Leucyl-tRNA Synthetase Inhibitors Research Articles

Insights into the Effects of Ligand Binding on Leucyl-tRNA Synthetase Inhibitors for Tuberculosis: In Silico Analysis and Isothermal Titration Calorimetry Validation.

Incidences of drug-resistant tuberculosis have become common and are rising at an alarming rate. Aminoacyl t-RNA synthetase has been validated as a newer target against Mycobacterium tuberculosis. Leucyl t-RNA synthetase (LeuRS) is ubiquitously found in all organisms and regulates transcription, protein synthesis, mitochondrial RNA cleavage, and proofreading of matured t-RNA. Leucyl t-RNA synthetase promotes growth and development and is the key enzyme needed for biofilm formation in Mycobacterium. Inhibition of this enzyme could restrict the growth and development of the mycobacterial population. A database consisting of 2734 drug-like molecules was screened against leucyl t-RNA synthetase enzymes through virtual screening. Based on the docking scores and MMGBSA energy values, the top three compounds were selected for molecular dynamics simulation. The druggable nature of the top three hits was confirmed by predicting their pharmacokinetic parameters. The top three hits-compounds 1035 (ZINC000001543916), 1054 (ZINC000001554197), and 2077 (ZINC000008214483)-were evaluated for their binding affinity toward leucyl t-RNA synthetase by an isothermal titration calorimetry study. The inhibitory activity of these compounds was tested against antimycobacterial activity, biofilm formation, and LeuRS gene expression potential. Compound 1054 (Macimorelin) was found to be the most potent molecule, with better antimycobacterial activity, enzyme binding affinity, and significant inhibition of biofilm formation, as well as inhibition of the LeuRS gene expression. Compound 1054, the top hit compound, has the potential to be used as a lead to develop successful leucyl t-RNA synthetase inhibitors.

A first-in-class leucyl-tRNA synthetase inhibitor, ganfeborole, for rifampicin-susceptible tuberculosis: a phase 2a open-label, randomized trial

New tuberculosis treatments are needed to address drug resistance, lengthy treatment duration and adverse reactions of available agents. GSK3036656 (ganfeborole) is a first-in-class benzoxaborole inhibiting the Mycobacterium tuberculosis leucyl-tRNA synthetase. Here, in this phase 2a, single-center, open-label, randomized trial, we assessed early bactericidal activity (primary objective) and safety and pharmacokinetics (secondary objectives) of ganfeborole in participants with untreated, rifampicin-susceptible pulmonary tuberculosis. Overall, 75 males were treated with ganfeborole (1/5/15/30 mg) or standard of care (Rifafour e-275 or generic alternative) once daily for 14 days. We observed numerical reductions in daily sputum-derived colony-forming units from baseline in participants receiving 5, 15 and 30 mg once daily but not those receiving 1 mg ganfeborole. Adverse event rates were comparable across groups; all events were grade 1 or 2. In a participant subset, post hoc exploratory computational analysis of 18F-fluorodeoxyglucose positron emission tomography/computed tomography findings showed measurable treatment responses across several lesion types in those receiving ganfeborole 30 mg at day 14. Analysis of whole-blood transcriptional treatment response to ganfeborole 30 mg at day 14 revealed a strong association with neutrophil-dominated transcriptional modules. The demonstrated bactericidal activity and acceptable safety profile suggest that ganfeborole is a potential candidate for combination treatment of pulmonary tuberculosis.ClinicalTrials.gov identifier: NCT03557281.

In Vitro Evaluation of Drug-Drug Interaction Potential of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor.

Epetraborole (EBO) is a boron-containing inhibitor of bacterial leucyl-tRNA synthetase, with potent activity against nontuberculous mycobacteria (NTM) and Gram-negative bacteria, including Burkholderia pseudomallei. EBO is being developed for the treatment of NTM lung disease and melioidosis, administered in combination with other therapeutic agents in both diseases. Therefore, EBO and its major circulating metabolite M3 were evaluated in comprehensive drug-drug interaction (DDI) in vitro studies. The CYP inhibitory and substrate potential of EBO and M3 were assessed using hepatic microsomes. Stably transfected cells that expressed individual efflux or uptake transporters were used to determine whether EBO or M3 were substrates or inhibitors for these receptors. Stability studies indicated that EBO is a poor substrate for major CYP enzymes. Neither EBO nor M3 was a potent reversible or time-dependent inhibitor of major CYP enzymes. EBO was not an inducer of CYP1A2 mRNA, while it was a weak inducer of CYP2B6 and CYP3A4. EBO was a substrate only for OCT2. At clinically relevant concentrations, neither EBO nor M3 inhibited major human efflux or uptake transporters. Based on these data, at clinically relevant concentrations of EBO and M3, there is a low risk of victim or perpetrator DDI.

Epetraborole, a leucyl-tRNA synthetase inhibitor, demonstrates murine efficacy, enhancing the in vivo activity of ceftazidime against Burkholderia pseudomallei, the causative agent of melioidosis.

Burkholderia pseudomallei is the causative agent of melioidosis, which is increasingly being reported worldwide. Mortality rates as high as 40% have been reported based on clinical patient outcomes in the endemic areas of Australia and Thailand. Novel therapies are needed to reduce treatment duration and adverse effects and improve treatment outcomes. Epetraborole, a novel antibiotic, targets leucyl-tRNA synthetase (LeuRS), an essential enzyme that catalyzes the attachment of leucine to transfer RNA. Epetraborole was evaluated for in vitro activity and efficacy in a murine model to assess clinical relevance against Burkholderia pseudomallei infections for possible treatment of melioidosis. Epetraborole was tested against 13 clinically derived and three reference B. pseudomallei strains that have a broad spectrum of susceptibilities to the standard-of-care (SoC) drugs for melioidosis, which showed that epetraborole exhibited minimal inhibitory concentrations of 0.5-4 μg/mL. Ex vivo studies using THP-1 macrophages confirmed the potency of epetraborole and demonstrated synergy between epetraborole and ceftazidime. In the acute pulmonary murine infection model of melioidosis, epetraborole demonstrated equivalent efficacy when delivered orally or subcutaneously, which compared well with the standard-of-care drug ceftazidime. In addition, adding epetraborole to ceftazidime significantly improved antimicrobial activity in this animal model. This work warrants further exploration of epetraborole as a candidate for treating melioidosis and substantiates LeuRS as a clinically relevant drug target in B. pseudomallei.

1698. Pharmacokinetics/pharmacodynamics of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, and High Intracellular Penetration in the Intracellular Hollow Fiber System Model of Mycobacterium avium Complex Lung Disease

Abstract Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. Dose-effect studies in the intracellular hollow fiber system model of Mycobacterium avium complex (MAC) lung disease, or HFS-MAC, have demonstrated that EBO is highly bactericidal. In this study we used dose fractionation in the HFS-MAC system to find the best pharmacokinetic/ pharmacodynamic (PK/PD) index that was associated with EBO microbial kill and acquired microbial resistance (AMR). Methods A dose-fractionation design was used to dose 30 HFS-MAC units. ED20, ED50 and ED80 doses were administered as one of 4 dose schedules of twice daily, once daily, twice weekly, or once weekly at a half-life of 10.4h. Total MAC burden and EBO-resistant MAC burden were determined every 7 days by culturing HFS-MAC contents on Middlebrook 7H10 agar. For drug concentrations, the central compartment of each HFS-MAC unit was sampled throughout the 28 days, and after the final dose, intracellular (IC) PK in the MAC-infected monocytes were established. EBO PK/PD index parameters including 0-168h area under the concentrations-time curve (AUC0-168), peak concentration, or % of time concentration persists above the intracellular MIC (%TMIC), which was 0.5 mg/L, were modeled versus total MAC burden using the inhibitory sigmoid maximal effect (Emax) model. The PK/PD index was chosen using Akaike Information Criteria (AIC). For AMR, EBO PK/PD index exposure value versus EBO-resistant MAC burden were modeled using a quadratic function, and the best model was chosen using AIC. Results The measured EBO concentrations demonstrated PKs shown in Figure 1. The median EBO IC versus extracellular (IC/EC) elimination rate constants were 0.018h-1 versus 0.061h-1 (p< 0.0001), which means slower IC EBO elimination, leading to IC/EC to AUC ratio of 11. Based on AIC scores in Table 1, the PK/PD driver for microbial kill was unequivocally AUC on each sampling day. For AMR the PK/PD driver was also AUC. Table 1.Corrected Akaike Information Criteria (AIC) Scores for PK/PD Indicesa-The intracellular EBO MIC was 0.5 mg/LFigure 1.Epetraborole concentrations achieved in the HFS-MAC during the 168-hour dosing interval. Epetraborole concentrations were measured for each of the four dosing schedules, and are shown together with compartmental pharmacokinetics modeling during the 168-hour dosing interval. A. Twice a day dosing. B. Once a day dosing. C. Once every 3.5 days. D. Once every 7 days. Conclusion The PK/PD index best associated with EBO microbial kill and AMR was AUC. Disclosures Shruti Athale, PhD, Praedicare Inc.: Stocks/Bonds Moti Chapagain, MD, PhD, Praedicare Inc.: Stocks/Bonds Jotam Pasipanodya, MD, PhD, Praedicare Inc.: Employee MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Tawanda Gumbo, MD, Hydronium Biopharma: Ownership Interest|Hydronium Biopharma: Stocks/Bonds|Praedicare Africa Pvt Ltd: Board Member|Praedicare Africa Pvt Ltd: Ownership Interest|Praedicare Africa Pvt Ltd: Stocks/Bonds|Praedicare Inc.: Patents|Praedicare Inc.: Ownership Interest|Praedicare Inc.: Stocks/Bonds.

1704. Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, Demonstrates Potent Efficacy and Improves Efficacy of Standard of Care Regimen Against Mycobacterium avium complex in a Chronic Mouse Lung Infection Model

Abstract Background Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. These studies evaluated oral doses (PO) of EBO against 5 M. avium complex (MAC) strains in a chronic mouse infection model either as monotherapy or in combination with standard of care [SOC; clarithromycin (CLR), rifabutin (RFB), ethambutol (EMB)]. Methods A pilot chronic efficacy study against M. avium 2285R evaluated EBO at 1, 10, 30, 100, 300 and 500 mg/kg PO once daily (QD) compared to 250 mg/kg CLR PO QD. C57BL/6 mice were infected with a pulmonary aerosol of 1x1011 CFU. Treatment was administered for 56 days starting on day 28 post-infection. The bacterial burden (CFU) in lungs was evaluated on days 1, 28 and 84 post-infection by plating serial dilutions of homogenates on Middlebrook 7H11 charcoal agar plates. An additional 4 strains of MAC were evaluated with EBO doses of 100, 200, 300 or 400 mg/kg QD compared with the SOC therapy for MAC (CLR 250 mg/kg, RFB 100 mg/kg, EMB 100 mg/kg) QD and SOC plus EBO 200mg/kg QD. Oral exposures of EBO were determined in a group of uninfected mice (Table 1).Table 1:C57BL/6 Murine Pharmacokinetic Parameters Results In a study with M. avium 2285R, a biofilm-forming strain, EBO at all doses tested was significantly better than CLR dosed at 250 mg/kg (Figure 1), and no CFU were detected on agar plates containing EBO (16 mg/L). In subsequent studies, SOC was compared to EBO in 4 additional MAC strains (Figure 2). Efficacy of EBO monotherapy was better than SOC against M. avium ATCC 700898, while it was as good as SOC with M. intracellulare 1956, M. intracellulareDNA00055, and M. intracellulare DNA00111 with CFU reductions ranging from 2 - 4.8 log10 compared to day 28 controls. In all four strains tested, 200 mg/kg EBO, which approximates the human oral equivalent dose of 500 mg, combined with SOC increased bacterial killing from 1.4 - 3.0 log10 CFU compared to SOC alone resulting in total lung CFU reductions of 4.6 - 5.6 log10. Figure 1Figure 2 Conclusion In this chronic mouse lung infection model, no EBO resistance development was detected with M. avium 2285R at day 84. EBO demonstrated potent in vivo efficacy against 5 MAC strains and significantly improved efficacy when combined with SOC, supporting further clinical development for EBO. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn Shoen, PhD, AN2 Therapeutics: Grant/Research Support Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds.

1697. Dose-response Studies of the Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, Epetraborole, in the Intracellular Hollow Fiber System Model of Mycobacterium avium complex Lung Disease

Abstract Background Based on meta-analyses the current standard-of-care regimen (SOC) for Mycobacterium avium complex (MAC) lung disease achieves a sustained sputum conversion rate of ∼54% at best. Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. To identify EBO exposure-effect parameters we used the intracellular hollow fiber system model of intracellular pulmonary MAC (HFS-MAC). Methods EBO was administered once daily at 8 different doses to HFS-MAC replicates for 28 days to achieve a half-life (t½) of 10.4h and the 0-24h area under the concentration-time curves (AUC0-24) that cover the observed AUC values in humans. The SOC combination of clarithromycin (CLR), ethambutol (EMB), and rifabutin (RFB) at human intrapulmonary pharmacokinetic (PK) concentrations was used. The central compartment of each HFS-MAC unit was sampled throughout the 28 days to assess PK parameters as was the peripheral compartment for total bacterial burden and the EBO-resistant bacterial burden. EBO AUC versus total MAC burden was modeled using the inhibitory sigmoid maximal effect (Emax) model. EBO AUC versus EBO-resistant MAC burden was modeled using a quadratic function. Results Measured EBO concentrations demonstrated a t1/2 of 10h. For SOC, the AUC for CLR, EMB, and RFB was 60 mg*h/L (t1/2=6h), 39 mg*h/L (t1/2=8h), and 1.5 mg*h/L (t1/2=45h), respectively, similar to human lung concentrations. Changes in MAC burden over 28 days (Fig. 1) show that highest EBO exposures matched SOC until day 14. The exposure versus effect on each sampling day is shown in Fig. 2. The EBO AUC mediating 50% of Emax (EC50) was an AUC of 22 mg*h/L (95% confidence interval [CI]: 16-70), and the EC80 was an AUC of 47.5 mg*h/L (CI: 34.6-151.2). The relationship between AUC and EBO-resistant subpopulation (Fig. 3) shows that an AUC0-24 of 47.5 mg*h/L, the same as the EC80 for microbial kill, was associated with resistance suppression, as was the addition of SOC to EBO. Figure 1.Changes in bacterial burden over the 28-day dosing period. Bacterial burden on serial samples from all HFS-MAC units and replicates over 28days are shown. The highest epetraborole exposures demonstrated the same effectiveness as the three drug SOC until day 14, after which it failed as resistance emerged. On the other hand adding epetraborole at an AUC of 43.6 mg*h/L to the SOC led to much faster kill (i.e, higher kill slopes) than SOC or the epetraborole monotherapy at the same AUC. Figure 2.Epetraborole exposure versus effect on bacterial burden. Shown are inhibitory sigmoid Emax curves for epetraborole AUC versus log10 CFU/mL on each sampling day, as well as various extents of microbial kill such as stasis (holding burden similar to day 0), 1 log CFU/mL kill and 2 log CFU/mL kill. Beyond day 3, epetraborole killed > 1log CFU/mL, which means it was rapidly bactericidal. Figure 3.Epetraborole exposure versus epetraborole-resistant subpopulation. Drug-resistant burden is shown as log10 CFU/mL. The addition of SOC to EBO totally suppressed emergence of any resistance on all days. Conclusion EBO monotherapy with an AUC0-24 > 16.9 mg*h/L killed > 1.0 log10 CFU/mL of MAC compared to day 0. At the EC80, EBO killed at least 2.0 log10 CFU/mL, thus was highly bactericidal. EBO plus SOC demonstrated resistance suppression. Disclosures Moti Chapagain, MD, PhD, Praedicare Inc.: Stocks/Bonds Shruti Athale, PhD, Praedicare Inc.: Stocks/Bonds Jotam Pasipanodya, MD, PhD, Praedicare Inc.: Employee MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Tawanda Gumbo, MD, Hydronium Biopharma: Ownership Interest|Hydronium Biopharma: Stocks/Bonds|Praedicare Africa Pvt Ltd: Board Member|Praedicare Africa Pvt Ltd: Ownership Interest|Praedicare Africa Pvt Ltd: Stocks/Bonds|Praedicare Inc.: Patents|Praedicare Inc.: Ownership Interest|Praedicare Inc.: Stocks/Bonds.

1712. In Vitro Activity of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, in Drug Combinations Against Nontuberculous Mycobacteria Including Resistance Frequency and MIC Characterization of Mycobacterium avium ATCC 700898 Epetraborole-resistant Mutants

Abstract Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria (NTM). The standard of care therapy for Mycobacterium avium complex (MAC) lung disease consists of a combination of a macrolide, ethambutol (EMB) and a rifamycin. A total of 5 strains of MAC, M. abscessus ATCC 19977 and M. peregrinum ATCC 700686 were tested in checkerboard assays to evaluate the effects of combining EBO with other agents. The spontaneous resistance frequency (RF) was determined for EBO singly and in combination against M. avium ATCC 700898. Methods The effects of combining EBO with clarithromycin (CLR), rifabutin (RBT), EMB, amikacin (AMK) or bedaquiline (BDQ) were evaluated in 7 strains of NTM. Synergy, additive effects, indifference or antagonism was characterized in the checkerboard assay using EUCAST criteria. The RF of M. avium ATCC 700898 at 2x, 4x and 8x the MIC (8 mg/L) of EBO was determined, as was the RF of EBO combined with CLR, RBT, AMK or EMB. MICs of selected EBO mutants were determined against AMK, BDQ, CLR, RBT, EMB, and clofazimine (CFZ) and the mutants were further characterized. Results The RF of EBO ranged from 1.58x10-7 to 8.48x10-9 when selected on 2 - 8x agar MIC. The MIC for EBO increased 32- > 256-fold for the resistant mutants; however, the MICs for the other drugs tested against EBO-resistant strains did not change significantly. The activity of EBO was not antagonized, and was mainly indifferent to the addition of CLR, RBT, AMK, or BDQ for all the NTM strains tested. Synergy with EMB was observed with 2 strains and additivity with 2 additional strains of MAC. The addition of EMB (24 mg/L), CLR (32 mg/L), RFB (2 mg/L) or AMK (128 mg/L) to agar plates containing 2x MIC of EBO lowered the RF to at least < 2.13x10-10 (Table 1). Table 1.In Vitro Resistance Frequency for the Components of SOC with and without EBO in Mycobacterium avium ATCC 700898 Conclusion In the checkerboard studies, no evidence of antagonism was observed with any strain or EBO combination; interactions were largely indifferent. EBO combined with EMB in MAC resulted in synergy or additive effects in the checkerboard assay. The addition of EMB, CLR, RFB or AMK to EBO led to a > 700-fold reduction in RF. Activity of other drugs was not impacted by EBO resistance suggesting that cross-resistance did not occur. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn M. Shoen, PhD, AN2 Therapeutics: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support.

A Novel Leucyl-tRNA Synthetase Inhibitor, MRX-6038, Expresses Anti-Mycobacterium abscessus Activity In Vitro and In Vivo.

ABSTRACTTherapeutic options for Mycobacterium abscessus infections are extremely limited, and new drugs are needed. The anti-M. abscessus activity of MRX-6038, a new leucyl-tRNA synthetase inhibitor, was evaluated in vitro and in vivo. Antimicrobial susceptibility testing was performed on 12 nontuberculosis mycobacteria (NTM) reference strains and 227 clinical NTM isolates. A minimum bactericidal concentration assay was conducted to distinguish the bactericidal versus bacteriostatic activity of MRX-6038. The synergy between MRX-6038 and 12 clinically important antibiotics was determined using a checkerboard assay. The activity of MRX-6038 against M. abscessus residing inside macrophages was also evaluated. Finally, the potency of MRX-6038 in vivo was determined in a neutropenic mouse model that mimicked a pulmonary M. abscessus infection. MRX-6038 exhibited high anti-M. abscessus activity against extracellular M. abscessus in culture, with a MIC50 of 0.063 mg/L and a MIC90 of 0.125 mg/L. Fifty percent of the activity was bactericidal, and fifty percent was bacteriostatic. A synergy between MRX-6038 and clarithromycin or azithromycin was found in 25% of strains. No antagonism was evident between MRX-6038 and 12 antibiotics commonly used to treat NTM infections. MRX-6038 also exhibited activity against intracellular NTM, which caused a significant reduction in the bacterial load in the lungs of M. abscessus-infected neutropenic mice. In conclusion, MRX-6038 was active against M. abscessus in vitro and in vivo, and it represents a potential candidate for incorporation into strategies by which M. abscessus infections are treated.

Leucyl-tRNA Synthetase Inhibitor, D-Norvaline, in Combination with Oxacillin, Is Effective against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium that causes severe diseases in humans. For decades, MRSA has acquired substantial resistance against conventional antibiotics through regulatory adaptation, thereby posing a challenge for treating MRSA infection. One of the emerging strategies to combat MRSA is the combinatory use of antibacterial agents. Based on the dramatic change in phospholipid fatty acid (PLFA) composition of MRSA in previous results, this study investigated branched-chain amino acid derivatives (precursors of fatty acid synthesis of cell membrane) and discovered the antimicrobial potency of D-norvaline. The compound, which can act synergistically with oxacillin, is among the three leucine-tRNA synthetase inhibitors with high potency to inhibit MRSA cell growth and biofilm formation. PLFA analysis and membrane properties revealed that D-norvaline decreased the overall amount of PLFA, increasing the fluidity and decreasing the hydrophobicity of the bacterial cell membrane. Additionally, we observed genetic differences to explore the response to D-norvaline. Furthermore, deletion mutants and clinically isolated MRSA strains were treated with D-norvaline. The study revealed that D-norvaline, with low concentrations of oxacillin, was effective in killing several MRSA strains. In summary, our findings provide a new combination of aminoacyl-tRNA synthetase inhibitor D-norvaline and oxacillin, which is effective against MRSA.

A calculation method for designing newTrypanosoma brucei leucyl-tRNA synthetase inhibitors: Combining QSAR and molecular docking technology

Leucyl-tRNA synthetase (LeuRS) is a promising target for novel antitrypanosomal agents. We conducted two-dimsional 9two2D-QSAR (HQSAR) and 3D-QSAR (Topomer CoMFA) studies on 37 Trypanosoma brucei LeuRS (TbLeuRS) inhibitors with different structures and potencies to identify the structural features for more LeuRS inhibitors. In the Topomer CoMFA model constructed based on the optimal fragment cutting mode, the cross-validation coefficient q 2 and the non-cross-validation coefficient r 2 are 0.624 and 0.774, respectively, and external validation parameters Q F 1 2 , Q F 2 2 , CCC and r pred 2 are 0.932, 0.931, 0.960 and 0.932, respectively. The best HQSAR model uses atoms, bonds, hydrogen atoms, and chirality as fragment distinctions, 7-8 as the fragment size, and three as the optimal principal component number. This model shows a cross-validated q 2 value of 0.721, a non-cross-validated r 2 value of 0.836, and r pred 2 of 0.923. The steric field maps, electrostatic field maps, and atomic contribution maps of the two models provide the structural characteristics of the inhibitory efficacy. Twelve promising new inhibitors were successfully designed and the models were validated by molecular docking. Finally, we used ADMET analysis to reveal that the designed inhibitors have excellent oral bioavailability and drugability. .

Identification of novel mycobacterium tuberculosis leucyl-tRNA synthetase inhibitor using a knowledge-based computational screening approach

ObjectivesTuberculosis is a chronic lung disease caused by Mycobacterium tuberculosis (MTB), whose thick cell envelope and drug metabolizing enzymes offering it multidrug resistance. Therefore, there is a need to identify new molecular targets, biologically active as well as clinically safe anti-MTB compounds from natural resources. MethodsIn this study, we performed high throughput computational screening of FDA listed natural bioactive compounds for identifying novel anti-MTB leucyl-tRNA (LeuRS) synthetase inhibitors. ResultsInitial virtual molecular docking of 136 bioactive compounds has identified Docetaxel, Reserpine and Irinotecan as promising lead molecules owing to their structural plasticity and binding affinity with the active site of MTB-LeuRS. Further, deep molecular docking and molecular dynamics (MD) simulation analysis (at 100 ns) of the above three test compounds along with Oxaborole compound (GSK656) has demonstrated the superior binding affinity and stability of Irinotecan in forming molecular complexes with LeuRS protein. Interestingly, it also showed comparable binding residues and affinity parameters (like flexibility, structural divergence) as the GSK656 inhibitor in binding the tRNASyn domain 2 of LeuRS. A good correlation of pharmacokinetic properties (ADME-Tox) like bioavailability, absorption, solubility, and low toxicity between Irinotecan and GSK656 was evident. Competitive binding of Irinotecan to tRNASyn domain 2 of LeuRS is likely to make it unavailable to bind Leucine amino acid, which may negatively impact the protein biosynthesis and eventually inhibit the bacterial growth and attenuate the pathogen’s virulence. ConclusionsOur findings pave the way for further experimental confirmation of Irinotecan in the quest for a novel anti-LeuRS specific inhibitor to combat drug resistant MTB infection.

Design and synthesis of leucylaniline derivatives as leucyl-tRNA synthetase inhibitors

By simulating the structure of Leu-AMP, 26 leu-arylamine derivatives were designed and synthesized as leucyl-tRNA Synthetase inhibitors; compounds 19 and 24 showed good anti-tuberculosis activity.

Epetraborole Is Active against Mycobacterium abscessus.

ABSTRACTBenzoxaboroles are a new class of leucyl-tRNA synthetase inhibitors. We recently reported that the antitubercular 4-halogenated benzoxaboroles are active against Mycobacterium abscessus. Here, we find that the nonhalogenated benzoxaborole epetraborole, a clinical candidate developed for Gram-negative infections, is also active against M. abscessus in vitro and in a mouse model of infection. This expands the repertoire of advanced lead compounds for the discovery of a benzoxaborole-based candidate to treat M. abscessus lung disease.

Design and synthesis of N-(3-sulfamoylphenyl)amides as Trypanosoma brucei leucyl-tRNA synthetase inhibitors.

The protozoan parasite Trypanosoma brucei (T.brucei) causes human African trypanosomiasis (HAT), which is a fatal and neglected disease in the tropic areas, and new treatments are urgently needed. Leucyl-tRNA synthetase (LeuRS) is an attractive target for the development of antimicrobial agents. In this work, starting from the hit compound thiourea ZCL539, we designed and synthesized a series of amides as effective T.brucei LeuRS (TbLeuRS) synthetic site inhibitors. The most potent compounds 74 and 91 showed IC50 of 0.24 and 0.25μM, which were about 700-fold more potent than the starting hit compound. The structure-activity relationship was also discussed. These compounds provided a new scaffold and lead compounds for further development of antitrypanosomal agents.

Development and Validation of an LC-MS/MS Method for Quantification of the Novel Antibacterial Candidate DA-7010 in Plasma and Application to a Preclinical Pharmacokinetic Study.

DA-7010 is a new candidate for an antibacterial agent that targets Gram-negative pathogens by acting as a leucyl-tRNA synthetase inhibitor. In this study, a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to determine DA-7010 levels in the plasma from mice, rats, and dogs. Plasma samples were mixed with methanol for protein precipitation. Chromatographic separation was carried out using a reversed-phase C18 column (Agilent Poroshell 120, 50 × 3.0 mm, 2.7 μm). An isocratic elution of the mobile phase consisting of 5 mM formic acid in water and acetonitrile at a ratio of 84:16 (v/v) was applied at a flow rate of 0.3 mL/min. The total chromatographic run time was 3.5 min. Multiple reaction monitoring (MRM) mode was used for mass spectrometric detection using an Agilent 6460 triple quadrupole coupled with an electrospray ionization (ESI) source operated in positive-ion mode. The MRM transitions analyzed were m/z 220.1→162.1 for DA-7010 and m/z 206.1→170.1 for the internal standard (structural analogue of DA-7010). Calibration curves were constructed in the range of 10–10,000 ng/mL. The intra- and interday precision and accuracy were within 11.3%, excluding those for the lower limit of quantification (LLOQ) samples, which were within 17.1%. The developed LC-MS/MS method was successfully validated and applied in preclinical pharmacokinetic studies of DA-7010 in mice, rats, and dogs following single oral administrations. The oral absorption of DA-7010 was rapid, and the systemic exposure was approximately four times higher in the dogs than in the mice or rats.

A Leucyl-tRNA Synthetase Inhibitor with Broad-Spectrum Anti-Mycobacterial Activity.

Global infections by non-tuberculous mycobacteria (NTM) are steadily rising. New drugs are needed to treat NTM infections, but the NTM drug pipeline remains poorly populated and focused on repurposing or reformulating approved antibiotics. We sought to accelerate de novo NTM drug discovery by testing advanced compounds with established activity against Mycobacterium tuberculosis 3-aminomethyl 4-halogen benzoxaboroles, a novel class of leucyl-tRNA synthetase inhibitors, were recently discovered as active against M. tuberculosis Here, we report that the benzoxaborole EC/11770 is not only a potent anti-tubercular agent but is active against the M. abscessus and M. avium complexes. Focusing on M. abscessus, which causes the most difficult-to-cure NTM disease, we show that EC/11770 retained potency against drug-tolerant biofilms in vitro and was effective in a mouse lung infection model. Resistant mutant selection experiments showed a low frequency of resistance and confirmed leucyl-tRNA synthetase as the target. This work establishes the benzoxaborole EC/11770 as a novel preclinical candidate for the treatment of NTM lung disease and tuberculosis and validates leucyl-tRNA synthetase as an attractive target for the development of broad-spectrum anti-mycobacterials.

Synthesis and structure-activity studies of novel anhydrohexitol-based Leucyl-tRNA synthetase inhibitors.

Leucyl-tRNA synthetase (LeuRS) is a clinically validated target for the development of antimicrobials. This enzyme catalyzes the formation of charged tRNALeu molecules, an essential substrate for protein translation. In the first step of catalysis LeuRS activates leucine using ATP, forming a leucyl-adenylate intermediate. Bi-substrate inhibitors that mimic this chemically labile phosphoanhydride-linked nucleoside have proven to be potent inhibitors of different members of the aminoacyl-tRNA synthetase family but, to date, they have demonstrated poor antibacterial activity. We synthesized a small series of 1,5-anhydrohexitol-based analogues coupled to a variety of triazoles and performed detailed structure-activity relationship studies with bacterial LeuRS. In an invitro assay, Kiapp values in the nanomolar range were demonstrated. Inhibitory activity differences between the compounds revealed that the polarity and size of the triazole substituents affect binding. X-ray crystallographic studies of N.gonorrhoeae LeuRS in complex with all the inhibitors highlighted the crucial interactions defining their relative enzyme inhibitory activities. We further examined their invitro antimicrobial properties by screening against several bacterial and yeast strains. While only weak antibacterial activity against M.tuberculosis was detected, the extensive structural data which were obtained could make these LeuRS inhibitors a suitable starting point towards further antibiotic development.

Design and synthesis of α-phenoxy-N-sulfonylphenyl acetamides as Trypanosoma brucei Leucyl-tRNA synthetase inhibitors

Human African trypanosomiasis (HAT), caused by the parasitic protozoa Trypanosoma brucei, is one of the fatal diseases in tropical areas and current medicines are insufficient. Thus, development of new drugs for HAT is urgently needed. Leucyl-tRNA synthetase (LeuRS), a recently clinically validated antimicrobial target, is an attractive target for development of antitrypanosomal drugs. In this work, we report a series of α-phenoxy-N-sulfonylphenyl acetamides as T. brucei LeuRS inhibitors. The most potent compound 28g showed an IC50 of 0.70 μM which was 250-fold more potent than the starting hit compound 1. The structure-activity relationship was also discussed. These acetamides provided a new scaffold and lead compounds for the further development of clinically useful antitrypanosomal agents.

First-Time-in-Human Study and Prediction of Early Bactericidal Activity for GSK3036656, a Potent Leucyl-tRNA Synthetase Inhibitor for Tuberculosis Treatment.

This first-time-in-human (FTIH) study evaluated the safety, tolerability, pharmacokinetics, and food effect of single and repeat oral doses of GSK3036656, a leucyl-tRNA synthetase inhibitor. In part A, GSK3036656 single doses of 5 mg (fed and fasted), 15 mg, and 25 mg and placebo were administered. In part B, repeat doses of 5 and 15 mg and placebo were administered for 14 days once daily. GSK3036656 showed dose-proportional increase following single-dose administration and after dosing for 14 days. The maximum concentration of drug in serum (Cmax) and area under the concentration-time curve from 0 h to the end of the dosing period (AUC0-τ) showed accumulation with repeated administration of approximately 2- to 3-fold. Pharmacokinetic parameters were not altered in the presence of food. Unchanged GSK3036656 was the only drug-related component detected in plasma and accounted for approximately 90% of drug-related material in urine. Based on total drug-related material detected in urine, the minimum absorbed doses after single (25 mg) and repeat (15 mg) dosing were 50 and 78%, respectively. Unchanged GSK3036656 represented at least 44% and 71% of the 25- and 15-mg doses, respectively. Clinical trial simulations were performed to guide dose escalation during the FTIH study and to predict the GSK3036656 dose range that produces the highest possible early bactericidal activity (EBA0-14) in the prospective phase II trial, with consideration of the predefined exposure limit. GSK3036656 was well tolerated after single and multiple doses, with no reports of serious adverse events. (This study has been registered at ClinicalTrials.gov under identifier NCT03075410.).