On the Importance of Rigidity in Designing Small Molecule Drugs to

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On the Importance of Rigidity in Designing Small Molecule Drugs to Tackle Protein-Protein Interactions (PPIs) through Stabilisation of Desired Confomers Alastair D.G. Lawson, Malcolm MacCoss, and Jag P. Heer J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01120 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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Journal of Medicinal Chemistry

On the Importance of Rigidity in Designing Small Molecule Drugs to Tackle Protein-Protein Interactions (PPIs) through Stabilisation of Desired Confomers

Miniperspective Alastair D.G. Lawson*, Malcolm MacCoss§, and Jag P. Heer UCB, 216 Bath Road, Slough, SL1 3WE §

Bohicket Pharma Consulting LLC, 2556 Seabrook Island Road, Seabrook Island, South Carolina 29455,

United States

ACS Paragon Plus Environment


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Abstract Tackling PPIs, particularly by stabilising clinically favoured conformations of target proteins, with orally available, bona fide small molecules remains a significant, but immensely worthwhile, challenge for the pharmaceutical industry. Success may be more likely through the application of nature’s learnings to build intrinsic rigidity into the design of clinical candidates.

Introduction Over the last thirty years we have witnessed the rise of therapeutic antibodies from not even existing to being among the world’s top selling drugs. This has resulted largely from their ability to bind with high affinity to specific, biologically relevant epitopes on target proteins, enabling the biomedical community to bring protein-protein interactions (PPIs) into play. For all the clinical success and corporate value that has been created, antibodies come with serious limitations of potential immunogenicity,1 tissue accessibility,2 supply chain complexity,3 and high cost of goods.4 Society is looking for the pharmaceutical industry to make orally dosed medicines with the efficacy of monoclonal antibodies for the cost of small molecules. Inhibition of PPIs with small molecules presents a special challenge for drug discovery, with 1:1 stoichiometry harshly exposing any shortcomings in potency,5 in contrast to inhibition of enzymes, where inhibiting one enzyme molecule can block the formation of many substrate molecules. However changes in protein function through natural sampling from an ensemble of confomers6,7 provides significant opportunities for allosteric pharmaceutical intervention.8 It should be noted that, even for natural ligands binding to their protein targets, the process of conformer selection (as opposed to induced fit mechanisms) is far more common than previously believed.9


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This is one of THE greatest opportunities for our time, and ‘PPI Professionals’ such as antibodies and natural products, can surely help us in this quest.

Guidance from Antibody Structures X-ray crystal structures of immune complexes show that antibodies use only a relatively small number of amino acids (typically in the range ten to twenty, and often including aromatic residues) in their paratopes to make the critical contacts with antigens.10 But nature then goes on to invest more than four hundred amino acids in a Fab fragment, for example, to rigidify the complementarity determining region (CDR) loops to provide precise three-dimensionality in presenting the key contact atoms, to enable fast association kinetics and high quality binding.11



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Figure 1 shows that in a typical Fab fragment of an antibody less than 5% of the amino acids interact directly with antigen, and that even with more ‘ligand efficient’ antibody formats, such as the single domain camelid VHH,12 only some 10% of the amino acid residues are committed to direct interaction with antigen, with the remaining 90% providing infrastructure.13 Another observation of note is the comparison between the association rate constants for the same anti-lysozyme antibody variable regions in Fab and Fv formats, showing the significant contribution provided by the relatively distant constant domains in the Fab to the stabilisation of the antibody-antigen complex.14 This relative investment in contact atoms and infrastructure by antibodies highlights the critical importance of reducing the entropic cost associated with the ligand adopting the required bound conformation in our small molecule drug designs. A recently described subset of bovine antibodies15,16 with greatly extended CDR3 loops (some sixty amino acids) provides a further structural lesson on the importance of rigidity. Binding is mediated through small, tight, disulphide-linked ‘knobs’ at the tips of the loops, a design concept reminiscent of the bicyclic peptide library approach.17 A complementary design is seen in a subset of single domain camelid VHH antibodies, which use extended and disulphide bond-supported CDR3 loops to drive much of their binding within small pockets on the target.18 Importance of the framework infrastructure in antibody design is likely to account for the general lack of success in the literal translation of antibody CDR loops into pharmacophores and peptidomimetics. The pioneering work of Mark Greene19 in the late 1980s has not led to the successful development of antibody-derived small peptide-based therapeutic candidates. When lifted from the context of the antibody’s framework, the identical amino acids, which make the critical contacts from crystal structures of complexes, and are no longer able to provide efficient binding or equivalent activity, even when incorporated into cyclic structures, which would generally be considered to offer a reasonable degree of conformational constraint. Instead, such peptidomimetics exist in an ensemble of conformational states,


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leading to both significant entropic penalties on binding and insufficient intrinsic rigidity to lock out the same conformational state of, for example a target GPCR, as the original VHH.20

Guidance from Antibody Composition Although constrained by the enforced use of only twenty amino acids in the sampling of chemical space, antibodies demonstrate that extreme, even ultimate, diversity in protein binding can be achieved from a combinatorial approach using a small number of versatile building blocks, as long as the framework infrastructure can provide sufficient diversity and precision. Indeed, the recovery of antibodies with nanomolar affinities from synthetic phage-displayed libraries with greatly restricted CDR diversity (using only Tyr, Ala, Asp and Ser)21 dramatically emphasises the diversity of binding that can be achieved by appropriate positioning of a versatile fragment, such as the tyrosine side chain, in the context of a suitable scaffold. Antibodies thus challenge the notion that exploration of chemical space far beyond current, clinically validated drug space will be particularly rewarding, especially in the early stages of fragment and hit identification in drug discovery.

Guidance from Antibody-defined Conformations of Target Proteins Antibodies which modify the function of target proteins, not by orthosterically occluding active sites, but by allosterically defining naturally sampled, structurally compromised, inactive conformations from the conformational ensemble, represent valuable tools in drug discovery. Structural information from complexes of targets with such antibodies gives direction to the design of allosteric small molecules that bind to and stabilise the same functionally validated protein conformers.22,23 An additional benefit of this approach is that new pockets, previously unseen in X-ray crystal structures of the apo target, may be revealed in the newly defined conformations. These new structural features can also confirm predictions



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from molecular dynamics simulations, providing ‘wet lab’ validation for the in silico methodology. Stabilisation of a relevant conformer allows for ready access to screening (e.g. with fragment libraries) of the antibody-protein complex for small molecules that bind to the appropriate biologically relevant state. This mimicking of antibodies’ functions with small molecules, not necessarily by binding at the same site, nor in the same way, but by stabilising the same, antibody-validated conformation of the target, may prove to be a productive way to de-risk PPI small molecule drug discovery.24

Guidance from Natural Products In contrast to antibodies, which initially may have only a few seconds to acquaint themselves with their target antigens, natural products represent structures validated from millions of years of co-evolution with specific target proteins, and presumably benefit from precision in complementary chemical design.25,26 Many important, biologically active natural products are low molecular weight compounds, which demonstrate an extremely high degree of chemical diversity, three-dimensionality, precise orientation through intrinsic rigidity,27,28 and overall higher ligand efficiency compared to antibodies. It seems that, in the absence of a high molecular weight scaffold, increased sampling of chemical space is recommended by nature in the structures of evolutionarily refined ligands, but both antibodies and natural products invest heavily to reduce the entropic cost of binding. Compared to synthetic small molecule drugs and antibodies, natural products display low abundance of aromatic ring systems, a greater number of chiral centres, cyclisation, more bridgehead tetrahedral carbon atoms, high numbers of hydrogen bond donors and acceptors, a higher proportion of oxygen over nitrogen atoms and perhaps most importantly, drug-like molecular properties.29 Such properties are generally under-represented in fragment libraries, so the focus on a natural product-derived fragment library in Herbert Waldmann’s laboratory, for example, is of particular interest.30 The compounds in this library resemble natural scaffolds rich in sp3-configured centres, but are also synthetically tractable. Similarly, a compound collection based on bicyclic scaffolds of natural products, such as the immunosuppressant


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FR901483, Elaeokanidine A, (-)-Lycoposerramine-R, (-)-8-Deoxyserratinine, Slaframine and Exiguaquinol has recently been described.31 A few indole-inspired natural product fragments, which also relate to Trp residues in antibodies, are shown in Figure 2, as examples and representing only a tiny fraction of the scope of natural productderived low molecular weight chemical space.

It can be argued that rigidity of fragments is likely to be less important in early hit finding, and indeed may be counter-productive in leading to the identification of fewer hits, due to the higher specificity of binding requirements. However it is interesting that ‘privileged structures’, with general applicability, are often inspired by or derived from natural products,32 and the work of Paul Hergenrother’s33,34 and Damien



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Young’s35 laboratories on the tractable synthesis of stereochemically and structurally complex and diverse compounds inspired by natural products is pertinent. Small fragments in libraries representing both clinically validated drug chemical space36,37 and biologically validated natural product-derived chemical space38 would seem to offer the best compromise between simplicity in probing the basic architecture of all proteins and sufficient complexity for hits to act as useful starting points, suitable for elaboration into lead compounds.39 Natural products are advocating increasing the rigidity of small molecule drug candidates, at least during the elaboration phase of drug discovery, to prosecute PPI targets through conformational stabilisation.

Opportunities for Allosteric Modulators While progress is being made with orthosteric blocking of PPIs,40 tackling these interactions ‘head on’ is not without inherent challenge, largely due to direct competition with the natural binding partner, and the small molecule lacking the footprint of an antibody to cover the relevant hot spots. In addition, with a few exceptions, such as the β-propeller Keap1 pocket for Nrf241, the shallow nature of many orthosteric PPI binding sites and the number of polar interactions is not readily addressed with a small molecule, where the cost of de-solvation cannot be met by the affinity of binding. An allosteric approach is likely to become more generally applicable, with the opportunity to modulate biology from protected sites,42 and the ASD Allosteric Database is proving to be a useful tool in tracking this trend, with information on structural mechanisms and networks of particular relevance.43

Novel allosteric sites are fickle in their pharmacological response due to the lack of evolutionary pressures to drive a particular response. Analysis of small molecule allosteric modulators, in particular of membrane receptors (notably GPCRs), ligand-gated ion channels and nuclear receptor targets, has shown


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these to have fewer rotatable bonds, more rings, and to be more conformationally constrained than orthosteric ligands.44,45

Following on the prediction of Richard Feynman in the early 1960s that ‘everything that living things do can be understood in terms of the jiggling and wiggling of atoms’,46 modulation of protein function through an effect on dynamics may be achieved through allosteric binding of small molecules.47 Molecular dynamics enable a protein to access different conformations and functional states among an ensemble of confomers, and the biology associated with the protein is modulated according to the residence time in each energy minimum.48 Small molecules can bind to the protein at allosteric sites and shift the equilibrium of conformer sampling, so that binding can influence the overall residence time that a protein spends in any particular conformer. However for this approach to be successful in the clinic, efficient and definitive stabilisation of desired confomers will be essential.

Target classes with greater conformational flexibility are likely to present more opportunity to find allosteric sites to modulate function. While such sites are not without inherent challenges including affinity-efficacy paradoxes,49 discussed below, they are also potentially more ligandable than orthosteric PPI sites due to a better balance of polar and hydrophobic interactions over a smaller footprint. Advances in computational techniques to identify cryptic allosteric sites will further shift attention to allosteric modulation of PPIs.50

Defining the Binding Site on the Protein The precise, specific and clinically desired conformation of the target may be far from obvious or stable, and data from a range of biophysical techniques need to be considered in the context of the limitations of each technique. For example differences in protein structures obtained from NMR and X-ray crystallography have been well documented and highlight the differences and complexity of conformational sampling in crystalline and solution environments.51,52 Crystal structures of multidomain



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proteins are at particular risk of distortion due to packing forces in a crystal lattice,53 and conformational equilibria can be modified in the crystal environment to favour more compact, less hydrated substrates.54 Data from X-ray crystallography, small-angle X-ray scattering (SAXS) and double electron-electron resonance (DEER) may be advantageously combined to obtain representative structures, as with the integrin α6β4.55 In our laboratory we have used distance data obtained from DEER to adjust the crystal structure of TNFα using constrained minimisation to create a working model of the binding site to guide small molecule drug discovery.56

Designing the Small Molecule In designing the therapeutic candidate, the critical issues of inefficient space-filling of the pocket and incomplete conformer definition need to be addressed. Figure 3 shows a hypothetical cartoon which illustrates the key points in the design of an allosteric antagonist of a PPI, but similar principles would apply to design of an allosteric agonist.


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Efficient filling of pocket space by the drug is a fundamental requirement, and in structure-based design we need to be mindful of the potential for artificial restriction in pocket volumes in crystal structure depictions, as discussed above. Rigid compounds, which appear to clash with protein surfaces, should be deliberately made to test real solution structures of targets. The kinetics of desirable compounds may also be atypical in displaying slow association and slow dissociation rates, reflecting the conformational sampling and subsequent stabilisation of the target, rather than being necessarily properties of the small molecules.



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When targeting PPIs, by seeking to stabilise clinically desirable confomations with small molecules binding at transient allosteric sites, divergence between binding/occupancy of the site and modulation of function can be observed if the small molecule is flexible and allows the protein to continue to ‘wiggle’ and still access active and clinically undesirable conformers, albeit at reduced frequency. This manifests as a disconnect in assay data, with 100% occupancy of the target with a small molecule giving

On the Importance of Rigidity in Designing Small Molecule Drugs to

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