Nailing Down a Notoriously Elusive Cancer Target: Direct Inhibition of MYC by a Covalent Small Molecule.

Direct inhibition of the transcription factor MYC is widely recognized as one of the thorniest challenges in cancer drug discovery. In this issue of Cell Chemical Biology, Boike et al. (2020) discover a covalent MYC inhibitor that selectively targets a single cysteine residue in an unstructured region of the protein.

Modulating Androgen Receptor-Driven Transcription in Prostate Cancer with Selective CDK9 Inhibitors.

Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.

A Small-Molecule Inhibitor to the Cytokine Interleukin-4.

Interleukin-4 (IL-4) is a multifunctional cytokine and an important regulator of inflammation. When deregulated, IL-4 activity is associated with asthma, allergic inflammation, and multiple types of cancer. While antibody-based inhibitors targeting the soluble cytokine have been evaluated clinically, they failed to achieve their end points in trials. Small-molecule inhibitors are an attractive alternative, but identifying effective chemotypes that inhibit the protein-protein interactions between cytokines and their receptors remains an active area of research. As a result, no small-molecule inhibitors to the soluble IL-4 cytokine have yet been reported. Here, we describe the first IL-4 small-molecule inhibitor identified and characterized through a combination of binding-based approaches and cell-based activity assays. The compound features a nicotinonitrile scaffold with micromolar affinity and potency for the cytokine and disrupts type II IL-4 signaling in cells. Small-molecule inhibitors of these important cell-signaling proteins have implications for numerous immune-related disorders and inform future drug discovery and design efforts for these challenging protein targets.

Structural and biophysical insights into the mode of covalent binding of rationally designed potent BMX inhibitors.

The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation. Molecular dynamic simulations also showed this interaction for two ligand/BMX complexes. Kinome selectivity profiling showed that the most potent compound is the strongest binder, displays intracellular target engagement in BMX-transfected cells with two-digit nanomolar inhibitory potency, and leads to BMX degradation PC3 in cells. The new inhibitors displayed anti-proliferative effects in androgen-receptor positive prostate cancer cells that where further increased when combined with known inhibitors of related signaling pathways, such as PI3K, AKT and Androgen Receptor. We expect these findings to guide development of new selective BMX therapeutic approaches.

An Array-Based Ligand Discovery Platform for Proteins With Short Half-Lives.

Many promising therapeutic protein targets were previously considered "undruggable" due to a deficit in structural information to guide drug design and/or a lack of an obvious binding pocket. Fortunately, array-based methods for evaluating protein binding against large chemical libraries, such as small-molecule microarray screening, have provided one of several emerging inroads to ligand discovery for these elusive targets. Despite the advance in the area of ligand discovery for poorly structured and intrinsically disordered proteins provided by array-based technologies involving cell lysates, the extension of this technology for screening proteins with short half-lives in physiologically relevant conformations has been technically challenging. In this chapter we present a protocol for leveraging in vitro translation strategies to enable array-based screening of short-lived proteins against large small-molecule libraries for ligand discovery.

Advances in discovering small molecules to probe protein function in a systems context.

High throughput screening (HTS) has historically been used for drug discovery almost exclusively by the pharmaceutical industry. Due to a significant decrease in costs associated with establishing a high throughput facility and an exponential interest in discovering probes of development and disease associated biomolecules, HTS core facilities have become an integral part of most academic and non-profit research institutions over the past decade. This major shift has led to the development of new HTS methodologies extending beyond the capabilities and target classes used in classical drug discovery approaches such as traditional enzymatic activity-based screens. In this brief review we describe some of the most interesting developments in HTS technologies and methods for chemical probe discovery.