RESUMO
Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional 'single target-single drug' approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3'-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC50 and EC50 values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.
Assuntos
Aminoacil-tRNA Sintetases , Toxoplasma , Toxoplasmose , Trifosfato de Adenosina/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Desenvolvimento de Medicamentos , Humanos , Toxoplasma/metabolismoRESUMO
Plasmodium falciparum lysyl-tRNA synthetase (PfKRS) represents a promising therapeutic anti-malarial target. Cladosporin was identified as a selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report chemical synthesis, biological evaluation and structural characterization of analogues where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine ring bearing functional group variations. Thermal binding, enzymatic, kinetic and parasitic assays complemented with X-ray crystallography reveal compounds that are moderate in potency. Co-crystals of Cla-B and Cla-C with PfKRS reveal key atomic configurations that allow drug binding to and inhibition of the enzyme. Collectively these piperidine ring scaffold inhibitors lay a framework for further structural editing and functional modifications of the cladosporin scaffold to obtain a potent lead.
Assuntos
Plasmodium falciparumRESUMO
Bicyclic azetidine compounds possess antimalarial activity via targeting of the cytoplasmic Plasmodium falciparum (Pf) protein translation enzyme phenylalanine-tRNA synthetase (cFRS). These drugs kill parasites both in vitro and in vivo, including the blood, liver, and transmission developmental stages. Here we present the co-crystal structure of PfcFRS with a potent inhibitor, the bicyclic azetidine BRD7929. Our studies reveal high-affinity binding of BRD7929 with PfcFRS along with exquisite specificity compared with the human enzyme, leading in turn to potent and selective inhibition of the parasite enzyme. Our co-crystal structure shows that BRD7929 binds in the active site in the α subunit of PfcFRS, where it occupies the amino acid site, an auxiliary site, and partially the ATP site. This structural snapshot of inhibitor-bound PfcFRS thus provides a platform for the structure-guided optimization of novel antimalarial compounds.
Assuntos
Aminoacil-tRNA Sintetases , Antimaláricos , Azetidinas , Antimaláricos/química , Antimaláricos/farmacologia , Humanos , Fenilalanina , Plasmodium falciparum/genéticaRESUMO
Here we have described a systematic structure activity relationship (SAR) of a set of compounds inspired from cladosporin, a tool compound that targets parasite (Plasmodium falciparum) lysyl tRNA synthetase (KRS). Four sets of analogues, synthesized based on point changes in the chemical scaffold of cladosporin and other logical modifications and hybridizations, were assessed using high throughput enzymatic and parasitic assays along with in vitro pharmacokinetics. Co-crystallization of the most potent compound in our series (CL-2) with PfKRS revealed its structural basis of enzymatic binding and potency. Further, we report that CL-2 has performed better than cladosporin in terms of metabolic stability. It thus represents a new lead for further optimization toward the development of antimalarial drugs. Collectively, along with a lead compound, the series offers insights on how even the slightest chemical modification might play an important role in enhancing or decreasing the potency of a chemical scaffold.
Assuntos
Malária , Parasitos , Animais , Isocumarinas , Plasmodium falciparumRESUMO
Aminoacyl-tRNA synthetases (aaRSs) are considered as important components in protein translation as they facilitate the attachment of specific transfer RNA (tRNA) to form aminoacyl-tRNAs. Our study focused on understanding the crystal structure of Glutaminyl-tRNA synthetase (GlnRS) from Thermus thermophilus HB8 (PDB ID:5ZDO) and mechanism of formation of enzyme-substrate complex using substrates and its analogs by applying molecular dynamics simulation (MDS) to investigate the conformational changes. Least energy structure of TtGlnRS was considered to dock the enzyme substrates such as glutamine (Gln), glutamic acid (Glu), adenosine monophosphate (AMP), adenosine triphosphate (ATP), QSI and various substrate analogs (2MA, 4SU and 5MU) onto the active site of the enzyme. We focused on comparative analysis of binding specificity between Gln and Glu; similarly, ATP and AMP. Active site organization as observed by MDS analysis showed interactive changes associated with substrate and catalytically important loops. Study found that when tRNAGln specific for GlnRS was docked into the active site of the TtGlnRS enzyme it interacts with 2' OH on the ribose acceptor end of the tRNA. Upon validation with 50 ns MDS, the maximum deviations and conformational changes of secondary structural elements were observed to be high in the loop regions of enzyme-substrate complexes. Binding affinity of ATP to TtGlnRS was further proved by isothermal titration calorimetry. AbbreviationsaaRSsaminoacyl-tRNA synthetasesAMPadenosine monophosphateATPadenosine triphosphateGlideGrid-based LIgand Docking with EnergeticGlnRSglutaminyl-tRNA synthetaseGRAVYGRand AVerage of hydropathicitYGROMACSGROingen Machine for Chemical SimulationsHADDOCKHigh Ambiguity Driven protein-protein DOCKingITCisothermal titration calorimetry2MA2-methyladenosine 5'-(dihydrogen phosphate)MDSmolecular dynamics simulation5MU5-methyluridine 5'-monophosphateNPTnumber of particles, pressure and temperatureNVTnumber of particles, volume and temperatureOPLS-AAoptimized potential for liquid simulation all atomPDBBrookhaven Protein DatabankPMEParticle-Mesh EwaldQSI5'-o-[n-(l-Glutaminyl)-sulfamoyl]adenosineRgradius of gyrationRMSDroot mean square deviationRMSFroot mean square fluctuation4SU4-thiouracil 5'-monophosphateSPCsimple point chargetRNAtransfer ribo nucleic acidTtThermus thermophilusXPextra precisionCommunicated by Ramaswamy H. Sarma.
Assuntos
Aminoacil-tRNA Sintetases , Simulação de Dinâmica Molecular , Aminoacil-tRNA Sintetases/metabolismo , Sítios de Ligação , Simulação de Acoplamento Molecular , RNA de Transferência , RNA de Transferência de GlutaminaRESUMO
Malaria remains a major infectious disease and, despite incidence reduction, it threatens resurgence in drug-resistant forms. Antimalarial drugs remain the mainstay of therapeutic options and hence there is a constant need to identify and validate new druggable targets. Plasmodium falciparum aminoacyl-tRNA synthetases (Pf-aaRSs) drive protein translation and are potent targets for development of next-generation antimalarials. Here, we detail advances made in structural-biology-based investigations in Pf-aaRSs and discuss their distribution of druggable pockets. This review establishes a platform for systematic experimental dissection of malarial parasite aaRSs as a new focus for sustained drug development efforts against malaria.