Novel-Type GABAB PAMs: Structure-Activity Relationship in Light of the Protein Structure.

Selecting a known HTS hit with the pyrazolo[1,5-a]pyrimidine core, our project was started from CMPPE, and its optimization was driven by a ligand-based pharmacophore model developed on the basis of published GABAB positive allosteric modulators (PAMs). Our primary goal was to improve the potency by finding new enthalpic interactions. Therefore, we included the lipophilic ligand efficiency (LLE or LipE) as an objective function in the optimization that led to a carboxylic acid derivative (34). This lead candidate offers the possibility to improve potency without drastically inflating the physicochemical properties. Although the discovery of the novel carboxyl feature was surprising, it turned out to be an important element of the GABAB PAM pharmacophore that can be perfectly explained based on the new protein structures. Rationalizing the binding mode of 34, we analyzed the intersubunit PAM binding site of GABAB receptor using the publicly available experimental structures.

Boronic acid inhibitors of penicillin-binding protein 1b: serine and lysine labelling agents.

Penicillin-binding proteins (PBPs) contribute to bacterial cell wall biosynthesis and are targets of antibacterial agents. Here, we investigated PBP1b inhibition by boronic acid derivatives. Chemical starting points were identified by structure-based virtual screening and aliphatic boronic acids were selected for further investigations. Structure-activity relationship studies focusing on the branching of the boron-connecting carbon and quantum mechanical/molecular mechanical simulations showed that reaction barrier free energies are compatible with fast reversible covalent binding and small or missing reaction free energies limit the inhibitory activity of the investigated boronic acid derivatives. Therefore, covalent labelling of the lysine residue of the catalytic dyad was also investigated. Compounds with a carbonyl warhead and an appropriately positioned boronic acid moiety were shown to inhibit and covalently label PBP1b. Reversible covalent labelling of the catalytic lysine by imine formation and the stabilisation of the imine by dative N-B bond is a new strategy for PBP1b inhibition.

Molecular Mechanism of Labelling Functional Cysteines by Heterocyclic Thiones.

Heterocyclic thiones have recently been identified as reversible covalent warheads, consistent with their mild electrophilic nature. Little is known so far about their mechanism of action in labelling nucleophilic sidechains, especially cysteines. The vast number of tractable cysteines promotes a wide range of target proteins to examine; however, our focus was put on functional cysteines. We chose the main protease of SARS-CoV-2 harboring Cys145 at the active site that is a structurally characterized and clinically validated target of covalent inhibitors. We screened an in-house, cysteine-targeting covalent inhibitor library which resulted in several covalent fragment hits with benzoxazole, benzothiazole and benzimidazole cores. Thione derivatives and Michael acceptors were selected for further investigations with the objective of exploring the mechanism of inhibition of the thiones and using the thoroughly characterized Michael acceptors for benchmarking our studies. Classical and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were carried out that revealed a new mechanism of covalent cysteine labelling by thione derivatives, which was supported by QM and free energy calculations and by a wide range of experimental results. Our study shows that the molecular recognition step plays a crucial role in the overall binding of both sets of molecules.

GproteinDb in 2024: new G protein-GPCR couplings, AlphaFold2-multimer models and interface interactions.

G proteins are the major signal proteins of ∼800 receptors for medicines, hormones, neurotransmitters, tastants and odorants. GproteinDb offers integrated genomic, structural, and pharmacological data and tools for analysis, visualization and experiment design. Here, we present the first major update of GproteinDb greatly expanding its coupling data and structural templates, adding AlphaFold2 structure models of GPCR-G protein complexes and advancing the interactive analysis tools for their interfaces underlying coupling selectivity. We present insights on coupling agreement across datasets and parameters, including constitutive activity, agonist-induced activity and kinetics. GproteinDb is accessible at https://gproteindb.org.

On-DNA Synthesis of Multisubstituted Indoles.

The increasing role of the DNA-encoded library technology in early phase drug discovery represents a significant demand for DNA-compatible synthetic methods for therapeutically relevant heterocycles. Herein, we report the first on-DNA synthesis of multisubstituted indoles via a cascade reaction of Sonogashira coupling and intramolecular ring closure. Further functionalization by Suzuki coupling at the third position exploits a diverse chemical space. The high fidelity of the method also enabled the construction of an indole-based mock library.

Electrophilic MiniFrags Revealed Unprecedented Binding Sites for Covalent HDAC8 Inhibitors.

Screening of ultra-low-molecular weight ligands (MiniFrags) successfully identified viable chemical starting points for a variety of drug targets. Here we report the electrophilic analogues of MiniFrags that allow the mapping of potential binding sites for covalent inhibitors by biochemical screening and mass spectrometry. Small electrophilic heterocycles and their N-quaternized analogues were first characterized in the glutathione assay to analyze their electrophilic reactivity. Next, the library was used for systematic mapping of potential covalent binding sites available in human histone deacetylase 8 (HDAC8). The covalent labeling of HDAC8 cysteines has been proven by tandem mass spectrometry measurements, and the observations were explained by mutating HDAC8 cysteines. As a result, screening of electrophilic MiniFrags identified three potential binding sites suitable for the development of allosteric covalent HDAC8 inhibitors. One of the hit fragments was merged with a known HDAC8 inhibitor fragment using different linkers, and the linker length was optimized to result in a lead-like covalent inhibitor.

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Covalent fragment approaches targeting non-cysteine residues.

Covalent fragment approaches combine advantages of covalent binders and fragment-based drug discovery (FBDD) for target identification and validation. Although early applications focused mostly on cysteine labeling, the chemistries of available warheads that target other orthosteric and allosteric protein nucleophiles has recently been extended. The range of different warheads and labeling chemistries provide unique opportunities for screening and optimizing warheads necessary for targeting non-cysteine residues. In this review, we discuss these recently developed amino-acid-specific and promiscuous warheads, as well as emerging labeling chemistries, which includes novel transition metal catalyzed, photoactive, electroactive, and noncatalytic methodologies. We also highlight recent applications of covalent fragments for the development of molecular glues and proteolysis-targeting chimeras (PROTACs), and their utility in chemical proteomics-based target identification and validation.

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging.

A novel family of julolidine-containing fluorescent rhodols equipped with a wide variety of substituents was synthesized in a versatile two-step process. The prepared compounds were fully characterized and exhibited excellent fluorescence properties for microscopy imaging. The best candidate was conjugated to the therapeutic antibody trastuzumab through a copper-free strain-promoted azide-alkyne click reaction. The rhodol-labeled antibody was successfully applied for in vitro confocal and two-photon microscopy imaging of Her2+ cells.

SH2db, an information system for the SH2 domain.

SH2 domains are key mediators of phosphotyrosine-based signalling, and therapeutic targets for diverse, mostly oncological, disease indications. They have a highly conserved structure with a central beta sheet that divides the binding surface of the protein into two main pockets, responsible for phosphotyrosine binding (pY pocket) and substrate specificity (pY + 3 pocket). In recent years, structural databases have proven to be invaluable resources for the drug discovery community, as they contain highly relevant and up-to-date information on important protein classes. Here, we present SH2db, a comprehensive structural database and webserver for SH2 domain structures. To organize these protein structures efficiently, we introduce (i) a generic residue numbering scheme to enhance the comparability of different SH2 domains, (ii) a structure-based multiple sequence alignment of all 120 human wild-type SH2 domain sequences and their PDB and AlphaFold structures. The aligned sequences and structures can be searched, browsed and downloaded from the online interface of SH2db (http://sh2db.ttk.hu), with functions to conveniently prepare multiple structures into a Pymol session, and to export simple charts on the contents of the database. Our hope is that SH2db can assist researchers in their day-to-day work by becoming a one-stop shop for SH2 domain related research.

Activation-Free Sulfonyl Fluoride Probes for Fragment Screening.

SuFEx chemistry is based on the unique reactivity of the sulfonyl fluoride group with a range of nucleophiles. Accordingly, sulfonyl fluorides label multiple nucleophilic amino acid residues, making these reagents popular in both chemical biology and medicinal chemistry applications. The reactivity of sulfonyl fluorides nominates this warhead chemotype as a candidate for an external, activation-free general labelling tag. Here, we report the synthesis and characterization of a small sulfonyl fluoride library that yielded the 3-carboxybenzenesulfonyl fluoride warhead for tagging tractable targets at nucleophilic residues. Based on these results, we propose that coupling diverse fragments to this warhead would result in a library of sulfonyl fluoride bits (SuFBits), available for screening against protein targets. SuFBits will label the target if it binds to the core fragment, which facilitates the identification of weak fragments by mass spectrometry.

Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.

Covalent fragment mapping of KRasG12C revealed novel chemotypes with in vivo potency.

G12C mutant KRas is considered druggable by allele-specific covalent inhibitors due to the nucleophilic character of the oncogenic mutant cysteine at position 12. Discovery of these inhibitors requires the optimization of both covalent and noncovalent interactions. Here, we report covalent fragment screening of our electrophilic fragment library of diverse non-covalent scaffolds equipped with 40 different electrophilic functionalities to identify fragments as suitable starting points targeting Cys12. Screening the library against KRasG12C using Ellman's free thiol assay, followed by protein NMR and cell viability assays, resulted in two potential inhibitor chemotypes. Characterization of these scaffolds in in vitro cellular- and in vivo xenograft models revealed them as promising starting points for covalent drug discovery programs.

GPCRdb in 2023: state-specific structure models using AlphaFold2 and new ligand resources.

G protein-coupled receptors (GPCRs) are physiologically abundant signaling hubs routing hundreds of extracellular signal substances and drugs into intracellular pathways. The GPCR database, GPCRdb supports >5000 interdisciplinary researchers every month with reference data, analysis, visualization, experiment design and dissemination. Here, we present our fifth major GPCRdb release setting out with an overview of the many resources for receptor sequences, structures, and ligands. This includes recently published additions of class D generic residue numbers, a comparative structure analysis tool to identify functional determinants, trees clustering GPCR structures by 3D conformation, and mutations stabilizing inactive/active states. We provide new state-specific structure models of all human non-olfactory GPCRs built using AlphaFold2-MultiState. We also provide a new resource of endogenous ligands along with a larger number of surrogate ligands with bioactivity, vendor, and physiochemical descriptor data. The one-stop-shop ligand resources integrate ligands/data from the ChEMBL, Guide to Pharmacology, PDSP Ki and PubChem database. The GPCRdb is available at https://gpcrdb.org.

[Farnesyltransferase inhibitors have antitumoral effects in mutant KRAS containing cancer cells in preclinical models]. / Farnezil-transzferáz-inhibitorok (FTI) szinergista módon potencírozzák a mutáns KRAS inhibitorai daganatellenes hatásait preklinikai modellekben.

In silico studies raised the possibility that farnesyltransferase inhibitors (FTIs) may have antitumoral effects on KRAS mutant cancer cells. Accordingly, we have tested FTIs (tipifarnib and lonafarnib) in G12C mutant human cancer cell lines in vitro and in vivo. We have discovered that the combination of the two drugs has a synergistic antitumoral effect. Next, we have tested FTIs on G12D mutant human cancer cell lines and found that the combination has antitumoral effect in various preclinical cancer models. At last, we have also tested FTIs on G12V mutant human cancer cells and again we have detected antitumoral effects. We suggest that FTIs may have clinical relevance outside the HRAS mutant cancers.

Next-Generation Heterocyclic Electrophiles as Small-Molecule Covalent MurA Inhibitors.

Heterocyclic electrophiles as small covalent fragments showed promising inhibitory activity on the antibacterial target MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase, EC:2.5.1.7). Here, we report the second generation of heterocyclic electrophiles: the quaternized analogue of the heterocyclic covalent fragment library with improved reactivity and MurA inhibitory potency. Quantum chemical reaction barrier calculations, GSH (L-glutathione) reactivity assay, and thrombin counter screen were also used to demonstrate and explain the improved reactivity and selectivity of the N-methylated heterocycles and to compare the two generations of heterocyclic electrophiles.

Conservation of Allosteric Ligand Binding Sites in G-Protein Coupled Receptors.

Despite the growing number of G protein-coupled receptor (GPCR) structures, only 39 structures have been cocrystallized with allosteric inhibitors. These structures have been studied by protein mapping using the FTMap server, which determines the clustering of small organic probe molecules distributed on the protein surface. The method has found druggable sites overlapping with the cocrystallized allosteric ligands in 21 GPCR structures. Mapping of Alphafold2 generated models of these proteins confirms that the same sites can be identified without the presence of bound ligands. We then mapped the 394 GPCR X-ray structures available at the time of the analysis (September 2020). Results show that for each of the 21 structures with bound ligands there exist many other GPCRs that have a strong binding hot spot at the same location, suggesting potential allosteric sites in a large variety of GPCRs. These sites cluster at nine distinct locations, and each can be found in many different proteins. However, ligands binding at the same location generally show little or no similarity, and the amino acid residues interacting with these ligands also differ. Results confirm the possibility of specifically targeting these sites across GPCRs for allosteric modulation and help to identify the most likely binding sites among the limited number of potential locations. The FTMap server is available free of charge for academic and governmental use at https://ftmap.bu.edu/.

Covalent inhibitors of bacterial peptidoglycan biosynthesis enzyme MurA with chloroacetamide warhead.

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the cytoplasmic part of peptidoglycan biosynthesis and is a validated target enzyme for antibacterial drug discovery; the inhibitor fosfomycin has been used clinically for decades. Like fosfomycin, most MurA inhibitors are small heterocyclic compounds that inhibit the enzyme by forming a covalent bond with the active site cysteine. The reactive chloroacetamide group was selected from a series of suitable electrophilic thiol-reactive warheads. The predominantly one-step synthesis led to the construction of the final library of 47 fragment-sized chloroacetamide compounds. Several new E. coli MurA inhibitors were identified, with the most potent compound having an IC50 value in the low micromolar range. The electrophilic reactivity of all chloroacetamide fragments in our library was evaluated by a high-throughput spectrophotometric assay using the reduced Ellman reagent as a surrogate for the cysteine thiol. LC-MS/MS experiments confirmed the covalent binding of the most potent inhibitor to Cys115 of the digested MurA enzyme. The covalent binding was further investigated by a biochemical time-dependent assay and a dilution assay, which confirmed the irreversible and time-dependent mode of action. The efficacy of chloroacetamide derivatives against MurA does not correlate with their thiol reactivity, making the active fragments valuable starting points for fragment-based development of new antibacterial agents targeting MurA.

Semisynthetic teicoplanin derivatives with dual antimicrobial activity against SARS-CoV-2 and multiresistant bacteria.

Patients infected with SARS-CoV-2 risk co-infection with Gram-positive bacteria, which severely affects their prognosis. Antimicrobial drugs with dual antiviral and antibacterial activity would be very useful in this setting. Although glycopeptide antibiotics are well-known as strong antibacterial drugs, some of them are also active against RNA viruses like SARS-CoV-2. It has been shown that the antiviral and antibacterial efficacy can be enhanced by synthetic modifications. We here report the synthesis and biological evaluation of seven derivatives of teicoplanin bearing hydrophobic or superbasic side chain. All but one teicoplanin derivatives were effective in inhibiting SARS-CoV-2 replication in VeroE6 cells. One lipophilic and three perfluoroalkyl conjugates showed activity against SARS-CoV-2 in human Calu-3 cells and against HCoV-229E, an endemic human coronavirus, in HEL cells. Pseudovirus entry and enzyme inhibition assays established that the teicoplanin derivatives efficiently prevent the cathepsin-mediated endosomal entry of SARS-CoV-2, with some compounds inhibiting also the TMPRSS2-mediated surface entry route. The teicoplanin derivatives showed good to excellent activity against Gram-positive bacteria resistant to all approved glycopeptide antibiotics, due to their ability to dually bind to the bacterial membrane and cell-wall. To conclude, we identified three perfluoralkyl and one monoguanidine analog of teicoplanin as dual inhibitors of Gram-positive bacteria and SARS-CoV-2.

Maximizing the integration of virtual and experimental screening in hit discovery.

INTRODUCTION: Experimental and virtual screening contributes to the discovery of more than 50% of clinical candidates. Considering the similar concept and goals, early-phase drug discovery would benefit from the effective integration of these approaches. AREAS COVERED: After reviewing the recent trends in both experimental and virtual screening, the authors discuss different integration strategies from parallel, focused, sequential, and iterative screening. Strategic considerations are demonstrated in a number of real-life case studies. EXPERT OPINION: Experimental and virtual screening are complementary approaches that should be integrated in lead discovery settings. Virtual screening can access extremely large synthetically feasible chemical space that can be effectively searched on GPU clusters or cloud architectures. Experimental screening provides reliable datasets by quantitative HTS applications, and DNA-encoded libraries (DEL) have enlarged the chemical space covered by these technologies. These developments, together with the use of artificial intelligence methods, represent new options for their efficient integration. The case studies discussed here demonstrate the benefits of complementary strategies, such as focused and iterative screening.