Efficient, multi-hundred-gram scale access to E3 ubiquitin ligase ligands for degrader development.

Small molecule heterobifunctional degraders (commonly also known as PROTACs) offer tremendous potential to deliver new therapeutics in areas of unmet medical need. To deliver on this promise, a new discipline directed at degrader design and optimization has emerged within medicinal chemistry to address a central challenge, namely how to optimize relatively large, heterobifunctional molecules for activity, whilst maintaining drug-like properties. This process involves simultaneous optimization of the three principle degrader components: E3 ubiquitin ligase ligand, linker, and protein of interest (POI) ligand. A substantial degree of commonality exists with the E3 ligase ligands typically used at the early stages of degrader development, resulting in demand for these compounds as chemical building blocks in degrader research programs. We describe herein a collation of large scale, high-yielding syntheses to access the most utilized E3 ligase ligands to support early-stage degrader development.

Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel.

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii.

Many aldehydes, such as furfural, are present in high quantities in lignocellulose lysates and are fermentation inhibitors, which makes biofuel production from this abundant carbon source extremely challenging. Cbei_3974 has recently been identified as an aldo-keto reductase responsible for partial furfural resistance in Clostridium beijerinckii Rational engineering of this enzyme could enhance the furfural tolerance of this organism, thereby improving biofuel yields. We report an extensive characterization of Cbei_3974 and a single-crystal X-ray structure of Cbei_3974 in complex with NADPH at a resolution of 1.75 Å. Docking studies identified residues involved in substrate binding, and an activity screen revealed the substrate tolerance of the enzyme. Hydride transfer, which is partially rate limiting under physiological conditions, occurs from the pro-R hydrogen of NADPH. Enzyme isotope labeling revealed a temperature-independent enzyme isotope effect of unity, indicating that the enzyme does not use dynamic coupling for catalysis and suggesting that the active site of the enzyme is optimally configured for catalysis with the substrate tested.IMPORTANCE Here we report the crystal structure and biophysical properties of an aldehyde reductase that can detoxify furfural, a common inhibitor of biofuel fermentation found in lignocellulose lysates. The data contained here will serve as a guide for protein engineers to develop improved enzyme variants that would impart furfural resistance to the microorganisms used in biofuel production and thus lead to enhanced biofuel yields from this sustainable resource.

Reviewing the toolbox for degrader development in oncology.

The field of targeted protein degradation encompasses a growing number of modalities that achieve potent and selective knockdown of target proteins at the post-translational level. Among the most clinically advanced are bifunctional small-molecule degraders, also referred to as PROteolysis Targeting Chimeras, Degronimids, SNIPERs, or uSMITEs. Although applicable to many disease indications, oncology stands to be the first to benefit from this promising therapeutic approach, with the first investigational new drugs (INDs) filed in 2019 and a proliferation of research specifically focused on harnessing degraders for cancer treatment. In this review, we consider the toolbox of guidelines, reagents, and technologies that has evolved alongside the field to support degrader research and development.

An unexpected co-crystal structure of the calpain PEF(S) domain with Hfq reveals a potential chaperone function of Hfq.

Calpain is a Ca2+-activated, heterodimeric cysteine protease consisting of a large catalytic subunit and a small regulatory subunit. Dysregulation of this enzyme is involved in a range of pathological conditions such as cancer, Alzheimer's disease and rheumatoid arthritis, and thus calpain I is a drug target with potential therapeutic applications. Difficulty in the production of this enzyme has hindered structural and functional investigations in the past, although heterodimeric calpain I can be generated by Escherichia coli expression in low yield. Here, an unexpected structure discovered during crystallization trials of heterodimeric calpain I (CAPN1C115S + CAPNS1ΔGR) is reported. A novel co-crystal structure of the PEF(S) domain from the dissociated regulatory small subunit of calpain I and the RNA-binding chaperone Hfq, which was likely to be overproduced as a stress response to the recombinant expression conditions, was obtained, providing unexpected insight in the chaperone function of Hfq.

Structure-based design of allosteric calpain-1 inhibitors populating a novel bioactivity space.

Dimeric calpains constitute a promising therapeutic target for many diseases such as cardiovascular, neurodegenerative and ischaemic disease. The discovery of selective calpain inhibitors, however, has been extremely challenging. Previously, allosteric inhibitors of calpains, such as PD150606, which included a specific α-mercaptoacrylic acid sub-structure, were reported to bind to the penta-EF hand calcium binding domain, PEF(S) of calpain. Although these are selective to calpains over other cysteine proteases, their mode of action has remained elusive due to their ability to inhibit the active site domain with and without the presence of PEF(S), with similar potency. These findings have led to the question of whether the inhibitory response can be attributed to an allosteric mode of action or alternatively to inhibition at the active site. In order to address this problem, we report a structure-based virtual screening protocol as a novel approach for the discovery of PEF(S) binders that populate a novel chemical space. We have identified compound 1, Vidupiprant, which is shown to bind to the PEF(S) domain by the TNS displacement method, and it exhibited specificity in its allosteric mode of inhibition. Compound 1 inhibited the full-length calpain-1 complex with a higher potency (IC50 = 7.5 µM) than the selective, cell-permeable non-peptide calpain inhibitor, PD150606 (IC50 = 19.3 µM), where the latter also inhibited the active site domain in the absence of PEF(S) (IC50 = 17.8 µM). Hence the method presented here has identified known compounds with a novel allosteric mechanism for the inhibition of calpain-1. We show for the first time that the inhibition of enzyme activity can be attributed to an allosteric mode of action, which may offer improved selectivity and a reduced side-effects profile.