Covalent drugs have proved to be successful therapies for various indications, but largely owing to safety concerns, they are rarely considered when initiating a target-directed drug discovery project. There is a need to reassess this important class of drugs, and to reconcile the discordance between the historic success of covalent drugs and the reluctance of most drug discovery teams to include them in their armamentarium. This review surveys the prevalence and pharmacological advantages of covalent drugs, discusses how potential risks and challenges may be addressed through innovative design, and presents the broad opportunities provided by targeted covalent inhibitors.

We have identified a small-molecule inhibitor of tumor necrosis factor alpha (TNF-alpha) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-alpha activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-alpha subunits.

The potential utility of synthetic macrocycles 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 macrocycles for good target protein-binding activity or bioavailability. To address this knowledge gap we analyze the binding modes of a representative set of macrocycle-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 macrocycles libraries possessing structural and physicochemical features likely to favor strong binding to protein targets and also good bioavailability. We additionally provide evidence that large, natural product derived macrocycles can bind to targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product inspired synthetic macrocycles 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.fragment-based drug discovery | ligand binding site | inhibitor design | side-chain adjustment

Key to the pharmaceutical utility of certain macrocyclic drugs is a “chameleonic” ability to change their conformation to expose polar groups in aqueous solution, but bury them when traversing lipid membranes. Based on analysis of the structures of 20 macrocyclic compounds that are approved oral drugs, we propose that good solubility requires a topological polar surface area (TPSA, in Å2) of ≥0.2 × MW. Meanwhile, good passive membrane permeability requires a molecular (i.e. 3D) PSA in nonpolar environments of ≤140 Å2. We show that one or other of these limits is almost invariably violated for compounds with MW > 600 Da., suggesting that some degree of chameleonic behavior is required for most high MW oral drugs.

Isoniazid (INH), a frontline antitubercular drug, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. Here, we report that the INH-NAD adduct is a slow, tight-binding competitive inhibitor of InhA. Demonstration that the adduct binds to WT InhA by a two-step enzyme inhibition mechanism, with initial, weak binding (K ؊1 ‫؍‬ 16 ؎ 11 nM) followed by slow conversion to a final inhibited complex (EI*) with overall K i ‫؍‬ 0. 75 A ttempts to treat tuberculosis, a disease that kills more than two million people every year, are hindered by the spread of multidrug-resistant strains of Mycobacterium tuberculosis (MDRTB), the causative agent, and by the increased susceptibility of HIV-positive individuals to this disease (1-4). Although isoniazid (INH; Scheme 1) has been the most effective and widely used drug for the treatment of tuberculosis since the 1950s, the mode of action of this compound is still not completely understood. INH is a prodrug that is activated by the mycobacterial catalase-peroxidase enzyme KatG (Scheme 1) (5-8) and a substantial fraction of all clinical isolates that are resistant to INH result from KatG mutations (2, 9-11). Consequently, compounds that inhibit the ultimate molecular target(s) of INH, but that do not require activation by KatG, have tremendous promise as novel drugs for combating MDRTB.Two enzymes, InhA and KasA, have been proposed as targets for INH. Both are members of the type II dissociated fatty acid biosynthesis pathway (FASII) in M. tuberculosis (Scheme 2), consistent with the observation that INH interferes with the biosynthesis of mycolic acids, very long chain fatty acid components of the mycobacterial cell wall. InhA, an enoyl reductase that catalyzes the NADH-dependent reduction of long chain trans-2-enoyl-acyl carrier proteins (ACPs), was first identified as a target by Jacobs and coworkers (6, 12) who observed mutations in the inhA gene in INH-resistant clinical isolates and identified a point mutant (S94A) that conferred resistance to INH and ethionamide in Mycobacterium smegmatis and in Mycobacterium bovis. Subsequently, Blanchard, Sacchettini, and coworkers (13-15) demonstrated that InhA was inhibited in vitro by a covalent adduct formed between activated INH and the nicotinamide head group of NAD (Scheme 1). InhA mutations observed in INH-resistant clinical isolates were found to be localized to the cofactor binding site and were shown to result in decreased affinity of the purified enzyme for NADH (12,16), consistent with the hypothesis that binding of NADH to the enzyme precedes adduct formation. In addition, Vilcheze et al. (17) used a temperature-sensitive mutation in the inhA gene to show that the phenotypic response to InhA inactivation in M. smegmatis was identical to that caused by treatment with INH, thereby validating InhA as a target for drug discovery. However, although there is convincing evidence that InhA is inhibited by INH, Barry and coworkers (18) have also proposed that KasA, on...

Cooperative binding effects pervade biology. Only a few basic principles are at play, but in different biological contexts cooperativity appears in distinct guises to achieve different ends. Here I discuss some of the manifestations of cooperativity that are most important in biology and drug discovery as they pertain to systems at different levels of complexity and also highlight aspects of this broadly important phenomenon that remain poorly understood.

According to the two-signal model of T cell activation, costimulatory molecules augment T cell receptor (TCR) signaling, whereas adhesion molecules enhance TCR-MHC-peptide recognition. The structure and binding properties of CD28 imply that it may perform both functions, blurring the distinction between adhesion and costimulatory molecules. Our results show that CD28 on naïve T cells does not support adhesion and has little or no capacity for directly enhancing TCR-MHC-peptide interactions. Instead of being dependent on costimulatory signaling, we propose that a key function of the immunological synapse is to generate a cellular microenvironment that favors the interactions of potent secondary signaling molecules, such as CD28.