Macrocycles have the potential to act on currently undruggable targets, but their discovery thus far has been unsystematic. A physicochemical analysis of all nonredundant co-crystal structures now sets out guidelines for macrocycle development. The potential utility of synthetic macrocycles (MCs) as drugs, particularly against low-druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of MCs for good target protein–binding activity or bioavailability. To address this knowledge gap, we analyze the binding modes of a representative set of MC–protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic MC libraries with structural and physicochemical features likely to favor strong binding to protein targets as well as good bioavailability. We additionally provide evidence that large, natural product–derived MCs can bind targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product–inspired synthetic MCs can expand the number of proteins that are druggable by synthetic small molecules.

Despite the growing number of examples of small-molecule inhibitors that disrupt protein–protein interactions (PPIs), the origin of druggability of such targets is poorly understood. To identify druggable sites in protein–protein interfaces we combine computational solvent mapping, which explores the protein surface using a variety of small “probe” molecules, with a conformer generator to account for side-chain flexibility. Applications to unliganded structures of 15 PPI target proteins show that the druggable sites comprise a cluster of binding hot spots, distinguishable from other regions of the protein due to their concave topology combined with a pattern of hydrophobic and polar functionality. This combination of properties confers on the hot spots a tendency to bind organic species possessing some polar groups decorating largely hydrophobic scaffolds. Thus, druggable sites at PPI are not simply sites that are complementary to particular organic functionality, but rather possess a general tendency to bind organic compounds with a variety of structures, including key side chains of the partner protein. Results also highlight the importance of conformational adaptivity at the binding site to allow the hot spots to expand to accommodate a ligand of drug-like dimensions. The critical components of this adaptivity are largely local, involving primarily low energy side-chain motions within 6 Å of a hot spot. The structural and physicochemical signature of druggable sites at PPI interfaces is sufficiently robust to be detectable from the structure of the unliganded protein, even when substantial conformational adaptation is required for optimal ligand binding.

Profiling the reactivity and stability of S^VI–F warheads towards nucleophilic amino acids for the development of biochemical probe compounds.

Significance: Cryptic binding sites are absent or occluded in unbound proteins but present in ligand-bound structures. They can provide druggable pockets in cases where the main functional site of the protein cannot be targeted with sufficient potency or specificity. We show that unbound structures of proteins with cryptic sites have strong binding hot spots and above-average flexibility around the incipient pockets. Binding hot spots can be easily identified computationally, and protein flexibility can often be assessed by the analysis of X-ray structures of the unbound protein, including structures with low resolution. The approach presented here helps to assess the potential druggability of cryptic sites prior to running expensive screening experiments and reveals information about how cryptic sites form.