Substrate Analogues Entering the Lipoic Acid Salvage Pathway via Lipoate-Protein Ligase 2 Interfere with Staphylococcus aureus Virulence.

Lipoic acid (LA) is an essential cofactor in prokaryotic and eukaryotic organisms, required for the function of several multienzyme complexes such as oxoacid dehydrogenases. Prokaryotes either synthesize LA or salvage it from the environment. The salvage pathway in Staphylococcus aureus includes two lipoate-protein ligases, LplA1 and LplA2, as well as the amidotransferase LipL. In this study, we intended to hijack the salvage pathway by LA analogues that are transferred via LplA2 and LipL to the E2 subunits of various dehydrogenases, thereby resulting in nonfunctional enzymes that eventually impair viability of the bacterium. Initially, a virtual screening campaign was carried out to identify potential LA analogues that bind to LplA2. Three selected compounds affected S. aureus USA300 growth in minimal medium at concentrations ranging from 2.5 to 10 µg/mL. Further analysis of the most potent compound (Lpl-004) revealed its transfer to E2 subunits of dehydrogenase complexes and a negative impact on its functionality. Growth impairment caused by Lpl-004 treatment was restored by adding products of the lipoate-dependent enzyme complexes. In addition, Caenorhabditis elegans infected with LpL-004-treated USA300 demonstrated a significantly expanded lifespan compared to worms infected with untreated bacteria. Our results provide evidence that LA analogues exploiting the LA salvage pathway represent an innovative strategy for the development of novel antimicrobial substances.

Structure-based identification of salicylic acid derivatives as malarial threonyl tRNA-synthetase inhibitors.

Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Threonyl t-RNA synthetase (ThrRS) is one of the enzymes involved in this pathway, and it has been validated as an anti-malarial drug target. Here, we present 9 structurally diverse low micromolar Plasmodium falciparum ThrRS inhibitors that were identified using high-throughput virtual screening (HTVS) and were verified in a FRET enzymatic assay. Salicylic acid-based compound (LE = 0.34) was selected as a most perspective hit and was subjected to hit-to-lead optimisation. A total of 146 hit analogues were synthesised or obtained from commercial vendors and were tested. Structure-activity relationship study was supported by the crystal structure of the complex of a salicylic acid analogue with a close homologue of the plasmodium target, E. coli ThrRS (EcThrRS). Despite the availability of structural information, the hit identified via virtual screening remained one of the most potent PfThrRS inhibitors within this series. However, the compounds presented herein provide novel scaffolds for ThrRS inhibitors, which could serve as starting points for further medicinal chemistry projects targeting ThrRSs or structurally similar enzymes.

Design, quality and validation of the EU-OPENSCREEN fragment library poised to a high-throughput screening collection.

The EU-OPENSCREEN (EU-OS) European Research Infrastructure Consortium (ERIC) is a multinational, not-for-profit initiative that integrates high-capacity screening platforms and chemistry groups across Europe to facilitate research in chemical biology and early drug discovery. Over the years, the EU-OS has assembled a high-throughput screening compound collection, the European Chemical Biology Library (ECBL), that contains approximately 100 000 commercially available small molecules and a growing number of thousands of academic compounds crowdsourced through our network of European and non-European chemists. As an extension of the ECBL, here we describe the computational design, quality control and use case screenings of the European Fragment Screening Library (EFSL) composed of 1056 mini and small chemical fragments selected from a substructure analysis of the ECBL. Access to the EFSL is open to researchers from both academia and industry. Using EFSL, eight fragment screening campaigns using different structural and biophysical methods have successfully identified fragment hits in the last two years. As one of the highlighted projects for antibiotics, we describe the screening by Bio-Layer Interferometry (BLI) of the EFSL, the identification of a 35 µM fragment hit targeting the beta-ketoacyl-ACP synthase 2 (FabF), its binding confirmation to the protein by X-ray crystallography (PDB 8PJ0), its subsequent rapid exploration of its surrounding chemical space through hit-picking of ECBL compounds that contain the fragment hit as a core substructure, and the final binding confirmation of two follow-up hits by X-ray crystallography (PDB 8R0I and 8R1V).

Electrochemical Formation of Oxazolines by 1,3-Oxyfluorination of Non-activated Cyclopropanes.

The C-C bond in non-activated cyclopropanes can be intramolecularly cleaved with an electrochemically generated amidyl radical forming oxazolines. In the presence of TBABF4, this provides 1,3-oxyfluorination products. C-C bond cleavage of cyclopropane proceeds with inversion of the configuration, suggesting an intramolecular homolytic substitution (SHi) mechanism. The performance of TBABF4 as an efficient fluoride source was explained by accumulation of the BF4- anion at the anode surface, at which a carbocation is formed by the oxidation of the C-centered radical.

Spiropyran-Based Photoisomerizable α-Amino Acid for Membrane-Active Peptide Modification.

Photoisomerizable peptides are promising drug candidates in photopharmacology. While azobenzene- and diarylethene-containing photoisomerizable peptides have already demonstrated their potential in this regard, reports on the use of spiropyrans to photoregulate bioactive peptides are still scarce. This work focuses on the design and synthesis of a spiropyran-derived amino acid, (S)-2-amino-3-(6'-methoxy-1',3',3'-trimethylspiro-[2H-1-benzopyran-2,2'-indolin-6-yl])propanoic acid, which is suitable for the preparation of photoisomerizable peptides. The utility of this amino acid is demonstrated by incorporating it into the backbone of BP100, a known membrane-active peptide, and by examining the photoregulation of the membrane perturbation by the spiropyran-containing peptides. The toxicity of the peptides (against the plant cell line BY-2), their bacteriotoxicity (E. coli), and actin-auxin oscillator modulation ability were shown to be significantly dependent on the photoisomeric state of the spiropyran unit.

Discovery of Malarial Threonyl tRNA Synthetase Inhibitors by Screening of a Focused Fragment Library.

While Plasmodium falciparum threonyl tRNA synthetase (PfThrRS) has clearly been validated as a prospective antimalarial drug target, the number of known inhbitors of this enzyme is still limited. In order to expand the chemotypes acting as inhibitors of PfThrRS, a set of fragments were designed which incorporated bioisosteres of the N-acylphosphate moiety of the aminoacyladenylate as an intermediate of an enzymatic reaction. N-Acyl sulfamate- and N-acyl benzenethiazolsulfonamide-based fragments 9a and 9k were identified as inhibitors of the PfThrRSby biochemical assay at 100 µM concentration. These fragments were then developed into potent PfThrRS inhibitors (10a,b and 11) by linking them with an amino pyrimidine as a bioisostere of adenine in the enzymatic reaction intermediate.

Semisynthesis of Linariophyllenes A-C and Rumphellolide H, Structure Revisions and Proposed Biosynthesis Pathways.

The first semisynthetic routes toward terrestrial anti-inflammatory natural products linariophyllene A-C and the refined route toward marine natural product rumphellolide H are presented. Among the synthesized target compounds, the correct structure of linariophyllene A was determined to be the diastereomer of the originally proposed structure with an inverted stereocenter at the secondary alcohol. The proposed structures of linariophyllene B and rumphellolide H were confirmed. However, the correct structure of linariophyllene C was found to be the diastereomer of the originally proposed structure with an inverted stereocenter at the tertiary carbon of the epoxide moiety. The structures of linariophyllenes A-C and rumphellolide H were unequivocally confirmed by single-crystal X-ray diffractometry. The obtained results enabled the proposal of the biosynthetic origins of the aforementioned natural products and bolstered the diversity of available sesquiterpenoids. Linariophyllenes A-C and rumphellolide H were obtained in sufficient amounts to further expand their bioactivity profile and utility as reference standards in future studies of chemical constituents of terrestrial and marine organisms.

Exploring the Binding Pathway of Novel Nonpeptidomimetic Plasmepsin V Inhibitors.

Predicting the interaction modes and binding affinities of virtual compound libraries is of great interest in drug development. It reduces the cost and time of lead compound identification and selection. Here we apply path-based metadynamics simulations to characterize the binding of potential inhibitors to the Plasmodium falciparum aspartic protease plasmepsin V (plm V), a validated antimalarial drug target that has a highly mobile binding site. The potential plm V binders were identified in a high-throughput virtual screening (HTVS) campaign and were experimentally verified in a fluorescence resonance energy transfer (FRET) assay. Our simulations allowed us to estimate compound binding energies and revealed relevant states along binding/unbinding pathways in atomistic resolution. We believe that the method described allows the prioritization of compounds for synthesis and enables rational structure-based drug design for targets that undergo considerable conformational changes upon inhibitor binding.

Assembling the Methanoindene Cage of Phragmalin-Type Natural Products.

Phragmalin-type limonoids are highly complex natural products based on an unusual octahydro-1H-2,4-methanoindene cage. The absence of feasible routes to sufficiently functionalized methanoindene cage building blocks impedes the total synthesis of these natural products. We have developed a short and robust route to methanoindene cage compounds from the Hajos-Parrish ketone (HPK). Several stereoselective modifications of the HPK provided a substrate that underwent aldol reaction as a key step for the cage formation.

Macrocyclic Peptidomimetic Plasmepsin X Inhibitors with Potent In Vitro and In Vivo Antimalarial Activity.

The Plasmodium falciparum aspartic protease plasmepsin X (PMX) is essential for the egress of invasive merozoite forms of the parasite. PMX has therefore emerged as a new potential antimalarial target. Building on peptidic amino alcohols originating from a phenotypic screening hit, we have here developed a series of macrocyclic analogues as PMX inhibitors. Incorporation of an extended linker between the S1 phenyl group and S3 amide led to a lead compound that displayed a 10-fold improved PMX inhibitory potency and a 3-fold improved half-life in microsomal stability assays compared to the acyclic analogue. The lead compound was also the most potent of the new macrocyclic compounds in in vitro parasite growth inhibition. Inhibitor 7k cleared blood-stage P. falciparum in a dose-dependent manner when administered orally to infected humanized mice. Consequently, lead compound 7k represents a promising orally bioavailable molecule for further development as a PMX-targeting antimalarial drug.

Synthesis and evaluation of an agrocin 84 toxic moiety (TM84) analogue as a malarial threonyl tRNA synthetase inhibitor.

An analogue of a toxic moiety (TM84) of natural product agrocin 84 containing threonine amide instead of 2,3-dihydroxy-4-methylpentanamide was prepared and evaluated as a putative Plasmodium falciparum threonyl t-RNA synthetase (PfThrRS) inhibitor. This TM84 analogue features submicromolar inhibitory potency (IC50 = 440 nM) comparable to that of borrelidin (IC50 = 43 nM) and therefore complements chemotypes known to inhibit malarial PfThrRS, which are currently limited to borrelidin and its analogues. The crystal structure of the inhibitor in complex with the E. coli homologue enzyme (EcThrRS) was obtained, revealing crucial ligand-protein interactions that will pave the way for the design of novel ThrRS inhibitors.

3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors.

SARS-CoV-2 nsp14 guanine-N7-methyltransferase plays an important role in the viral RNA translation process by catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to viral mRNA cap. We report a structure-guided design and synthesis of 3-(adenosylthio)benzoic acid derivatives as nsp14 methyltransferase inhibitors resulting in compound 5p with subnanomolar inhibitory activity and improved cell membrane permeability in comparison with the parent inhibitor. Compound 5p acts as a bisubstrate inhibitor targeting both SAM and mRNA-binding pockets of nsp14. While the selectivity of 3-(adenosylthio)benzoic acid derivatives against human glycine N-methyltransferase was not improved, the discovery of phenyl-substituted analogs 5p,t may contribute to further development of SARS-CoV-2 nsp14 bisubstrate inhibitors.

Subtilisin-like Serine Protease 1 (SUB1) as an Emerging Antimalarial Drug Target: Current Achievements in Inhibitor Discovery.

Widespread resistance to many antimalarial therapies currently in use stresses the need for the discovery of new classes of drugs with new modes of action. The subtilisin-like serine protease SUB1 controls egress of malaria parasites (merozoites) from the parasite-infected red blood cell. As such, SUB1 is considered a prospective target for drugs designed to interrupt the asexual blood stage life cycle of the malaria parasite. Inhibitors of SUB1 have potential as wide-spectrum antimalarial drugs, as a single orthologue of SUB1 is found in the genomes of all known Plasmodium species. This mini-perspective provides a short overview of the function and structure of SUB1 and summarizes all of the published SUB1 inhibitors. The inhibitors are classified by the methods of their discovery, including both rational design and screening.

Functionalization of Tetrazoles Bearing the Electrochemically Cleavable 1N-(6-Methylpyridyl-2-methyl) Protecting Group.

6-Methylpyridyl-2-methyl protected tetrazoles can be C-H deprotonated using the turbo-Grignard reagent and involved in the reactions with aldehydes and ketones. The protecting group can be cleaved under reductive electrochemical conditions using Pb bronze as a cathode and Zn as a sacrificial anode.

Exploring Aspartic Protease Inhibitor Binding to Design Selective Antimalarials.

Selectivity is a major issue in the development of drugs targeting pathogen aspartic proteases. Here, we explore the selectivity-determining factors by studying specifically designed malaria aspartic protease (plasmepsin) open-flap inhibitors. Metadynamics simulations are used to uncover the complex binding/unbinding pathways of these inhibitors and describe the critical transition states in atomistic resolution. The simulation results are compared with experimentally determined enzymatic activities. Our findings demonstrate that plasmepsin inhibitor selectivity can be achieved by targeting the flap loop with hydrophobic substituents that enable ligand binding under the flap loop, as such a behavior is not observed for several other aspartic proteases. The ability to estimate the selectivity of compounds before they are synthesized is of considerable importance in drug design; therefore, we expect that our approach will be useful in selective inhibitor designs against not only aspartic proteases but also other enzyme classes.

Convergent biomimetic semisynthesis of disesquiterpenoid rumphellolide J.

The convergent biomimetic gram-scale synthesis of disesquiterpenoid ester rumphellolide J is described. 4ß,8ß-Epoxycaryophyllan-5-ol was prepared in 67% yield (1.4 g) from naturally ambudant (-)-ß-caryophyllene. (+)-Rumphellaoic acid A was obtained in 46% yield (2.2 g) from (-)-caryophyllene oxide. The synthesised (+)-rumphellaoic acid had an opposite specific rotation compared to that of (-)-rumphellaoic acid A isolated from nature, indicating possible occurrence of (+)-ß-caryophyllene in Rumphella antipathies and Psidium guajava. Esterification of (+)-rumphellaoic acid A via acyl fluoride and alkoxide of 4ß,8ß-epoxycaryophyllan-5-ol gave rumphellolide J in 70% yield (1.65 g). The same structure for the synthesized product and natural isolate was proven despite the opposite specific rotation value of the intermediate acid. The short access to the terpenoids provides a material for further investigations of biological activities and valuable reference standards for the analysis of the chemical composition of various natural sources.

Functionalization of 1N-Protected Tetrazoles by Deprotonation with the Turbo Grignard Reagent.

1N-PMB-protected tetrazole undergoes C-H deprotonation with the turbo Grignard reagent, providing a metalated intermediate with increased stability. This can be used for the reaction with electrophiles such as aldehydes, ketones, Weinreb amides, and iodine. C-H deprotonation with the turbo Grignard reagent is compatible with the PMB-protecting group at the tetrazole, which can be cleaved using oxidative hydrogenolysis and acidic conditions. The method enables the tetrazole functionalization at the fifth position by overcoming the difficulties associated with retro [2 + 3] cycloaddition of the metalated intermediates.

Discovery of SARS-CoV-2 Nsp14 and Nsp16 Methyltransferase Inhibitors by High-Throughput Virtual Screening.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses mRNA capping to evade the human immune system. The cap formation is performed by the SARS-CoV-2 mRNA cap methyltransferases (MTases) nsp14 and nsp16, which are emerging targets for the development of broad-spectrum antiviral agents. Here, we report results from high-throughput virtual screening against these two enzymes. The docking of seven million commercially available drug-like compounds and S-adenosylmethionine (SAM) co-substrate analogues against both MTases resulted in 80 virtual screening hits (39 against nsp14 and 41 against nsp16), which were purchased and tested using an enzymatic homogeneous time-resolved fluorescent energy transfer (HTRF) assay. Nine compounds showed micromolar inhibition activity (IC50 < 200 µM). The selectivity of the identified inhibitors was evaluated by cross-checking their activity against human glycine N-methyltransferase. The majority of the compounds showed poor selectivity for a specific MTase, no cytotoxic effects, and rather poor cell permeability. Nevertheless, the identified compounds represent good starting points that have the potential to be developed into efficient viral MTase inhibitors.

Potent SARS-CoV-2 mRNA Cap Methyltransferase Inhibitors by Bioisosteric Replacement of Methionine in SAM Cosubstrate.

Viral mRNA cap methyltransferases (MTases) are emerging targets for the development of broad-spectrum antiviral agents. In this work, we designed potential SARS-CoV-2 MTase Nsp14 and Nsp16 inhibitors by using bioisosteric substitution of the sulfonium and amino acid substructures of the cosubstrate S-adenosylmethionine (SAM), which serves as the methyl donor in the enzymatic reaction. The synthetically accessible target structures were prioritized using molecular docking. Testing of the inhibitory activity of the synthesized compounds showed nanomolar to submicromolar IC50 values for five compounds. To evaluate selectivity, enzymatic inhibition of the human glycine N-methyltransferase involved in cellular SAM/SAH ratio regulation was also determined, which indicated that the discovered compounds are nonselective inhibitors of the studied MTases with slight selectivity for Nsp16. No cytotoxic effects were observed; however, this is most likely a result of the poor cell permeability of all evaluated compounds.

Peptidic boronic acids are potent cell-permeable inhibitors of the malaria parasite egress serine protease SUB1.

Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle. Egress is regulated by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and optimization of substrate-based peptidic boronic acids that inhibit Plasmodium falciparum SUB1 with low nanomolar potency. Structural optimization generated membrane-permeable, slow off-rate inhibitors that prevent Pfalciparum egress through direct inhibition of SUB1 activity and block parasite replication in vitro at submicromolar concentrations. Our results validate SUB1 as a potential target for a new class of antimalarial drugs designed to prevent parasite replication and disease progression.