Covalent binders in drug discovery.

Covalent modulation of protein function can have multiple utilities including therapeutics, and probes to interrogate biology. While this field is still viewed with scepticism due to the potential for (idiosyncratic) toxicities, significant strides have been made in terms of understanding how to tune electrophilicity to selectively target specific residues. Progress has also been made in harnessing the potential of covalent binders to uncover novel biology and to provide an enhanced utility as payloads for Antibody Drug Conjugates. This perspective covers the tenets and applications of covalent binders.

Development of Orally Efficacious Allosteric Inhibitors of TNFα via Fragment-Based Drug Design.

Tumor necrosis factor α (TNFα) is a soluble cytokine that is directly involved in systemic inflammation through the regulation of the intracellular NF-κB and MAPK signaling pathways. The development of biologic drugs that inhibit TNFα has led to improved clinical outcomes for patients with rheumatoid arthritis and other chronic autoimmune diseases; however, TNFα has proven to be difficult to drug with small molecules. Herein, we present a two-phase, fragment-based drug discovery (FBDD) effort in which we first identified isoquinoline fragments that disrupt TNFα ligand-receptor binding through an allosteric desymmetrization mechanism as observed in high-resolution crystal structures. The second phase of discovery focused on the de novo design and optimization of fragments with improved binding efficiency and drug-like properties. The 3-indolinone-based lead presented here displays oral, in vivo efficacy in a mouse glucose-6-phosphate isomerase (GPI)-induced paw swelling model comparable to that seen with a TNFα antibody.

Construction and validation of an automated flow hydrogenation instrument for application in high-throughput organic chemistry.

This manuscript details the construction of a fully automated flow hydrogenation apparatus for use in high-throughput organic synthesis. The instrument comprises of a Bohdan robot platform coupled with a ThalesNano H-cube hydrogenator and a series of solvent valves and pumping mechanisms. Using this instrument, we have been able to fully automate a number of key transformations that could not otherwise be conveniently undertaken in a high-throughput manner.

Merits and Pitfalls in the Characterization of Covalent Inhibitors of Bruton's Tyrosine Kinase.

In vitro analysis of covalent inhibitors requires special consideration, due to the time-dependent and typically irreversible nature of their target interaction. While many analyses are reported for the characterization of a final candidate, it is less clear which are most useful in the lead optimization phase of drug discovery. In the context of identifying covalent inhibitors of Bruton's tyrosine kinase (BTK), we evaluated multiple techniques for characterizing covalent inhibitors. Several methods qualitatively support the covalent mechanism of action or support a particular aspect of interaction but were not otherwise informative to differentiate inhibitors. These include the time dependence of IC50, substrate competition, mass spectrometry, and recovery of function after inhibitor removal at the biochemical and cellular level. A change in IC50 upon mutation of the targeted BTK C481 nucleophile or upon removal of the electrophilic moiety of the inhibitor was not always a reliable indicator of covalent inhibition. Determination of kinact and KI provides a quantitative description of covalent interactions but was challenging at scale and frequently failed to provide more than the ratio of the two values, kinact/KI. Overall, a combination of approaches is required to assess time-dependent, covalent, and irreversible inhibitors in a manner suitable to reliably advance drug candidates.

Recent trends in microwave-assisted synthesis.

Microwave-assisted organic synthesis streamlines a wide variety of reactions that require thermal reaction conditions. The popularity of this method has made an impact among synthetic chemists, greatly increasing the productivity of many medicinal chemistry laboratories and allowing difficult synthetic transformations to be achieved under milder conditions. This field is rapidly growing, and a number of comprehensive accounts and significant achievements have been published recently. This review will focus on some emerging key areas.

Hit-to-lead studies on benzimidazole inhibitors of ITK: discovery of a novel class of kinase inhibitors.

Benzimidazole 1 was identified as a selective inhibitor of ITK by high throughput screening. Hit-to-lead studies defined the SAR at all three substituents. Reversing the amide linkage at C6 led to 16, with a fivefold improvement of potency. This enhancement is rationalized by the conformational preference of the substituent. A model for the binding of the benzimidazoles to the ATP-binding site of ITK is proposed.