James B. Murray

Evasion of apoptosis is a hallmark of cancer. Deregulation of BCL-XL, a member of the BCL-2 family of proteins, has been linked to the development of various tumor types. This study presents the design and synthesis of BCL-XL inhibitors with novel mono- and bicyclic cores. The new structural features were optimized to combine high binding efficiency with the opening of diverse novel vectors for additional modifications. The lead compounds exhibited picomolar affinities and significant cellular potency in the BCL-XL-dependent MOLT-4 cell line, which also translated into marked tumor growth inhibition in a xenograft study. These findings highlight the potential of BCL-XL inhibitors as therapeutic agents in cancer treatment by targeting the apoptotic intrinsic pathway.

Antibacterial resistance is a major global health problem, causing an increasing number of deaths worldwide. DsbA, a bacterial oxidoreductase enzyme, is pivotal for the correct folding and activity of virulence factors in bacteria. Inhibiting DsbA presents a promising avenue for developing antivirulence compounds and combating bacterial resistance. The enzyme's structure features two ligand-binding sites: a hydrophobic groove that is the binding site for natural peptide substrates and a "cryptic pocket" enclosed within the protein, which has recently been identified as a target for ligand design. In this study, we report the elaboration of a fragment from within the enclosed cryptic pocket into the hydrophobic groove of Escherichia coli DsbA, using X-ray crystallography-guided structure-based design and parallel synthesis coupled with crude reaction mixture screening (direct-to-biology). This effort yielded the most potent small-molecule EcDsbA inhibitors reported to date and exemplifies a productive strategy for exploiting a cryptic pocket for drug development.

Despite the exciting progress of bifunctional degrader molecules, also known as proteolysis-targeting chimeras (PROTACs), the rapidly expanding field is still significantly hampered by the lack of available E3 ligase ligands. Our research bridges this gap by uncovering a series of small-molecule ligands to the E3 ligase TRIM21 through DNA-Encoded Library (DEL) technology. We confirmed their interaction with TRIM21 using crystallography and demonstrated their antiproliferative effects across various cancer cell types. Furthermore, proteomic studies identified that the mRNA Export Factor GLE1 and the Nuclear Pore Complex Protein NUP155 were significantly downregulated on TRIM21 ligand treatment. This degradation required TRIM21 and was ubiquitin-proteasome-dependent. More specifically, NUP155 was the primary target for the TRIM21 ligands, while GLE1 was considered a passenger target on initial degradation of NUP155. Using immunofluorescence techniques, we further demonstrated that the degradation of GLE1 and NUP155 proteins impaired the integrity of the nuclear envelope, leading to cell death. Highlighted by this research, a novel mode of action has been discovered for the TRIM21 E3 ligase ligand, acting as a monovalent degrader that triggers de novo interaction with functional complex proteins and induces their degradation.

Small molecules that covalently modify proteins typically contain an electrophile that selectively reacts with nucleophilic residues in a protein target, such as cysteine, serine, and threonine. Targeting other amino acids is an emerging strategy in covalent probe design. This study reports the discovery and characterization of the covalent reaction between a novel allene warhead and a histidine residue in the active site of the bacterial thiol–disulfide oxidoreductase enzyme Escherichia coli DsbA (EcDsbA). Allenes have not been widely reported for their use as covalent warheads. The interaction was characterized by X-ray crystallography, nuclear magnetic resonance spectroscopy, and mass spectrometry. This analysis provided insights into the structure, reaction rate, and selectivity of the allene. Investigation of the reactivity with nucleophilic amino acids revealed that the reaction with the allene warhead shows some specificity for the histidine in the active site of EcDsbA. Thus, the allene represents a novel histidine-modifying warhead.

Inhibition of ubiquitin-specific protease 7, USP7, has been proposed as a mechanism to affect many disease processes, primarily those implicated in oncology. The bound crystal structure of a published high-throughput screening hit with low-micromolar affinity for USP7 identified three regions of the compound for structure-guided optimization. Replacing one side of the compound with different aromatic moieties gave little improvement in affinity, and the central piperidine could not be improved. However, the binding site for the other side of the compound was poorly defined in the crystal structure, which suggested a wide variety of synthetically accessible options for optimization. These were assessed by screening reaction mixtures that introduced different substituents to this other side. Subsequent optimization led to a compound with low-nanomolar affinity for USP7, which showed target engagement in tumors, was tolerated in mice, and showed efficacy in xenograft models.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down’s syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases. Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency, although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and selective DYRK1A inhibitors. Exploiting the structural differences in DYRK1A and its close homologue DYRK2, we were able to fine-tune the selectivity of our inhibitors. Our best compounds potently inhibited DYRK1A in the cell culture and in vivo and demonstrated drug-like properties. The inhibition of DYRK1A in vivo translated into dose-dependent tumor growth inhibition in a model of ovarian carcinoma.

Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.

Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation when observed in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy, has emerged as an attractive target for cancer therapy. Here we report the discovery of selective small molecule inhibitors of Mcl-1 that inhibit cellular activity. Fragment screening identified thienopyrimidine amino acids as a promising but non-selective hit that were optimized using NMR and X-ray derived structural information. The introduction of hindered rotation along a biaryl axis has conferred high selectivity to the compounds and cellular activity was brought on scale by offsetting the negative charge of the anchoring carboxylate group. The obtained compounds exhibit nanomolar binding affinity and mechanism-based cellular efficacy, caspase induction and growth inhibition. Thus, they are considered as potential leads toward the discovery of anti-Mcl-1 therapeutics.