BCL-XL-targeting antibody-drug conjugates are active in preclinical models and mitigate on-mechanism toxicity of small-molecule inhibitors.

Overexpression of the antiapoptotic protein B-cell lymphoma-extra large (BCL-XL) is associated with drug resistance and disease progression in numerous cancers. The compelling nature of this protein as a therapeutic target prompted efforts to develop selective small-molecule BCL-XL inhibitors. Although efficacious in preclinical models, we report herein that selective BCL-XL inhibitors cause severe mechanism-based cardiovascular toxicity in higher preclinical species. To overcome this liability, antibody-drug conjugates were constructed using altered BCL-XL-targeting warheads, unique linker technologies, and therapeutic antibodies. The epidermal growth factor receptor-targeting antibody-drug conjugate AM1-15 inhibited growth of tumor xenografts and did not cause cardiovascular toxicity nor dose-limiting thrombocytopenia in monkeys. While an unprecedented BCL-XL-mediated toxicity was uncovered in monkey kidneys upon repeat dosing of AM1-15, this toxicity was mitigated via further drug-linker modification to afford AM1-AAA (AM1-25). The AAA drug-linker has since been incorporated into mirzotamab clezutoclax, the first selective BCL-XL-targeting agent to enter human clinical trials.

Enabling, Decagram-Scale Synthesis of Macrocyclic MCL-1 Inhibitor ABBV-467.

ABBV-467 is a highly potent and selective MCL-1 inhibitor that was advanced to a phase I clinical trial for the treatment of multiple myeloma. Due to its large size and structural complexity, ABBV-467 is a challenging synthetic target. Herein, we describe the synthesis of ABBV-467 on a decagram scale, which enabled preclinical characterization. The strategy is convergent and stereoselective, featuring a hindered biaryl cross coupling, enantioselective hydrogenation, and conformationally preorganized macrocyclization by C-O bond formation as key steps.

Selective MCL-1 inhibitor ABBV-467 is efficacious in tumor models but is associated with cardiac troponin increases in patients.

BACKGROUND: MCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902). METHODS: Binding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy. RESULTS: Here we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings. CONCLUSIONS: The selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients.

Apoptosis is a type of cell death that removes abnormal cells from the body. Cancer cells can have increased levels of MCL-1, a protein that helps cells survive and prevents apoptosis. ABBV-467 is a new drug that blocks the action of MCL-1 (an MCL-1 inhibitor) and could promote apoptosis. In animal models, ABBV-467 led to cancer cell death and delayed tumor growth. ABBV-467 was also studied in a clinical trial in 8 patients with multiple myeloma, a blood cancer. In 1 patient, ABBV-467 treatment prevented the cancer from getting any worse for 8 months. However, in 4 out of 8 patients ABBV-467 increased the levels of troponin, a protein associated with damage to the heart. This concerning side effect may impact the future development of MCL-1 inhibitors as anticancer drugs.

Novel chemical entities inhibiting Mycobacterium tuberculosis growth identified by phenotypic high-throughput screening.

We performed a high-throughput phenotypic whole cell screen of Mycobacterium tuberculosis against a diverse chemical library of approximately 100,000 compounds from the AbbVie corporate collection and identified 24 chemotypes with anti-tubercular activity. We selected two series for further exploration and conducted structure-activity relationship studies with new analogs for the 4-phenyl piperidines (4PP) and phenylcyclobutane carboxamides (PCB). Strains with mutations in MmpL3 demonstrated resistance to both compound series. We isolated resistant mutants for the two series and found mutations in MmpL3. These data suggest that MmpL3 is the target, or mechanism of resistance for both series.

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-XL Inhibitor.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.

Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts.

Discovery of an orally bioavailable small molecule inhibitor of prosurvival B-cell lymphoma 2 proteins.

Overexpression of prosurvival proteins such as Bcl-2 and Bcl-X L has been correlated with tumorigenesis and resistance to chemotherapy, and thus, the development of antagonists of these proteins may provide a novel means for the treatment of cancer. We recently described the discovery of 1 (ABT-737), which binds Bcl-2, Bcl-X L, and Bcl-w with high affinity, shows robust antitumor activity in murine tumor xenograft models, but is not orally bioavailable. Herein, we report that targeted modifications at three key positions of 1 resulted in a 20-fold improvement in the pharmacokinetic/pharmacodynamic relationship (PK/PD) between oral exposure (AUC) and in vitro efficacy in human tumor cell lines (EC 50). The resulting compound, 2 (ABT-263), is orally efficacious in an established xenograft model of human small cell lung cancer, inducing complete tumor regressions in all animals. Compound 2 is currently in multiple phase 1 clinical trials in patients with small cell lung cancer and hematological malignancies.

Discovery and design of novel HSP90 inhibitors using multiple fragment-based design strategies.

The molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme. Second site leads were also identified which bound to HSP90 in two distinct conformations, an 'open' and 'closed' form. Intriguingly, linked fragment approaches targeting both of these conformations were successful in producing novel, micromolar inhibitors. Overall, this study shows that, with only a few fragment hits, multiple lead series can be generated for HSP90 due to the inherent flexibility of the active site. Thus, ample opportunities exist to use these lead series in the development of clinically useful HSP90 inhibitors for the treatment of cancers.

Discovery of a novel small molecule binding site of human survivin.

Survivin is one of the most tumor-specific genes in the human genome and is an attractive target for cancer therapy. However, small-molecule ligands for survivin have not yet been described. Thus, an interrogation of survivin which could potentially both validate a small-molecule therapy approach, and determine the biochemical nature of any of survivin's functions has not been possible. Here we describe the discovery and characterization of a small molecule binding site on the survivin surface distinct from the Smac peptide-binding site. The new site is located at the dimer interface and exhibits many of the features of highly druggable, biologically relevant protein binding sites. A variety of small hydrophobic compounds were found that bind with moderate affinity to this binding site, from which one lead was developed into a group of compounds with nanomolar affinity. Additionally, a subset of these compounds are adequately water-soluble and cell-permeable. Thus, the structural studies and small molecules described here provide tools that can be used to probe the biochemical role(s) of survivin, and may ultimately serve as a basis for the development of small molecule therapeutics acting via direct or allosteric disruption of binding events related to this poorly understood target.

Studies leading to potent, dual inhibitors of Bcl-2 and Bcl-xL.

Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.

Discovery and structure-activity relationship of antagonists of B-cell lymphoma 2 family proteins with chemopotentiation activity in vitro and in vivo.

Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X(L) function, is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-microM binding but were strongly deactivated in the presence of serum. The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of inhibitors bound to both Bcl-X(L) and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-X(L) binding and greatly increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X(L) with a K(i) of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X(L) overexpression against cytokine deprivation in FL5.12 cells with an EC(50) of 0.47 microM. 73R showed little effect on the viability of the human non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model.

An inhibitor of Bcl-2 family proteins induces regression of solid tumours.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

A facile one-carbon homologation of aryl aldehydes to amides.

The easily accessible 2-aryl-1,1-dibromo-1-alkenes can be converted to amides under unusually mild conditions in good to excellent yields. Both electron-donating and electron-withdrawing substitutions on the aromatic rings are tolerated, and the reaction works well with hindered alkylamines. This simple homologation could find broad applications. [reaction: see text]