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.

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.

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.

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.

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.

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.

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.

Design of beta-lactams with mechanism based nonantibacterial activities.

The majority of nonantibacterial activities discovered for beta-lactam derivatives during the last 15 years are based on their ability to form a stable covalent complex with nucleophile in the active site of enzymes regulating fundamental physiological processes in mammalian organism such as serine and cysteine proteases, LDL phospholipase A(2), A-independent transacylase and some still indeciphered enzymes. Regulation of their catalytic activity both in vitro and in vivo by compounds designed on the cephalosporin, penicillin and 2-azetidinone base was successfully exploited in the treatment of inflammatory, respiratory, cardiovascular disorders, cancer and other pathologic processes. Availability of X-ray crystallographic data for target enzymes and computational molecular modelling in combination with wide possibilities of structural modifications for commercial natural and synthetic beta-lactams and the chiral blocks allow to consider this class of organic compounds as a perspective source of mechanism based nonantibacterial drugs.

Doxorubicin prodrug on the basis of tert-butyl cephalosporanate sulfones.

Doxorubicin-cephalosporin prodrug adapted to the development of elastases for the liberation of parent drug was synthesized on the basis of cephalosporanate sulfone esters.

Synthesis of antitumor 6-alkylidenepenicillanate sulfones and related 3-alkylidene-2-azetidinones.

6-alkylidenepenicillanate sulfoxides and sulfones were synthesized on the base of 6-oxopenicillanate esters. The targeted splitting of their thiazolidine ring led to the formation of 3-alkylidene substituted 4-heteryldithio and 4-methylsulfonyl azetidin-2-ones. Some of mono and bicyclic beta-lactams revealed potent cytotoxic properties towards monolayer tumor cells in <10-microM concentrations.

Synthesis and cytotoxicity of silicon and germanium containing pyridine oxime O-ethers.

Silicon and germanium containing pyridine aldoxime, ketoxime and amidoxime O-ethers have been prepared using phase transfer catalytic systems oxime alkyl halide solid KOH 18-crown-6 benzene and oxime alkyl halide solid K(2)CO(3) or Cs(2)CO(3) 18-crown-6 toluene. Cytotoxic activity of silicon and germanium containing pyridine oxime O-ethers was tested in vitro on two monolayer tumor cell lines: MG- 22A (mouse hepatoma) and HT-1080 (human fibrosarcoma). O-[3-Yriethylsilylpropyl]- and O-[3-(1-methyl- 1-silacyclopentyl)propyl] oximes of pyridine aldehydes and ketones exhibit high cytotoxicity. Presence of methyl group in the pyridine ring considerably decreased activity of amidoxime O-ethers. Oxime ethers containing two elements are essentially inactive. For 2-acetylpyridine oxime ethers the activity increases in order of alkyl substituents: Et(3)GeCH(2)CH(2)SiMe(2)CH(2) < Et(3)SiCH(2)CH(2)CH(2) < (CH(2))(4)SiCH(2)CH(2)CH(2). Cytotoxicity of ketoxime O-ethers is considerably lower in comparison with aldoxime O-ethers.