Front-line Drug Research Articles

Herbo-vitamin medicine Livogrit Vital ameliorates isoniazid induced liver injury (IILI) in human liver (HepG2) cells by decreasing isoniazid accumulation and oxidative stress driven hepatotoxicity

BackgroundTuberculosis (TB) is a leading cause of infection related mortality. Isoniazid is one of the frontline drugs for anti-TB therapy. Hepatotoxicity induced by isoniazid is a major cause of drug-discontinuation which may lead to development of resistant TB or increased mortality.PurposeTo characterize pharmacological properties of plant-based prescription medicine, Livogrit Vital (LVV) against isoniazid-induced liver injury (IILI) using HepG2 cells.MethodPhytometabolite characterization of LVV was performed by High-performance liquid chromatography (HPLC). The effects of LVV on cytosafety, IC50 shift, oxidative stress, ER stress, apoptosis, liver injury markers, and accumulation of isoniazid and hydrazine was performed on HepG2 cells induced with isoniazid. Silymarin was used as the positive control.ResultsHPLC based phytometabolite characterization of LVV revealed the presence of several anti-oxidant, anti-apoptotic, and hepatoprotective compounds. In isoniazid-induced HepG2 cells, LVV reduced cytotoxicity of isoniazid and shifted its IC50 value. Treatment with LVV reduced ROS generation and lipid peroxidation; enhanced GSH enzyme levels in isoniazid-induced HepG2 cells. As per the mechanistic evaluation, LVV modulated gene expression level of Caspase-3, FGF21, and IRE-1α. LVV treatment also normalized isoniazid-induced elevated Caspase-3 activity and cPARP1 protein levels, indicating its potentials to regulate liver cell apoptosis. Concomitantly, biomarkers of hepatotoxicity, ALT and GGT, also decreased by LVV treatment. Interestingly, LVV treatment reduced intracellular accumulation of isoniazid and its toxic metabolite hydrazine, in isoniazid-stimulated HepG2 cells.ConclusionTreatment of hepatic cells with the herbo-vitamin medicine, Livogrit Vital, regulates IILI by modulation of oxidative and ER stress, apoptosis, and bioaccumulation of isoniazid and hydrazine. Collectively, Livogrit Vital could well be explored as an adjuvant hepatoprotective agent alongwith anti-TB medicines.

Targeting PfCLK3 with Covalent Inhibitors: A Novel Strategy for Malaria Treatment.

Malaria still causes over 600,000 deaths annually, with rising resistance to frontline drugs by Plasmodium falciparum increasing this number each year. New medicines with novel mechanisms of action are, therefore, urgently needed. In this work, we solved the cocrystal structure of the essential malarial kinase PfCLK3 with the reversible inhibitor TCMDC-135051 (1), enabling the design of covalent inhibitors targeting a unique cysteine residue (Cys368) poorly conserved in the human kinome. Chloroacetamide 4 shows nanomolar potency and covalent inhibition in both recombinant protein and P. falciparum assays. Efficacy in parasites persisted after a 6 h washout, indicating an extended duration of action. Additionally, 4 showed improved kinase selectivity and a high selectivity index against HepG2 cells, with a low propensity for resistance (log MIR > 8.1). To our knowledge, compound 4 is the first covalent inhibitor of a malarial kinase, offering promising potential as a lead for a single-dose malaria cure.

Computational approach for decoding malaria drug targets from single-cell transcriptomics and finding potential drug molecule

Malaria is a deadly disease caused by Plasmodium parasites. While potent drugs are available in the market for malaria treatment, over the years, Plasmodium parasites have successfully developed resistance against many, if not all, front-line drugs. This poses a serious threat to global malaria eradication efforts, and the continued discovery of new drugs is necessary to tackle this debilitating disease. With recent unprecedented progress in machine learning techniques, single-cell transcriptomic in Plasmodium offers a powerful tool for identifying crucial proteins as a drug target and subsequent computational prediction of potential drugs. In this study, We have implemented a mutual-information-based feature reduction algorithm with a classification algorithm to select important proteins from transcriptomic datasets (sexual and asexual stages) for Plasmodium falciparum and then constructed the protein-protein interaction (PPI) networks of the proteins. The analysis of this PPI network revealed key proteins vital for the survival of Plasmodium falciparum. Based on the function and identification of a few strong binding sites on a couple of these key proteins, we computationally predicted a set of potential drug molecules using a deep learning-based technique. Lead drug molecules that satisfy ADMET and drug-likeliness properties are finally reported out of the generated drugs. The study offers a general computational pipeline to identify crucial proteins using scRNA-seq data sets and further development of potential new drugs.

Therapeutic targeting of RNA for neurological and neuromuscular disease.

Neurological and neuromuscular diseases resulting from familial, sporadic, or de novo mutations have devasting personal, familial, and societal impacts. As the initial product of DNA transcription, RNA transcripts and their associated ribonucleoprotein complexes provide attractive targets for modulation by increasing wild-type or blocking mutant allele expression, thus relieving downstream pathological consequences. Therefore, it is unsurprising that many existing and under-development therapeutics have focused on targeting disease-associated RNA transcripts as a frontline drug strategy for these genetic disorders. This review focuses on the current range of RNA targeting modalities using examples of both dominant and recessive neurological and neuromuscular diseases.

Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1.

Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of Leishmania donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.

Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter.

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs using non-traditional approaches. Pseudomonas aeruginosa, one of the most common pathogens involved in nosocomial infections, is extremely difficult to treat even with the last resort frontline drug, the carbapenems. As the pathogen has the ability to acquire resistance to new small-molecule antibiotics, being deployed, a novel biological approach has been tried using antibody fragments in combination with carbapenems and β-lactams as adjunct therapy for an enduring solution to the problem. In this study, we developed a camelid antibody fragment (VHH) library against P. aeruginosa and isolated a highly potent hit, PsC23. Mass spectrometry identified the target to be a component of the C4-dicarboxylate transporter that feeds metabolites to the glyoxylate shunt particularly under conditions of oxidative stress. PsC23 is bacteriostatic at a concentration of 1.66 µM (25 µg ml-1) and shows a synergistic effect with both the classes of drugs at an effective concentration of 100-200 nM (1.5-3.0 µg ml-1) when co administered with them. In combination with meropenem the VHH completely cleared the infection from a neutropenic mouse with a carbapenem-resistant P. aeruginosa systemic infection. Blocking the glyoxylate shunt by PsC23 resulted in disruption of energy transduction due to a respiratory shift to the oxygen-depleted TCA cycle causing inhibition of efflux and increased free radical generation from carbapenems and β-lactams exerting a strong bactericidal effect that reversed the resistance to multiple unrelated drugs.

Cancer nuclear envelope rupture and repair in taxane resistance

Abstract Taxanes, including paclitaxel, docetaxel, and cabazitaxel, are key agents in cancer treatment, often used as front-line chemotherapy drugs in combination with other agent(s) (commonly carboplatin) and as second-line treatments alone. Generally, taxanes are highly effective, but drug resistance unavoidably develops following repeated treatment. Taxanes work by binding to and stabilizing microtubules, leading to mitotic arrest, mitotic catastrophe, and micronucleation. The long-recognized mechanisms of drug resistance generally can be classified into three categories: drug efflux, microtubule polymerization, and apoptotic pathway. A recent new addition to this list is a mechanism related to the nuclear envelope, as cancer cells undergo micronucleation and nuclear membrane rupture when treated with taxanes. All these mechanisms may operate simultaneously as taxane resistance is multi-factorial. Here, we review the cell biology understanding of nuclear envelope breaking in production of micronucleation, and nuclear membrane rupture and repair, and propose that these processes are involved in taxane resistance.

Immunoinhibitory effects of anti-tuberculosis therapy induce the host vulnerability to tuberculosis recurrence.

The host immune responses play a pivotal role in the establishment of long-term memory responses, which effectively aids in infection clearance. However, the prevailing anti-tuberculosis therapy, while aiming to combat tuberculosis (TB), also debilitates innate and adaptive immune components of the host. In this study, we explored how the front-line anti-TB drugs impact the host immune cells by modulating multiple signaling pathways and subsequently leading to disease relapse. Administration of these drugs led to a reduction in innate immune activation and also the cytokines required to trigger protective T cell responses. Moreover, these drugs led to activation-induced cell death in the mycobacterial-specific T cell leading to a reduced killing capacity. Furthermore, these drugs stalled the T cell differentiation into memory subsets by modulating the activation of STAT3, STAT4, FOXO1, and NFκB transcription factors and hampering the Th1 and Th17-mediated long-term host protective memory responses. These findings suggest the urgent need to augment directly observed treatment, short-course (DOTS) therapy with immunomodulatory agents to mitigate the adverse effects linked to the treatment.IMPORTANCEAs a central component of TB eradication initiatives, directly observed treatment, short-course (DOTS) therapy imparts immune-dampening effects during the course of treatment. This approach undermines the host immune system by delaying the activation process and lowering the immune response. In our investigation, we have unveiled the impact of DOTS on specific immune cell populations. Notably, the signaling pathways involving STAT3 and STAT4 critical for memory responses and NFκβ associated with pro-inflammation were substantially declined due to the therapy. Consequently, these drugs exhibit limited effectiveness in preventing recurrence of the disease. These observations highlight the imperative integration of immunomodulators to manage TB infection.

Mechanistic insights into the conformational changes and alterations in residual communications due to the mutations in the pncA Gene of Mycobacterium tuberculosis: A computational perspective for effective therapeutic solutions

Due to its emerging resistance to first-line anti-TB medications, tuberculosis (TB) is one of the most contagious illness in the world. According to reports, the effectiveness of treating TB is severely impacted by drug resistance, notably resistance caused by mutations in the pncA gene-encoded pyrazinamidase (PZase) to the front-line drug pyrazinamide (PZA). The present study focused on investigating the resistance mechanism caused by the mutations D12N, T47A, and H137R to better understand the structural and molecular events responsible for the resistance acquired by the pncA gene of Mycobacterium tuberculosis (MTB) at the structural level. Bioinformatics analysis predicted that all three mutations were deleterious and located near the active centre of the pncA, affecting its functional activity. Furthermore, molecular dynamics simulation (MDS) results established that mutations significantly reduced the structural stability and caused the rearrangement of FE2+ in the active centre of pncA. Moreover, essential dynamics analysis, including principal component analysis (PCA) and free energy landscape (FEL), concluded variations in the protein motion and decreased conformational space in the mutants. Additionally, the mutations potentially impacted the network topologies and altered the residual communications in the network. The complex simulation study results established the significant movement of the flap region from the active centre of mutant complexes, further supporting the flap region’s significance in developing resistance to the PZA drug. This study advances our knowledge of the primary cause of the mechanism of PZA resistance and the structural dynamics of pncA mutants, which will help us to design new and potent chemical scaffolds to treat drug-resistant TB (DR-TB).

TcrXY is an acid-sensing two-component transcriptional regulator of Mycobacterium tuberculosis required for persistent infection

The ability of Mycobacterium tuberculosis (Mtb) to persist in the host complicates and prolongs tuberculosis (TB) patient chemotherapy. Here we demonstrate that a neglected two-component system (TCS) of Mtb, TcrXY, is an autoregulated acid-sensing TCS that controls a functionally diverse 70-gene regulon required for bacterial persistence. Characterisation of two representatives of this regulon, Rv3706c and Rv3705A, implicate these genes as key determinants for the survival of Mtb in vivo by serving as important effectors to mitigate redox stress at acidic pH. We show that genetic silencing of the response regulator tcrX using CRISPR interference attenuates the persistence of Mtb during chronic mouse infection and improves treatment with the two front-line anti-TB drugs, rifampicin and isoniazid. We propose that targeting TcrXY signal transduction blocks the ability of Mtb to sense and respond to acid stress, resulting in a disordered program of persistence to render the organism vulnerable to existing TB chemotherapy.

Metformin Treatment Leads to Increased HIV Transcription and Gene Expression through Increased CREB Phosphorylation and Recruitment to the HIV LTR Promoter.

Antiretroviral therapy has effectively suppressed HIV infection and replication and prolonged the lifespan of HIV-infected individuals. In the meantime, various complications including type 2 diabetes associated with the long-term antiviral therapy have shown steady increases. Metformin has been the front-line anti-hyperglycemic drug of choice and the most widely prescribed medication for the treatment of type 2 diabetes. However, little is known about the effects of Metformin on HIV infection and replication. In this study, we showed that Metformin treatment enhanced HIV gene expression and transcription in HIV-transfected 293T and HIV-infected Jurkat and human PBMC. Moreover, we demonstrated that Metformin treatment resulted in increased CREB expression and phosphorylation, and TBP expression. Furthermore, we showed that Metformin treatment increased the recruitment of phosphorylated CREB and TBP to the HIV LTR promoter. Lastly, we showed that inhibition of CREB phosphorylation/activation significantly abrogated Metformin-enhanced HIV gene expression. Taken together, these results demonstrated that Metformin treatment increased HIV transcription, gene expression, and production through increased CREB phosphorylation and recruitment to the HIV LTR promoter. These findings may help design the clinical management plan and HIV cure strategy of using Metformin to treat type 2 diabetes, a comorbidity with an increasing prevalence, in people living with HIV.

Artesunate in glioblastoma therapy: Case reports and review of clinical studies

BackgroundArtesunate, a derivative of the active ingredient artemisinin from Artemisia annua L. used for centuries in the traditional Chinese medicine, is being applied as front-line drug in malaria treatment. As it is cytotoxic for cancer cells, trials are ongoing to include this drug as supplement in cancer therapy. In glioblastoma cells, artesunate was shown to induce oxidative stress, DNA base damage and double-strand breaks (DSBs), apoptosis, and necroptosis. It also inhibits DNA repair functions and bears senolytic activity. Compared to ionizing radiation, DNA damages accumulate over the whole exposure period, which makes the agent unique in its genotoxic profile. Artesunate has been used in adjuvant therapy of various cancers. PurposeAs artesunate has been used in adjuvant therapy of different types of cancer and clinical trials are lacking in brain cancer, we investigated its activity in glioma patients with focus on possible side effects. Study designBetween 2014 and 2020, twelve patients were treated with artesunate for relapsing glioma and analyzed retrospectively: 8 males and 4 females, median age 45 years. Histology: 4 glioblastomas WHO grade 4, 5 astrocytomas WHO grade 3, 3 oligodendrogliomas grade 2 or 3. All patients were pretreated with radiation and temozolomide-based chemotherapy. Artesunate 100 mg was applied twice daily p.o. combined with dose-dense temozolomide alone (100 mg/m2 day 1–5/7, 10 patients) or with temozolomide (50 mg/m2 day 1–5/7) plus lomustine (CCNU, 40 mg day 6/7). Blood count, C-reactive protein (CRP), liver enzymes, and renal parameters were monitored weekly. ResultsApart from one transient grade 3 hematological toxicity, artesunate was well tolerated. No liver toxicity was observed. While 8 patients with late stage of the disease had a median survival of 5 months after initiation of artesunate treatment, 4 patients with treatment for remission maintenance showed a median survival of 46 months. We also review clinical trials that have been performed in other cancers where artesunate was included in the treatment regimen. ConclusionsArtesunate administered at a dose of 2 × 100 mg/day was without harmful side effects, even if combined with alkylating agents used in glioma therapy. Thus, the phytochemical, which is also utilized as food supplement, is an interesting, well tolerated supportive agent useful for long-term maintenance treatment. Being itself cytotoxic on glioblastoma cells and enhancing the cytotoxicity of temozolomide as well as in view of its senolytic activity, artesunate has clearly a potential to enhance the efficacy of malignant brain cancer therapy.

Co-amorphous mixture of erlotinib hydrochloride and gallic acid for enhanced antitumor effects

Erlotinib hydrochloride (ERL) is a frontline drug approved by FDA for the treatment of several types of cancers, while its clinical application is seriously compromised by poor water solubility. In this study, gallic acid (GA) was utilized as a bioactive co-former, and the co-amorphous system of ERL and GA (ERL-GA CM) was constructed to improve the water solubility and bioavailability of ERL as well as produce synergistic antitumor effects. After being prepared by solvent evaporation method, ERL-GA CM was characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and molecular dynamics (MD) simulations demonstrated that the intermolecular hydrogen bonds were formed between ERL and GA in ERL-GA CM. The concentration of ERL in ERL-GA CM was 6.7 times (25 °C) and 5.1 times (37 °C) higher than that of ERL after shaking for 72 h at pH 2.0, and the dissolution rate of ERL in ERL-GA CM was also significantly increased. Pharmacokinetic assay demonstrated that ERL-GA CM obviously enhanced the oral bioavailability of ERL, and pharmacodynamic studies indicated that ERL-GA CM exerted synergistic antitumor effects characterized by Q value of 1.3. Besides, ERL-GA CM showed negligible toxicity to the healthy tissues. Taken together, co-amorphization of ERL using GA provides a promising strategy to improve the solubility and bioavailability of ERL for enhanced antitumor effects.

Benznidazole treatment leads to DNA damage in Trypanosoma cruzi and the persistence of rare widely dispersed non-replicative amastigotes in mice.

Benznidazole is the front-line drug used to treat infections with Trypanosoma cruzi, the causative agent of Chagas disease. However, for reasons that are unknown, treatment failures are common. When we examined parasites that survived benznidazole treatment in mice using highly sensitive in vivo and ex vivo bioluminescence imaging, we found that recrudescence is not due to persistence of parasites in a specific organ or tissue that preferentially protects them from drug activity. Surviving parasites are widely distributed and located in host cells where the vast majority contained only one or two amastigotes. Therefore, infection relapse does not arise from a small number of intact large nests. Rather, persisters are either survivors of intracellular populations where co-located parasites have been killed, or amastigotes in single/low-level infected cells exist in a state where they are less susceptible to benznidazole. To better assess the nature of parasite persisters, we exposed infected mammalian cell monolayers to a benznidazole regimen that reduces the intracellular amastigote population to <1% of the pre-treatment level. Of host cells that remained infected, as with the situation in vivo, the vast majority contained only one or two surviving intracellular amastigotes. Analysis, based on non-incorporation of the thymidine analogue EdU, revealed these surviving parasites to be in a transient non-replicative state. Furthermore, treatment with benznidazole led to widespread parasite DNA damage. When the small number of parasites which survive in mice after non-curative treatment were assessed using EdU labelling, this revealed that these persisters were also initially non-replicative. A possible explanation could be that triggering of the T. cruzi DNA damage response pathway by the activity of benznidazole metabolites results in exit from the cell cycle as parasites attempt DNA repair, and that metabolic changes associated with non-proliferation act to reduce drug susceptibility. Alternatively, a small percentage of the parasite population may pre-exist in this non-replicative state prior to treatment.

RES-Seq-a barcoded library of drug-resistant Leishmania donovani allowing rapid assessment of cross-resistance and relative fitness.

Visceral leishmaniasis (VL) remains the third largest parasitic killer worldwide, responsible for 20,000-30,000 deaths each year. Control and ultimate elimination of VL will require a range of therapeutic options with diverse mechanisms of action to combat drug resistance. One approach to ensure that compounds in development exploit diverse mechanisms of action is to screen them against highly curated cell lines resistant to drugs already in the VL pipeline. The identification of cross-resistant cell lines indicates that test compounds are likely acting via previously established mechanisms. Current cross-resistance screens are limited by the requirement to profile individual resistant cell lines one at a time. Here, we introduce unique DNA barcodes into multiple resistant cell lines to facilitate parallel profiling. Utilizing the power of Illumina sequencing, growth kinetics and relative fitness under compound selection can be monitored revolutionizing our ability to identify and prioritize compounds acting via novel mechanisms.

Structural and functional characterization of 6-phosphogluconate dehydrogenase in Plasmodium falciparum (3D7) and identification of its potent inhibitors

The malarial parasite Plasmodium falciparum predominantly causes severe malaria and deaths worldwide. Moreover, resistance developed by P. falciparum to frontline drugs in recent years has markedly increased malaria-related deaths in South Asian Countries. Ribulose 5-phosphate and NADPH synthesized by Pentose Phosphate Pathway (PPP) act as a direct precursor for nucleotide synthesis and P. falciparum survival during oxidative challenges in the intra-erythrocytic growth phase . In the present study, we have elucidated the structure and functional characteristics of 6-phosphogluconate dehydrogenase (6PGD) in P. falciparum and have identified potent hits against 6PGD by pharmacophore-based virtual screening with ZINC and ChemBridge databases. Molecular docking and Molecular dynamics simulation, binding free energies (MMGBSA & MMPBSA), and Density Functional Theory (DFT) calculations were integratively employed to validate and prioritize the most potential hits. The 6PGD structure was found to have an open and closed conformation during MD simulation. The apo form of 6PGD was found to be in closed conformation, while a open conformation attributed to facilitating binding of cofactor. It was also inferred from the conformational analysis that the small domain of 6PGD has a high influence in altering the conformation that may aid in open/closed conformation of 6PGD. The top three hits identified using pharmacophore hypotheses were ChemBridge_11084819, ChemBridge_80178394, and ChemBridge_17912340. Though all three hits scored a high glide score, MMGBSA, and favorable ADMET properties, ChemBridge_11084819 and ChemBrdige_17912340 showed higher stability and binding free energy. Moreover, these hits also featured stable H-bond interactions with the active loop of 6PGD with binding free energy comparable to substrate-bound complex. Therefore, the ChemBridge_11084819 and ChemBridge_17912340 moieties demonstrate to have high therapeutic potential against 6PGD in P. falciparum. Communicated by Ramaswamy H. Sarma

Evolutionarily conserved heat shock protein, HtpX, as an adjunct target against antibiotic-resistant Neisseria gonorrhoeae.

The emergence of multiple drug resistance and extreme drug resistance pathogens necessitates the continuous evaluation of the pathogenic genome to identify conserved molecular targets and their respective inhibitors. In this study, we mapped the global mutational landscape of Neisseria gonorrhoeae (an intracellular pathogen notoriously known to cause the sexually transmitted disease gonorrhoea). We identified highly variable amino acid positions in the antibiotic target genes like the penA, ponA, 23s rRNA, rpoB, gyrA, parC, mtrR and porB. Some variations are directly reported to confer resistance to the currently used front-line drugs like ceftriaxone, cefixime, azithromycinand ciprofloxacin. Further, by whole genome comparison and Shannon entropy analysis, we identified a completely conserved protein HtpX in the drug-resistant as well as susceptible isolates of N. gonorrhoeae (NgHtpX). Comparison with the only available information of Escherichia coli HtpX suggested it to be a transmembrane metalloprotease having a role in stress response. The critical zinc-binding residue of NgHtpX was mapped to E141. By applying composite high throughput screening followed by MD simulations, we identified pemirolast and thalidomide as high-energy binding ligands of NgHtpX. Following cloning and expression of the purified metal-binding domain of NgHtpX (NgHtpXd), its Zn2+ -binding (Kd = 0.4 µM) and drug-binding (pemirolast, Kd = 3.47 µM; and thalidomide, Kd = 1.04 µM) potentials were determined using in-vitro fluorescence quenching experiment. When tested on N. gonorrhoeae cultures, both the ligands imposed a dose-dependent reduction in viability. Overall, our results provide high entropy positions in the targets of presently used antibiotics, which can be further explored to understand the AMR mechanism. Additionally, HtpX and its specific inhibitors identified can be utilised effectively in managing gonococcal infections.

Fluorescent immunochromatographic test strip for therapeutic drug monitoring of methotrexate with high sensitivity and wide dynamic range.

As a front-line chemotherapeutic drug for maintenance and consolidation therapy, methotrexate (MTX) has widely been applied to treat various tumors and some inflammatory diseases. However, because of its severe toxicity ascribed to low selectivity, it is necessary to monitor therapeutic drugs in high-dose MTX therapeutic regimens to ensure treatment safety. In this work, we developed a fluorescent immunochromatographic test strip (FITS) for monitoring MTX by employing time-resolved fluorescent microspheres as signal probes. With a competitive immunoassay mode, the FITS for MTX shows a super-wide dynamic range of 10pM-10μM, covering the entire clinical therapeutic concentration range of MTX. Therapeutic drug monitoring of MTX can be achieved within 7min with high specificity, facilitating the timely rescue of drug poisoning led by high-dose MTX treatment. The methodwasemployedfor monitoring MTX in the spiked human serum, urine, and milk, showing acceptable recoveriesranging from 94.0 to 110.0%. The established FITS has been applied toMTX detection in serum obtained from high-dose MTX treatment. The results from FITS and enzyme multiplied immunoassay technique showed no significant difference, suggesting its reliability for usage in real biological samples. The device shows promise in point-of-caretherapeutic drug monitoring for resource-limited countries and institutes, which significantly facilitates overcoming the lag time between sampling and results.

Nanotherapeutic delivery of antibiotic cocktail enhances intra-macrophage killing of Mycobacterium marinum

Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis-like infections in cold-blooded animals and is an opportunistic pathogen in humans. M. marinum is the closest genetic relative of the Mycobacterium tuberculosis complex and is a reliable surrogate for drug susceptibility testing. We synthesized and evaluated two nanoparticle (NP) formulations for compatibility with rifampicin, isoniazid, pyrazinamide, and ethambutol (PIRE), the front-line antimycobacterial drugs used in combination against active tuberculosis infections. Improved in vitro antimicrobial activity was observed with encapsulated rifampicin alone or in a cocktail of drugs formulated through co-encapsulation in amphiphilic polyanhydride NPs. Broth antimicrobial testing revealed that the encapsulation of PIRE in NP resulted in a significant increase in antimicrobial activity, with the benefit over soluble formulations at biologically relevant concentrations ranging from &amp;gt;10 to &amp;gt;3,000 fold. M. marinum-infected human macrophages treated with NP-PIRE were cleared of viable bacteria in 48 h following a single treatment, representing a &amp;gt;4 log reduction in colony-forming units and a &amp;gt;2,000-fold increase in antimicrobial activity. The amphiphilic polyanhydride nanoparticles demonstrated the ability to co-encapsulate PIRE antibiotics and enhance their antimicrobial activity against M. marinum in infected macrophages in culture and in vitro. These data suggest that polyanhydride nanoparticles are a promising nanotherapeutic for combatting Mycobacterium infections through improved intracellular targeting of encapsulated antibiotics.

Crystal structure of dihydrofolate reductase from the emerging pathogenic fungus Candida auris.

Candida auris has emerged as a global health problem with a dramatic spread by nosocomial transmission and a high mortality rate. Antifungal therapy for C.auris infections is currently limited due to widespread resistance to fluconazole and amphotericin B and increasing resistance to the front-line drug echinocandin. Therefore, new treatments are urgently required to combat this pathogen. Dihydrofolate reductase (DHFR) has been validated as a potential drug target for Candida species, although no structure of the C. auris enzyme (CauDHFR) has been reported. Here, crystal structures of CauDHFR are reported as an apoenzyme, as a holoenzyme and in two ternary complexes with pyrimethamine and cycloguanil, which are common antifolates, at near-atomic resolution. Preliminary biochemical and biophysical assays and antifungal susceptibility testing with a variety of classical antifolates were also performed, highlighting the enzyme-inhibition rates and the inhibition of yeast growth. These structural and functional data might provide the basis for a novel drug-discovery campaign against this global threat.