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.

Enantioselective Bioreduction of Medicinally Relevant Nitrogen-Heteroaromatic Ketones.

We herein report an enantioselective bioreduction of ketones that bear the most frequently used nitrogen-heteroaromatics in FDA-approved drugs. Ten varieties of these nitrogen-containing heterocycles were systematically investigated. Eight categories were studied for the first time and seven types were tolerated, significantly expanding the substrate scope of plant-mediated reduction. By use of purple carrots in buffered aqueous media with a simplified reaction setup, this biocatalytic transformation was achieved within 48 h at ambient temperature, offering medicinal chemists a pragmatic and scalable tool to access a broad variety of nitrogen-heteroaryl-containing chiral alcohols. With multiple reactive sites, the structurally diverse set of chiral alcohols can be used for library compound preparation, early route-scouting activities, and synthesis of other pharmaceutical molecules, favorably accelerating medicinal chemistry campaigns.

Performance of a 50-gene next generation sequencing panel with post-centrifuge supernatant cytology fluid in non-small-cell lung cancer.

BACKGROUND: Liquid based cytology (LBC) specimens are increasingly utilized for molecular analysis, as results are comparable to molecular analysis performed on traditional specimens (biopsy or cell block). However, there are few studies demonstrating the long-term viability of DNA in LBC samples. METHODS: In this study, a 50-gene next generation sequencing (NGS) panel was performed on DNA isolated from post-centrifuged supernatant LBC samples of cases of non-small-cell lung carcinoma. Comparison was made to results of an identical NGS panel performed on a concurrent clinical sample (biopsy or cell block). Quality parameters including DNA concentration, total reads, amplicons with reads under 450 and 350, and variant allele fraction were also compared. For a subset of LBC samples, DNA was isolated after being held for varying extended lengths of time after collection (up to 41 days) at 5°C and results compared. RESULTS: Results of NGS mutation analysis were concordant between LBC samples and clinical samples. DNA concentration was on average higher in the LBC samples compared to the clinical samples. The remaining metrics were more variable, but illustrated the adequacy of LBC samples for NGS testing. DNA isolated from LBC samples held for longer periods of time was of good concentration. NGS analysis was successfully performed on all samples, with concordance with results of clinical samples. CONCLUSION: DNA isolated directly from LBC fluid is suitable for NGS analysis. DNA is also stable in LBC preservative for extended periods of time before isolation and NGS analysis can subsequently be successfully performed.

Structure-Based Design of A-1293102, a Potent and Selective BCL-XL Inhibitor.

BCL-XL, an antiapoptotic member of the BCL-2 family of proteins, drives tumor survival and maintenance and thus represents a key target for cancer treatment. Herein we report the rational design of a novel series of selective BCL-XL inhibitors exemplified by A-1293102. This molecule contains structural elements of selective BCL-XL inhibitor A-1155463 and the dual BCL-XL/BCL-2 inhibitors ABT-737 and navitoclax, while representing a distinct pharmacophore as assessed by an objective cheminformatic evaluation. A-1293102 exhibited picomolar binding affinity to BCL-XL and both efficiently and selectively killed BCL-XL-dependent tumor cells. X-ray crystallographic analysis demonstrated a key hydrogen bonding network in the P2 binding pocket of BCL-XL, while the bent-back moiety achieved efficient occupancy of the P4 pocket in a manner similar to that of navitoclax. A-1293102 represents one of the few distinct structural series of selective BCL-XL inhibitors, and thus serves as a useful tool for biological studies as well as a lead compound for further optimization.

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.

Comparison of Tissue Molecular Biomarker Testing Turnaround Times and Concordance Between Standard of Care and the Biocartis Idylla Platform in Patients With Colorectal Cancer.

OBJECTIVES: Management of colorectal cancer warrants mutational analysis of KRAS/NRAS when considering anti-epidermal growth factor receptor therapy and BRAF testing for prognostic stratification. In this multicenter study, we compared a fully integrated, cartridge-based system to standard-of-care assays used by participating laboratories. METHODS: Twenty laboratories enrolled 874 colorectal cancer cases between November 2017 and December 2018. Testing was performed on the Idylla automated system (Biocartis) using the KRAS and NRAS-BRAF cartridges (research use only) and results compared with in-house standard-of-care testing methods. RESULTS: There were sufficient data on 780 cases to measure turnaround time compared with standard assays. In-house polymerase chain reaction (PCR) had an average testing turnaround time of 5.6 days, send-out PCR of 22.5 days, in-house Sanger sequencing of 14.7 days, send-out Sanger of 17.8 days, in-house next-generation sequencing (NGS) of 12.5 days, and send-out NGS of 20.0 days. Standard testing had an average turnaround time of 11 days. Idylla average time to results was 4.9 days with a range of 0.4 to 13.5 days. CONCLUSIONS: The described cartridge-based system offers rapid and reliable testing of clinically actionable mutation in colorectal cancer specimens directly from formalin-fixed, paraffin-embedded tissue sections. Its simplicity and ease of use compared with other molecular techniques make it suitable for routine clinical laboratory testing.

Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria.

Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism.

Structural Aspects of Bacterial Outer Membrane Protein Assembly.

The outer membrane of Gram-negative bacteria is predominantly populated by ß-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The ß-barrel assembly machinery (Bam) complex is responsible for the proper assembly of ß-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

Structural basis for the recruitment of glycogen synthase by glycogenin.

Glycogen is a primary form of energy storage in eukaryotes that is essential for glucose homeostasis. The glycogen polymer is synthesized from glucose through the cooperative action of glycogen synthase (GS), glycogenin (GN), and glycogen branching enzyme and forms particles that range in size from 10 to 290 nm. GS is regulated by allosteric activation upon glucose-6-phosphate binding and inactivation by phosphorylation on its N- and C-terminal regulatory tails. GS alone is incapable of starting synthesis of a glycogen particle de novo, but instead it extends preexisting chains initiated by glycogenin. The molecular determinants by which GS recognizes self-glucosylated GN, the first step in glycogenesis, are unknown. We describe the crystal structure of Caenorhabditis elegans GS in complex with a minimal GS targeting sequence in GN and show that a 34-residue region of GN binds to a conserved surface on GS that is distinct from previously characterized allosteric and binding surfaces on the enzyme. The interaction identified in the GS-GN costructure is required for GS-GN interaction and for glycogen synthesis in a cell-free system and in intact cells. The interaction of full-length GS-GN proteins is enhanced by an avidity effect imparted by a dimeric state of GN and a tetrameric state of GS. Finally, the structure of the N- and C-terminal regulatory tails of GS provide a basis for understanding phosphoregulation of glycogen synthesis. These results uncover a central molecular mechanism that governs glycogen metabolism.

Identification and preliminary characterization of a potent, safe, and orally efficacious inhibitor of acyl-CoA:diacylglycerol acyltransferase 1.

A high-throughput screen against human DGAT-1 led to the identification of a core structure that was subsequently optimized to afford the potent, selective, and orally bioavailable compound 14. Oral administration at doses ≥0.03 mg/kg significantly reduced postprandial triglycerides in mice following an oral lipid challenge. Further assessment in both acute and chronic safety pharmacology and toxicology studies demonstrated a clean profile up to high plasma levels, thus culminating in the nomination of 14 as clinical candidate ABT-046.

Inhibitors of diacylglycerol acyltransferase: a review of 2008 patents.

BACKGROUND: Postprandial hypertriglyceridemia has been identified as a major independent risk factor for future cardiovascular events. Therefore, inhibition of triglyceride synthesis has enormous therapeutic potential in the treatment of metabolic disorders. Diacylglycerol acyltransferase (DGAT) enzymes catalyze the final and only committed step in triglyceride biosynthesis and have thus been identified as potential therapeutic targets to combat human cardio-metabolic diseases. OBJECTIVE/METHOD: Significant interest in DGAT-1 inhibitors has emerged in the last several years. To provide a perspective on the exciting features of this enzyme for targeting metabolic diseases, a summary of the biology and pharmacology surrounding the DGAT enzymes is presented. Following this is a discussion of the various chemotypes that have been disclosed within relevant patent applications published in 2008. Specifically, the similarities and differences of the chemical structures and the biological data that are provided to support the corresponding claims are presented. CONCLUSION: Small molecule and biologic-based DGAT inhibitors have been successfully used for the preclinical validation of DGAT enzymes as targets for the treatment of metabolic diseases. Given the advanced stage in which some of the chemical matter resides, it is expected that DGAT inhibitors will enter the clinic in the coming years.

Lead optimization of melanin concentrating hormone receptor 1 antagonists with low hERG channel activity.

The discovery of small molecule melanin concentrating hormone receptor (MHCr1) antagonists as novel therapeutic agents has been widely pursued across the pharmaceutical industry. While multiple chemotypes of small molecule MCHr1 antagonists have been identified and shown to induce weight loss in rodent models of obesity, many of these lead compounds have been found to cross react with the hERG channel. This review describes efforts that led to the identification of two sub-series of MCHr1 antagonists with low affinity for the hERG channel. Ultimately, however, the modifications introduced to thwart hERG channel activity resulted in lead compounds with sub-optimal CNS behavior.

Validation of diacyl glycerolacyltransferase I as a novel target for the treatment of obesity and dyslipidemia using a potent and selective small molecule inhibitor.

A highly potent and selective DGAT-1 inhibitor was identified and used in rodent models of obesity and postprandial chylomicron excursion to validate DGAT-1 inhibition as a novel approach for the treatment of metabolic diseases. Specifically, compound 4a conferred weight loss and a reduction in liver triglycerides when dosed chronically in DIO mice and depleted serum triglycerides following a lipid challenge in a dose-dependent manner, thus, reproducing major phenotypical characteristics of DGAT-1(-/-) mice.

Concise construction of novel bridged bicyclic lactams by sequenced Ugi/RCM/Heck reactions.

Herein we report a concise protocol for the diastereoselective synthesis of novel bridged bicyclic lactams from commercially available components by the sequence of Ugi, ring-closing metathesis (RCM), and Heck reactions. X-ray diffraction studies revealed that the bicyclic products contain varying degrees of pyramidalization of the bridgehead nitrogen atom.

Lead optimization strategies and tactics applied to the discovery of melanin concentrating hormone receptor 1 antagonists.

The discovery of small molecule melanin concentrating hormone receptor (MCHr1) antagonists as novel therapeutic agents for the treatment of obesity has been actively pursued across the pharmaceutical industry. While multiple chemotypes of small molecule MCHr1 antagonists have been identified and shown to deliver weight loss in animal models of obesity, many of these lead compounds have been found to cross-react with the hERG channel and/or demonstrate deleterious effects on cardiovascular hemodynamic parameters. This review describes an approach to rapidly identifying safer MCHr1 antagonists by placing assays to assess cardiovascular safety early in the lead optimization compound prioritization process. Ultimately, despite putting significant effort toward the discovery of a MCHr1 antagonist for the treatment of obesity, we were unable to deliver a candidate compound that attained an acceptable therapeutic index (TI = 30-100) in our in vivo models. Our inability to identify a compound with an acceptable therapeutic index was driven by two primary factors: 1) high levels of sustained drug exposure in the brain was required to achieve efficacy; and 2) many small molecule MCHR1 receptor antagonists suffer from receptor cross-reactivity that leads to cardiovascular toxicity at low multiples of their therapeutic plasma concentration.

Discovery and SAR development of thienopyridones: a class of small molecule AMPK activators.

AMP-activated protein kinase (AMPK) is well established as a sensor and regulator of intracellular and whole-body energy metabolism. A high-throughput screen was performed in order to identify chemotypes that are bound by AMPK. A novel thienopyridone compound (1) was identified and subsequently optimized. The structure-activity relationships that emerged from this effort are described.

Identification of diamino chromone-2-carboxamides as MCHr1 antagonists with minimal hERG channel activity.

A series of potent 2-carboxychromone-based melanin-concentrating hormone receptor 1 (MCHr1) antagonists were synthesized and evaluated for hERG (human Ether-a-go-go Related Gene) channel affinity and functional blockade. Basic dialkylamine-terminated analogs were found to weakly bind the hERG channel and provided marked improvement in a functional patch-clamp assay versus previously reported antagonists of the series.

An evaluation of 3,4-methylenedioxy phenyl replacements in the aminopiperidine chromone class of MCHr1 antagonists.

The optimization of potent MCHr1 antagonist 1 with respect to improving its in vitro profile by replacement of the 3,4-methylenedioxy phenyl (piperonyl) moiety led to the discovery of 19, a compound that showed excellent MCHr1 binding and functional potencies in addition to possessing superior hERG separation, CYP3A4 profile, and receptor cross-reactivity profiles.

Constrained 7-fluorocarboxychromone-4-aminopiperidine based Melanin-concentrating hormone receptor 1 antagonists: the effects of chirality on substituted indan-1-ylamines.

The incorporation of constrained tertiary amines into an existing class of N-benzyl-4-aminopiperidinyl chromone-based MCHr1 antagonists led to the identification of a series of chiral racemic compounds that displayed good to excellent functional potency, binding affinity, and selectivity over the hERG channel. Further separation of two distinct chiral racemic compounds into their corresponding pairs of enantiomers revealed a considerable selectivity for MCHr1 for one configuration, in addition to a striking difference in oral exposure between one pair of enantiomers in diet-induced obese mice. Oral administration of the most potent compound in this class in the same animal model led to significant reduction of fat mass in a semi-chronic model for weight loss.