Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, while its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify novel CFTR modulators. We docked ~155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered novel mid-nanomolar potentiators as well as inhibitors that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Docking for EP4R antagonists active against inflammatory pain.

The lipid prostaglandin E2 (PGE2) mediates inflammatory pain by activating G protein-coupled receptors, including the prostaglandin E2 receptor 4 (EP4R). Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce nociception by inhibiting prostaglandin synthesis, however, the disruption of upstream prostanoid biosynthesis can lead to pleiotropic effects including gastrointestinal bleeding and cardiac complications. In contrast, by acting downstream, EP4R antagonists may act specifically as anti-inflammatory agents and, to date, no selective EP4R antagonists have been approved for human use. In this work, seeking to diversify EP4R antagonist scaffolds, we computationally dock over 400 million compounds against an EP4R crystal structure and experimentally validate 71 highly ranked, de novo synthesized molecules. Further, we show how structure-based optimization of initial docking hits identifies a potent and selective antagonist with 16 nanomolar potency. Finally, we demonstrate favorable pharmacokinetics for the discovered compound as well as anti-allodynic and anti-inflammatory activity in several preclinical pain models in mice.

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors.

Antiviral therapeutics to treat SARS-CoV-2 are needed to diminish the morbidity of the ongoing COVID-19 pandemic. A well-precedented drug target is the main viral protease (MPro ), which is targeted by an approved drug and by several investigational drugs. Emerging viral resistance has made new inhibitor chemotypes more pressing. Adopting a structure-based approach, we docked 1.2 billion non-covalent lead-like molecules and a new library of 6.5 million electrophiles against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 and 20 µM, respectively. Several series were optimized, resulting in low micromolar inhibitors. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. While the new chemotypes may aid further optimization of MPro inhibitors for SARS-CoV-2, the modest success rate also reveals weaknesses in our approach for challenging targets like MPro versus other targets where it has been more successful, and versus other structure-based techniques against MPro itself.

C-C Coupling through Nitrogen Deletion: Application to Library Synthesis.

Invited for the cover of this issue are Oleksandr Grygorenko and his Ukrainian colleagues at Enamine Ltd., Taras Shevchenko National University of Kyiv, National Academy of Sciences of Ukraine, and ChemSpace, as well as Mark Levin at the University of Chicago. The image depicts application of a nitrogen-deleting anomeric amide to parallel C(sp3 )-C(sp3 ) coupling. Read the full text of the article at 10.1002/chem.202203470.

C-C Coupling through Nitrogen Deletion: Application to Library Synthesis.

A protocol for parallel C(sp3 )-C(sp3 ) coupling of (hetero)aromatic aldehydes and (hetero)arylmethyl amines based on a reductive amination - "nitrogen deletion" reaction sequence has been developed. After preliminary validation experiments, an illustrative compound library of 25 members was prepared with 76 % synthetic efficiency. The estimated chemical space accessible by the proposed approach covers almost 600 000 representatives that are scarcely represented in current compound databases.

Challenges for chemistry in Ukraine after the war: Ukrainian science requires rebuilding and support.

The unprovoked Russian invasion has created considerable challenges for Ukrainian science. In this article, we discuss actions needed to support and rebuild Ukrainian science and educational systems. The proposed actions take into account past Ukrainian scientific achievements including developments in organic chemistry.

Impact of ß-perfluoroalkyl substitution of proline on the proteolytic stability of its peptide derivatives.

A series of all stereoisomers of ß-CF3 or ß-C2F5 substituted prolines and their dipeptide derivatives were synthesized. Mouse plasma stability assay was carried out to study the impact of fluoroalkyl substituents on the proteolytic stability of proline-derived peptides. The effect of the (R)-/(S)-configuration at the C-2 atom in combination with electronic and steric effects imposed by fluoroalkyl groups was addressed to rationalize the difference in the half-life stability of diastereomeric ß-CF3-Pro-Gly and ß-C2F5-Pro-Gly derivatives and compared to those of parent (S)-Pro-Gly and (R)-Pro-Gly dipeptides. The steric effect was predominant when the ß-CF3 or ß-C2F5 group was placed properly to create a spatial interference within the pockets of proteases, thereby protecting the substances from degradation (e.g., for cis-isomeric derivatives). Otherwise, a smaller electronic effect accelerating proteolysis was in charge (i.e., for the (2S,3S) isomers).

The Ukrainian Factor in Early-Stage Drug Discovery in the Context of Russian Invasion: The Case of Enamine Ltd.

Ukrainian companies occupy an important niche in the global drug discovery process; however, before the Russian invasion, the role of Ukraine was not obvious. The biggest Ukrainian fine chemical supplier, Enamine Ltd, had to stop operation for more than a month, which significantly affected various early-stage drug discovery projects. The role of Enamine in drug discovery and the company's past and future in the context of the Russian invasion are described in this Viewpoint.

Computationally driven discovery of SARS-CoV-2 Mpro inhibitors: from design to experimental validation.

We report a fast-track computationally driven discovery of new SARS-CoV-2 main protease (Mpro) inhibitors whose potency ranges from mM for the initial non-covalent ligands to sub-µM for the final covalent compound (IC50 = 830 ± 50 nM). The project extensively relied on high-resolution all-atom molecular dynamics simulations and absolute binding free energy calculations performed using the polarizable AMOEBA force field. The study is complemented by extensive adaptive sampling simulations that are used to rationalize the different ligand binding poses through the explicit reconstruction of the ligand-protein conformation space. Machine learning predictions are also performed to predict selected compound properties. While simulations extensively use high performance computing to strongly reduce the time-to-solution, they were systematically coupled to nuclear magnetic resonance experiments to drive synthesis and for in vitro characterization of compounds. Such a study highlights the power of in silico strategies that rely on structure-based approaches for drug design and allows the protein conformational multiplicity problem to be addressed. The proposed fluorinated tetrahydroquinolines open routes for further optimization of Mpro inhibitors towards low nM affinities.

Synthesis, biological evaluation, and modeling studies of 1,3-disubstituted cyclobutane-containing analogs of combretastatin A4.

With the aim of circumventing the adverse cis/trans-isomerization of combretastatin A4 (CA4), a naturally occurring tumor-vascular disrupting agent, we designed novel CA4 analogs bearing 1,3-cyclobutane moiety instead of the cis-stilbene unit of the parent compound. The corresponding cis and trans cyclobutane-containing derivatives were prepared as pure diastereomers. The structure of the target compounds was confirmed by X-ray diffraction study. The title compounds were evaluated for their cytotoxic properties in human cancer cell lines HepG2 (hepatocarcinoma) and SK-N-DZ (neuroblastoma), and the overall activity was found in micromolar range. Molecular docking studies and molecular dynamics simulation within the colchicine binding site of tubulin were in good agreement with the obtained cytotoxicity data.

Last of the gem-Difluorocycloalkanes: Synthesis and Characterization of 2,2-Difluorocyclobutyl-Substituted Building Blocks.

An efficient approach to synthesis of previously unavailable 2-substituted difluorocyclobutane building blocks was developed and applied on a multigram scale. The key step of the synthetic sequence included deoxofluorination of O-protected 2-(hydroxylmethyl)cyclobutanone. Dissociation constants (p Ka) and log P values for 2,2-difluorocyclobutaneamine and 2,2-difluorocyclobutanecarboxylic acid or their derivatives were measured and compared with the values obtained for the corresponding 3,3-difluorocyclobutane derivatives and nonfluorinated counterparts. Three-dimensional structures of 2,2- and 3,3-difluorocyclobutanamines were compared using exit vector plot analysis of X-ray crystallographic data.

3-((Hetera)cyclobutyl)azetidines, "Stretched" Analogues of Piperidine, Piperazine, and Morpholine: Advanced Building Blocks for Drug Discovery.

Four 3-((hetera)cyclobutyl)azetidine-based isosteres of piperidine, piperazine, and morpholine were designed and synthesized on up to gram scale. The key step of the synthetic sequence included cyclization of N-protected 2-(azetidin-3-yl)propane-1,3-diol or the corresponding 1,3-dibromide. X-ray diffraction studies of the products obtained, followed by exit vector plot analysis of their molecular geometry, demonstrated their larger size and increased conformational flexibility as compared to the parent heterocycles and confirmed their potential utility as building blocks for lead optimization programs.

Synthesis of new fluorinated proline analogues from polyfluoroalkyl ß-ketoacetals and ethyl isocyanoacetate.

The reaction of trifluoroaldol acetal and other polyfluoroalkyl ß-ketoacetals with ethyl isocyanoacetate was applied for the preparation of hitherto unknown fluorinated amino acids, cis- and trans-3-CF3/C2F5-prolines as well as trans-3-CF2Br/CF2Cl/CHF2-3-hydroxyprolines.

Straightforward hit identification approach in fragment-based discovery of bromodomain-containing protein 4 (BRD4) inhibitors.

A combination approach of a fragment screening and "SAR by catalog" was used for the discovery of bromodomain-containing protein 4 (BRD4) inhibitors. Initial screening of 3695-fragment library against bromodomain 1 of BRD4 using thermal shift assay (TSA), followed by initial hit validation, resulted in 73 fragment hits, which were used to construct a follow-up library selected from available screening collection. Additionally, analogs of inactive fragments, as well as a set of randomly selected compounds were also prepared (3 × 3200 compounds in total). Screening of the resulting sets using TSA, followed by re-testing at several concentrations, counter-screen, and TR-FRET assay resulted in 18 confirmed hits. Compounds derived from the initial fragment set showed better hit rate as compared to the other two sets. Finally, building dose-response curves revealed three compounds with IC50 = 1.9-7.4 µM. For these compounds, binding sites and conformations in the BRD4 (4UYD) have been determined by docking.

Cyclobutyl-Containing Rigid Analogues of Threonine: Synthesis and Physical Chemical Properties.

Hitherto unknown cis- and trans-1-amino-3-hydroxy-3-methylcyclobutanecarboxylic acids were synthesized in multigram scale. The obtained compounds can be considered as achiral conformationally restricted analogues of threonine with fixed spatial orientation of functional groups. pKa values are noticeably different for both amino acids. According to the X-ray data the cyclobutane rings in both compounds are almost planar (the corresponding torsion angles are below 7°).

Synthesis and physical chemical properties of 2-amino-4-(trifluoromethoxy)butanoic acid - a CF3O-containing analogue of natural lipophilic amino acids.

2-Amino-2-(trifluoromethoxy)butanoic acid (O-trifluoromethyl homoserine) was synthesized as a racemate and in both enantiomeric forms. The measured pKa and log D values establish the compound as a promising analogue of natural aliphatic amino acids.

Synthesis of diverse pyrazole-4-sulfonyl chlorides starting from 2-(benzylthio)malonaldehyde.

A series of pyrazole-4-sulfonyl chlorides was obtained by a convenient 2-step method starting from synthetically available 2-(benzylthio)malonaldehyde. The method can be applied for the effective multi-gram synthesis of diverse pyrazole-containing sulfonyl chlorides which are mostly not available by other methods.

Radical Reactions of Alkyl 2-Bromo-2,2-difluoroacetates with Vinyl Ethers: "Omitted" Examples and Application for the Synthesis of 3,3-Difluoro-GABA.

Addition reactions of perfluoroalkyl radicals to ordinary or polyfluorinated alkenes have been frequently used to synthesize perfluoroalkylated organic compounds. Here ethyl/methyl 2-bromo-2,2-difluoroacetate, diethyl (bromodifluoromethyl)phosphonate, [(bromodifluoromethyl)sulfonyl]benzene, and ethyl 2-bromo-2-fluoroacetate were involved in Na2S2O4-mediated radical additions to vinyl ethers in the presence of alcohols to give difluoro or monofluoroacetyl-substituted acetals or corresponding difluoromethylphosphonate- and (difluoromethylphenyl)sulfonyl-substituted alkyl acetals. This methodology has also been applied as a key step in the synthesis of hitherto unknown 3,3-difluoro-GABA, completing the series of isomeric difluoro GABAs. Comparison of the pKa values of 3-fluoro- and 3,3-difluoro-GABA with that of the fluorine free parent compound showed that introduction of each fluorine lead to acidification of both the amino and the carboxyl functions by approximately one unit.