targets of selective helix mimetics (a) terphenyls (b) oligooxopiperazine (c) 3-O-alkylated aromatic oligoamides (d) hybrid oligomides (e) triazineepiperazineetriazine (f) spiroligomer.

8. Conclusions Helix mimetics represent potential generic templates for development or proteineprotein interaction inhibitors. The last 5e10 years have seen impressive progress in terms of developing

synthetic methods that allow access to libraries rich in compositional diversity and in screening such compounds against an increasingly broad spectrum of proteineprotein interactions relevant to disease giving some impressive results. For instance the first small molecules shown to target the HIF-1a/p300, Bcl-2 family and



nuclear receptor/co-activator interactions, interfere with these pathways in cells and inhibit tumour growth in mouse models have been described. Similarly, this class of ligand have been shown to meet many of the criteria identified for good chemical probes. More widespread use of this approach for multiple different targets is now needed to realise the promise of small molecule helix mimetics. Acknowledgement The author wishes to acknowledge support of his research program by the European Research Council [ERC-StG-240324]. References Azzarito, V., Prabhakaran, P., Bartlett, A.I., Murphy, N.S., Hardie, M.J., Kilner, C.A., Edwards, T.A., Warriner, S.L., Wilson, A.J., 2012. 2-O-Alkylated para-benzamide aHelix mimetics: the role of scaffold curvature. Org. Biomol. Chem. 10, 6469e6472. Azzarito, V., Long, K., Murphy, N.S., Wilson, A.J., 2013. Inhibition of a-helix-mediated protein-protein interactions using designed molecules. Nat. Chem. 5, 161e173. 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