Genetically encoded fragment-based discovery

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This opinion describes recent advances of molecular discovery technology dubbed Genetically Encoded Fragment-Based Discovery (GE-FBD). GE-FBD starts from a known ligand or ‘fragment’ that binds to a desired target weakly and often with low specificity. Covalent incorporation of fragment into a diverse, genetically encoded library of peptides yields a library of peptide–fragment combinations. Selection from such a library has a high likelihood to identify ligands, in which the peptides bind to distinct adjacent pockets of the target in synergy with the fragment and exhibits enhanced affinity and specificity when compared to the fragment itself. GE-FBD could employ fragments that bind non-covalently as well as reversible covalent warheads. The key advances in GE-FBD include (i) synthetic chemistry that enables incorporation of diverse fragments into both linear and cyclic peptide libraries; (ii) quantification of multi-step modifications in million-to-billion library members, (iii) and chemical transformations that permit incorporation of fragments with concurrent topological change from linear to macrocyclic topologies.

Introduction

Fragment-based drug design (FBDD) is an important process in medicinal chemistry that gives rise to specific, and selective binding ligands to a protein of interest by linking weak and often promiscuous synthetic molecules termed ‘molecular fragments’ (Figure 1a) [1,2]. This review focuses on technology that combines the basic principles of FBDD with the power of genetically encoded (GE) discovery of peptides or macrocyclic peptide ligands [3••]. Covalent incorporation of a fragment F into a GE-library of peptides, denoted as {P} yields a new library, denoted as {FP} in which most members contain the fragment of interest. This opinion focuses on chemical post-translational incorporation of fragments (Figure 1b). Alternatively, {FP} libraries can be built from unnatural amino acids (UAA) that contain fragments as part of their side-chain (Figure 1c). Selection from {FP} libraries has been shown to identify ligands in which the fragment covalently linked to peptide segment bind the target with improved affinity and specificity when compared to the original fragment (Figure 1d). Improvement can be defined as r = FKD/FPKD > 1 or ΔΔGbind ∼ −log(r) < 0, where FKD is the affinity of the fragment towards the target of interest (Figure 1e). Figure 1d summarizes up-to-date outcomes from published GE-FBD reports and shows that r = 10–100 can be found across diverse fragment with FKD ranging from 100 nM to 1 mM [3••,4, 5, 6,7••,8,9,10,11,12,13,14]. GE-FBD was also reported to be successful with reversible covalent fragments.

Historically, peptides are considered as the suboptimal drug modality due to their poor stability and pharmacological properties. However, peptides are increasingly recognized as potential starting point for lead optimization through systematic medicinal chemistry studies. Chemical modification of peptides—cyclization, N-methylation, capping of N-terminus and C-terminus and substitution of l-amino acid with unnatural or d-amino acid—can yield peptide derivatives with excellent stability and cell permeability that are suitable for pre-clinical and ‘investigational new drug’ (IND) studies [15,16]. These efforts have translated to a stapled peptide (ALRN-6924) that has entered the clinical trial (NCT02264613). The encouraging developments show the potential of the peptide modality, especially for targeting protein–protein interactions that are historically intractable to small-molecule approach [17,18].

Section snippets

Biophysical considerations of GE-FDB

GE-FBD offers a unique opportunity to study genetically encoded ligand discovery. Unlike traditional selection from a random library of molecules where the vast majority of ligands exhibit non-detectable binding for the target, members of the {FP} library contain the fragment that has a measurable affinity FKD towards the target. Specifically, {FP} library can be demarcated into members in which the affinity of ith peptide–fragment combination (FPiKD) are stronger (ΔΔGbind < 0), similar (ΔΔGbind

Chemical synthesis of libraries for GE-FBD

Predicting a priori which molecular topology provides the best selection outcome for a particular target is challenging. Solving this question practically necessitates building libraries of diverse architectures. Nature builds diverse proteins from the canonical 20 amino acids and further diversifies them via hundreds of different PTMs (Figure 1b) [25,26]. The same approach can build diverse libraries for GE-FBD. Chemical post-translational modifications (cPTM) are attractive because one

cPTM of N-terminus

Oxidation of native N-terminal Ser/Thr residue yields a bio-orthogonal aldehyde handle for conjugation of fragments (Figure 2a) [27]. We employed oxime ligation to attach a mannose (Man) onto the N-terminus of the random heptapeptide library SX7 (Figure 2b). Selection from Man-X7 library against protein ConA identified a conserved peptide segment (WYD) that binds in synergy with the Man [3••]. X-ray structure (PDB code: 4CZS) confirmed interaction of Man-WYDLF with ConA; KD of this binding was

GE-FBD through cPTM of cys residue

Historically important examples of modification of GE libraries used Cys as handle: (i) SN2 alkylation of a Cys-containing antibody libraries with a fluorophore [30]; (ii) native chemical ligation of a 32-amino-acid eglin ‘fragment’ to the N-terminal Cys of a peptide library [31]; and (iii) SN2 alkylation of Cys-containing mRNA library with 6-bromoacetyl penicillin [11]. Screening the penicillin-modified library successfully identified LRNSNC(penicillin)IRHFF that exhibited 100-fold stronger

Enabling chemistry for cPTM

Growing interest in macrocyclic peptides makes synthetic strategies that can introduce a fragment and cyclize the peptide library highly desirable. Cys-alkylation with dichloroacetone-derived oxime cyclizes the peptide library and introduces the fragments concomitantly [36] (Figure 4a). An alternative approach cyclizes a peptide and introduces an orthogonal reactive group first, followed by ligation of the fragments in the second step. Phage decorated with aldehyde or ketone are stable in

Conclusions and outlook

Innovations in molecular discovery are driven by unsolved demands. One of such unsolved challenges is the discovery of ligands that elicit a defined mechanism of action such as antagonism or agonism. De novo screening with libraries of small molecules or peptides has a likelihood of finding binders occupying a binding site that does not elicit any biological effect. In contrast, GE-FBD incorporates a well-characterized and functional binding fragment into the library and guides the selection

Conflict of interest statement

R.D. is the Founder and Chief Executive Officer of 48Hour Discovery Inc., the company that commercializes genetically-encoded, chemically-modified peptide library technologies.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgement

R.D. gratefully acknowledges support from the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (#402511), NSERC Discovery Accelerator Supplements Program, GlycoNet Canadian Glycomics Network and Alberta Glycomics Centre.

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