Challenges with the Synthesis of a Macrocyclic Thioether Peptide: From Milligram to Multigram Using Solid Phase Peptide Synthesis (SPPS).

We describe an optimization and scale-up of the 45-membered macrocyclic thioether peptide BMS-986189 utilizing solid-phase peptide synthesis (SPPS). Improvements to linear peptide isolation, macrocyclization, and peptide purification were demonstrated to increase the throughput and purification of material on scale and enabled the synthesis and purification of >60 g of target peptide. Taken together, not only these improvements resulted in a 28-fold yield increase from the original SPPS approach, but also the generality of this newly developed SPPS purification sequence has found application in the synthesis and purification of other macrocyclic thioether peptides.

Annotation of 2,507 Saccharomyces cerevisiae genomes.

Saccharomyces cerevisiae (baker's yeast, budding yeast) is one of the most important model organisms for biological research and is a crucial microorganism in industry. Currently, a huge number of Saccharomyces cerevisiae genome sequences are available at the public domain. However, these genomes are distributed at different websites and a large number of them are released without annotation information. To provide one complete annotated genome data resource, we collected 2,507 Saccharomyces cerevisiae genome assemblies and re-annotated 2,506 assemblies using a custom annotation pipeline, producing a total of 15,407,164 protein-coding gene models. With a custom pipeline, all these gene sequences were clustered into families. A total of 1,506 single-copy genes were selected as marker genes, which were then used to evaluate the genome completeness and base qualities of all assemblies. Pangenomic analyses were performed based on a selected subset of 847 medium-high-quality genomes. Statistical comparisons revealed a number of gene families showing copy number variations among different organism sources. To the authors' knowledge, this study represents the largest genome annotation project of S. cerevisiae so far, providing rich genomic resources for the future studies of the model organism S. cerevisiae and its relatives.IMPORTANCESaccharomyces cerevisiae (baker's yeast, budding yeast) is one of the most important model organisms for biological research and is a crucial microorganism in industry. Though a huge number of Saccharomyces cerevisiae genome sequences are available at the public domain, these genomes are distributed at different websites and most are released without annotation, hindering the efficient reuse of these genome resources. Here, we collected 2,507 genomes for Saccharomyces cerevisiae, performed genome annotation, and evaluated the genome qualities. All the obtained data have been deposited at public repositories and are freely accessible to the community. This study represents the largest genome annotation project of S. cerevisiae so far, providing one complete annotated genome data set for S. cerevisiae, an important workhorse for fundamental biology, biotechnology, and industry.

Discovery of novel pyridinones as MGAT2 inhibitors for the treatment of metabolic disorders.

Inhibition of monoacylglycerol transferase 2 (MGAT2) has recently emerged as a potential therapeutic strategy for the treatment of metabolic diseases such as obesity, diabetes and non-alcoholic steatohepatitis (NASH). Metabolism studies with our clinical lead (1) suggested variability in in vitro glucuronidation rates in liver microsomes across species, which made projection of human doses challenging. In addition, the observation of deconjugation of the C3-C4 double bond in the dihydropyridinone ring of 1 in solution had the potential to complicate its clinical development. This report describes our lead optimization efforts in a novel pyridinone series, exemplified by compound 33, which successfully addressed both of these potential issues.

Analysis of CRISPR-Cas Loci and their Targets in Levilactobacillus brevis.

The CRISPR‒Cas system acts as a bacterial defense mechanism by conferring adaptive immunity and limiting genetic reshuffling. However, under adverse environmental hazards, bacteria can employ their CRISPR‒Cas system to exchange genes that are vital for adaptation and survival. Levilactobacillus brevis is a lactic acid bacterium with great potential for commercial purposes because it can be genetically manipulated to enhance its functionality and nutritional value. Nevertheless, the CRISPR‒Cas system might interfere with the genetic modification process. Additionally, little is known about the CRISPR‒Cas system in this industrially important microorganism. Here, we investigate the prevalence, diversity, and targets of CRISPR‒Cas systems in the genus Levilactobacillus, further focusing on complete genomes of L. brevis. Using the CRISPRCasFinder webserver, we identified 801 putative CRISPR-Cas systems in the genus Levilactobacillus. Further investigation focusing on the complete genomes of L. brevis revealed 54 putative CRISPR-Cas systems. Of these, 46 were orphan CRISPRs, and eight were CRISPR‒Cas systems. The type II-A CRISPR‒Cas system is the most common in Levilactobacillus and L. brevis complete genomes. Analysis of the spacer's target showed that the CRISPR‒Cas systems of L. brevis mainly target the enterococcal plasmids. Comparative analysis of putative CRISPR-Cas loci in Levilactobacillus brevis.

The Discovery of GSK3640254, a Next-Generation Inhibitor of HIV-1 Maturation.

GSK3640254 is an HIV-1 maturation inhibitor (MI) that exhibits significantly improved antiviral activity toward a range of clinically relevant polymorphic variants with reduced sensitivity toward the second-generation MI GSK3532795 (BMS-955176). The key structural difference between GSK3640254 and its predecessor is the replacement of the para-substituted benzoic acid moiety attached at the C-3 position of the triterpenoid core with a cyclohex-3-ene-1-carboxylic acid substituted with a CH2F moiety at the carbon atom α- to the pharmacophoric carboxylic acid. This structural element provided a new vector with which to explore structure-activity relationships (SARs) and led to compounds with improved polymorphic coverage while preserving pharmacokinetic (PK) properties. The approach to the design of GSK3640254, the development of a synthetic route and its preclinical profile are discussed. GSK3640254 is currently in phase IIb clinical trials after demonstrating a dose-related reduction in HIV-1 viral load over 7-10 days of dosing to HIV-1-infected subjects.

Reduced sensitivity of lager brewing yeast to premature yeast flocculation via adaptive evolution.

Malt-induced premature yeast flocculation (PYF) is a sporadic problem within the brewing industry. The use of PYF malts is concomitant with a number of negative impacts on beer quality, including incomplete fermentation and/or flavor defects. Although malt-induced PYF is widely acknowledged, actions taken so far have proved insufficient to solve the PYF-related issues. To limit the detrimental effects of PYF malts on beer production, an adaptive laboratory evolution (ALE) process was applied in this study to an industrial lager brewing yeast strain (TT02), in an attempt to generate variant strains with improved fermentation performance in PYF wort. Through a batch fermentation-based adaptation process, evolved variants were isolated and screened for their phenotypic and metabolic traits. The investigation focused mainly on the tendency to remain in suspension, fermentation capacity and final acetaldehyde concentration. We successfully obtained a variant (TT02-30 T) with improved fermentation properties. The improvement was seen in worts prepared from different types of PYF malt as well as normal malt. Furthermore, ALE of lager brewing yeast in PYF wort yielded a wide array of mutations. Several changes in the genomes (copy number variation in flocculin encoding gene FLO1 and a missense SNP in a putative mitochondrial membrane protein coding gene FMP10) of the variant strains relative to the original strain were observed. These could potentially contribute to the improved yeast suspension during fermentation. Importantly, mutational enrichment in genes related to ion binding in PYF-evolved strains suggests the involvement of the yeast ion transportation process in dealing with the PYF stress. Our study demonstrates the possibility of attenuating yeast sensitivity to PYF malts over time through adaptive laboratory evolution via spontaneous mutation.

Discovery of Milvexian, a High-Affinity, Orally Bioavailable Inhibitor of Factor XIa in Clinical Studies for Antithrombotic Therapy.

Factor XIa (FXIa) is an enzyme in the coagulation cascade thought to amplify thrombin generation but has a limited role in hemostasis. From preclinical models and human genetics, an inhibitor of FXIa has the potential to be an antithrombotic agent with superior efficacy and safety. Reversible and irreversible inhibitors of FXIa have demonstrated excellent antithrombotic efficacy without increased bleeding time in animal models (Weitz, J. I., Chan, N. C. Arterioscler. Thromb. Vasc. Biol. 2019, 39 (1), 7-12). Herein, we report the discovery of a novel series of macrocyclic FXIa inhibitors containing a pyrazole P2' moiety. Optimization of the series for (pharmacokinetic) PK properties, free fraction, and solubility resulted in the identification of milvexian (BMS-986177/JNJ-70033093, 17, FXIa Ki = 0.11 nM) as a clinical candidate for the prevention and treatment of thromboembolic disorders, suitable for oral administration.

Screening Hit to Clinical Candidate: Discovery of BMS-963272, a Potent, Selective MGAT2 Inhibitor for the Treatment of Metabolic Disorders.

MGAT2 inhibition is a potential therapeutic approach for the treatment of metabolic disorders. High-throughput screening of the BMS internal compound collection identified the aryl dihydropyridinone compound 1 (hMGAT2 IC50 = 175 nM) as a hit. Compound 1 had moderate potency against human MGAT2, was inactive vs mouse MGAT2 and had poor microsomal metabolic stability. A novel chemistry route was developed to synthesize aryl dihydropyridinone analogs to explore structure-activity relationship around this hit, leading to the discovery of potent and selective MGAT2 inhibitors 21f, 21s, and 28e that are stable to liver microsomal metabolism. After triaging out 21f due to its inferior in vivo potency, pharmacokinetics, and structure-based liabilities and tetrazole 28e due to its inferior channel liability profile, 21s (BMS-963272) was selected as the clinical candidate following demonstration of on-target weight loss efficacy in the diet-induced obese mouse model and an acceptable safety and tolerability profile in multiple preclinical species.

Long-Acting Tumor-Activated Prodrug of a TGFßR Inhibitor.

Inhibition of TGFß signaling in concert with a checkpoint blockade has been shown to provide improved and durable antitumor immune response in mouse models. However, on-target adverse cardiovascular effects have limited the clinical use of TGFß receptor (TGFßR) inhibitors in cancer therapy. To restrict the activity of TGFßR inhibitors to tumor tissues and thereby widen the therapeutic index, a series of tumor-activated prodrugs of a selective small molecule TGFßR1 inhibitor 1 were prepared by appending 1 to a serine protease substrate and a half-life extension fatty acid carbon chain. The prodrugs were shown to be selectively metabolized in tumor tissues relative to the heart and blood and demonstrated a prolonged favorable increase in the tumor-to-heart ratio of the active drug in tissue distribution studies. Once-weekly administration of the most tissue-selective compound 10 provided anti-tumor efficacy comparable to the parent compound and reduced systemic exposure of the active drug.

Large-scale supercritical fluid chromatography purification of unstable STING agonist intermediates.

A regioisomeric mixture of the nucleoside derivative, Intermediate 1, required resolution by preparative supercritical fluid chromatography (SFC) in order to obtain the desired regioisomer as a key intermediate in a STING agonist program. Various chiral columns and solvents including methanol, acetonitrile, isopropanol, and the mixture of acetonitrile and isopropanol as organic modifiers in carbon dioxide at different temperatures were screened to obtain the best regioisomeric resolution. A key issue associated with interconversion between the regioisomers via silyl migration during purification was investigated in methanol, acetonitrile, and the mixture of acetonitrile and isopropanol, and the optimal organic modifier in CO2 was established to mitigate the interconversion to an acceptable level (<5%). Taking into account peak resolution, throughput, interconversion and operation robustness, an efficient SFC method for large-scale purification was successfully developed and scaled up onto a 5 cm I. D. Chiralcel OJ-H column using 25% acetonitrile: isopropanol [1:1 (v/v)] with 0.1% ammonium hydroxide as the modifier in CO2 at a total flow rate of 270 mL/min and a temperature of 30°C. In addition, continual evaporation (i.e. every hour) of the desired isomer fraction stream post-separation ensured minimal further interconversion. A total of 258 grams were separated at a high throughput of 8.6 g/h. Regioisomeric purity of the desired isomer of Intermediate 1 was ≥98.2% and the recovery was ≥90.2%. A similar purification strategy was applied to the regioisomeric resolution of Intermediate 2, an analog of Intermediate 1. In total, 1028 grams of Intermediate 2 were processed at a high throughput of 12.5 g/h on a Viridis BEH 2-EP column. The regioisomeric purity of the desired isomer was ≥96.8% and the recovery was ≥90.7%.

A Stereocontrolled Synthesis of a Phosphorothioate Cyclic Dinucleotide-Based STING Agonist.

We describe a stereodefined synthesis of the newly identified non-natural phosphorothioate cyclic dinucleotide (CDN) STING agonist, BMT-390025. The new route avoids the low-yielding racemic approach using P(III)-based reagents, and the stereospecific assembly of the phosphorothioate linkages are forged via the recently invented P(V)-based platform of the so-called PSI (Ψ) reagent system. This P(V) approach allows for the complete control of chirality of the P-based linkages and enabled conclusive evidence of the absolute configuration. The new approach offers robust procedures for preparing the stereodefined CDN in eight steps starting from advanced nucelosides, with late-stage direct drop isolations and telescoped steps enabling an efficient scale-up that proceeded in an overall 15% yield to produce multigram amounts of the CDN.

The effect of water on the large-scale supercritical fluid chromatography purification of two factor XIa active pharmaceutical ingredients.

BMS-962212, a parenteral Factor XIa inhibitor, was scaled-up for toxicity studies. Two steps of supercritical fluid chromatography (SFC) were developed for the chiral resolution of the penultimate and achiral purification of final active pharmaceutical ingredient (API), BMS-962212. A robust SFC process using Chiralcel OD-H with methanol-acetonitrile as modifier in CO2 was established to achieve a stable and uninterrupted operation with reduced mobile phase viscosity and system pressure drop. More than 230 g of the racemic penultimate was chirally resolved to reach >99% chiral purity, ready for final tert-butyl ester deprotection to provide the API. There were a significant number of impurities in BMS-962212 generated from the final step that needed to be removed. In contrast to conventional SFC conditions, an SFC method exploiting water and ammonia as additives in both the mobile phase and sample solution was developed to accomplish purification and desalting (i.e. removing TFA) of the zwitterionic API in one step. Water as an additive eliminated salt precipitation and improved the resolution while ammonia contributed to the desalting, details of which will be discussed in this article. A throughput of 2 g/h was achieved, and >80 g of the crude API was purified. The same strategy was applied to another Factor XIa API (compound A) and its penultimate.

Driving Potency with Rotationally Stable Atropisomers: Discovery of Pyridopyrimidinedione-Carbazole Inhibitors of BTK.

Bruton's tyrosine kinase (BTK) has been shown to play a key role in the pathogenesis of autoimmunity. Therefore, the inhibition of the kinase activity of BTK with a small molecule inhibitor could offer a breakthrough in the clinical treatment of many autoimmune diseases. This Letter describes the discovery of BMS-986143 through systematic structure-activity relationship (SAR) development. This compound benefits from defined chirality derived from two rotationally stable atropisomeric axes, providing a potent and selective single atropisomer with desirable efficacy and tolerability profiles.

Discovery of a Parenteral Small Molecule Coagulation Factor XIa Inhibitor Clinical Candidate (BMS-962212).

Factor XIa (FXIa) is a blood coagulation enzyme that is involved in the amplification of thrombin generation. Mounting evidence suggests that direct inhibition of FXIa can block pathologic thrombus formation while preserving normal hemostasis. Preclinical studies using a variety of approaches to reduce FXIa activity, including direct inhibitors of FXIa, have demonstrated good antithrombotic efficacy without increasing bleeding. On the basis of this potential, we targeted our efforts at identifying potent inhibitors of FXIa with a focus on discovering an acute antithrombotic agent for use in a hospital setting. Herein we describe the discovery of a potent FXIa clinical candidate, 55 (FXIa Ki = 0.7 nM), with excellent preclinical efficacy in thrombosis models and aqueous solubility suitable for intravenous administration. BMS-962212 is a reversible, direct, and highly selective small molecule inhibitor of FXIa.

Discovery of Highly Potent Liver X Receptor ß Agonists.

Introducing a uniquely substituted phenyl sulfone into a series of biphenyl imidazole liver X receptor (LXR) agonists afforded a dramatic potency improvement for induction of ATP binding cassette transporters, ABCA1 and ABCG1, in human whole blood. The agonist series demonstrated robust LXRß activity (>70%) with low partial LXRα agonist activity (<25%) in cell assays, providing a window between desired blood cell ABCG1 gene induction in cynomolgus monkeys and modest elevation of plasma triglycerides for agonist 15. The addition of polarity to the phenyl sulfone also reduced binding to the plasma protein, human α-1-acid glycoprotein. Agonist 15 was selected for clinical development based on the favorable combination of in vitro properties, excellent pharmacokinetic parameters, and a favorable lipid profile.

Discovery of Indazoles as Potent, Orally Active Dual Neurokinin 1 Receptor Antagonists and Serotonin Transporter Inhibitors for the Treatment of Depression.

Combination studies of neurokinin 1 (NK1) receptor antagonists and serotonin-selective reuptake inhibitors (SSRIs) have shown promise in preclinical models of depression. Such a combination may offer important advantages over the current standard of care. Herein we describe the discovery and optimization of an indazole-based chemotype to provide a series of potent dual NK1 receptor antagonists/serotonin transporter (SERT) inhibitors to overcome issues of ion channel blockade. This effort culminated in the identification of compound 9, an analogue that demonstrated favorable oral bioavailability, excellent brain uptake, and robust in vivo efficacy in a validated depression model. Over the course of this work, a novel heterocycle-directed asymmetric hydrogenation was developed to facilitate installation of the key stereogenic center.

Association study of angiotensin converting enzyme gene polymorphism with elderly diabetic hypertension and lipids levels.

OBJECTIVE: To investigate the relationship between angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and diabetic essential hypertension in elderly population. METHODS: Polymerase chain reaction (PCR) technique was used in 260 elderly normal control patients, 205 elderly hypertensive patients and 138 elderly diabetic hypertensive patients to detect the I/D polymorphism in ACE gene. RESULTS: DD genotype frequency (0.352) and D allele frequency (0.543) in elderly hypertensive patients were higher than those in the normal control patients. DD genotype (0.421) and D allele frequency (0.579) in elderly diabetic hypertensive patients were significantly higher than those in the control patients (0.133 and 0.250). The differences of DD genotype and D allele frequency between the elderly hypertensive patients and the elderly diabetic hypertensive patients were not significant (P > 0.05). CONCLUSION: ACE gene deletion is a risk factor for hypertension but is not a risk factor for diabetes in elderly population.

Reciprocal regulation between SigK and differentiation programs in Streptomyces coelicolor.

Here we reported that deletion of SigK (SCO6520), a sigma factor in Streptomyces coelicolor, caused an earlier switch from vegetative mycelia to aerial mycelia and higher expression of chpE and chpH than that in the wild type. Loss of SigK also resulted in accelerated and enhanced production of antibiotics, actinorhodin, and undecylprodigiosin and increased expression of actII-orf4 and redD. These results suggested that SigK had a negative role in morphological transition and secondary metabolism. Furthermore, the sigK promoter (sigKp) activity gradually increased and sigK expression was partially dependent on SigK, but this dependence decreased during the developmental course of substrate mycelia. Meanwhile, two potentially nonspecific cleavages occurred between SigK and green fluorescent protein, and the SigK fusion proteins expressed under the constitutive promoter ermEp* sharply decreased and disappeared when aerial mycelia emerged. If expressed under sigKp, 3FLAG-SigK showed similar dynamic patterns but did not decrease as sharply as SigK expressed under ermEp*. These data suggested that the climbing expression of sigK might reduce the prompt degradation of SigK during vegetative hypha development for the proper timing of morphogenesis and that SigK vanished to remove the block for the emergence of aerial mycelia. Thus, we proposed that SigK had inhibitory roles on developmental events and that these inhibitory effects may be released by SigK degradation.

Involvement of SigT and RstA in the differentiation of Streptomyces coelicolor.

SigT is an ECF sigma factor in Streptomyces coelicolor. sigT and its putative anti-sigma factor gene rstA are located in one putative operon, and SigT could physically interact with RstA. Deletion of sigT or rstA caused accelerated morphological development and enhanced production of antibiotics, concomitant with over-expression of chpE, chpH, actII-orf4 and redD. Furthermore, SigT was undetectable after loss of rstA. These data suggested that SigT has a negative role on differentiation and that RstA negatively regulates the SigT activity through a putative antagonistic mechanism and at the post-transcriptional level.

Discovery of novel aminothiadiazole amides as selective EP(3) receptor antagonists.

This Letter discloses a series of 2-aminothiadiazole amides as selective EP(3) receptor antagonists. SAR optimization resulted in compounds with excellent functional activity in vitro. In addition, efforts to optimize DMPK properties in the rat are discussed. These efforts have resulted in the identification of potent, selective EP(3) receptor antagonists with excellent DMPK properties suitable for in vivo studies.