Protective efficacy of nafronyl in diabetic retinopathy through targeted inhibition of key enzymes.

Diabetic retinopathy is governed by abnormal apoptosis, increased capillary pressure, and other linked pathology that needs an efficient treatment by multitargeted approaches. Thus, the current study aimed to explore the potential of inhibition of targeted enzymes (DPP4, ACE-2, and aldose reductase) and free radical scavenging capabilities of selected compounds (nafronyl or naftidrofuryl) through in silico and in vivo investigations. Significant binding energies were observed in complexes of aldolase reductase, angiotensin type 1 receptor, and DPP4 against the nafronyl and sitagliptin more than -7.5 kcal/mol. Further validation of free energy was confirmed by calculations of molecular mechanics Poisson-Boltzmann surface area (MMPBSA), and configurational stabilities examined by PCA (principal component analysis). Additionally, drug-likeness was examined by the Swiss ADME web tool, which showed significant findings. Consequently, in vivo experimentations showed significant inflammation and alterations in retinal layers of inner plexiform (inner limiting membrane, nerve fibers, and ganglionic cells), inner nuclear layer (bipolar cells and horizontal cells), and photoreceptors cells. Whereas the treatments (nafronyl and sitagliptin) caused significant improvements in the histoarchitecture of the retina. Additionally, the HOMA indices (IR-insulin resistance, sensitivity, and ß cells functioning) and levels of free radicals were significantly altered in the diabetic control group in comparison to intact control. Nafronyl administration showed significant ameliorations in HOMA indices as well as antioxidant levels. Based on the results, it can be concluded that nafronyl efficiently interacts with target enzymes, which may result in potent inhibition and ameliorations in retinal histology as well as glucose homeostasis and antioxidants.

Antimicrobial effect of pimozide by targeting ROS-mediated killing in Staphylococcus aureus.

In spite of the higher nosocomial and community-acquired infections caused by Staphylococcus aureus, emerging drug resistance is a leading cause of increased mortality and morbidity associated with the overuse of antimicrobials. It is an emergent need to find out new molecules to combat such infections. In the present study, we analyzed the antibacterial effect of pimozide (PMZ) against gram-positive and gram-negative bacterial strains, including methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) S. aureus. The growth of MSSA and MRSA was completely inhibited at concentrations of 12.5 and 100 µg/mL, respectively, which is referred to as 1× minimum inhibitory concentration (MIC). The cell viability was completely eliminated within 90 min of PMZ treatment (2× MIC) through reactive oxygen species (ROS)-mediated killing without affecting cell membrane permeability. It suppressed α-hemolysin production and biofilm formation of different S. aureus strains by almost 50% at 1× MIC concentration, and was found to detach matured biofilm. PMZ treatment effectively eliminates S. aureus infection in Caenorhabditis elegans and improves its survival by 90% and is found safe to use with no hemolytic effect on human and chicken blood tissues. Taken together, it is concluded that PMZ may turn out to be an effective antibacterial for treating bacterial infections including MSSA and MRSA.

Mucormycosis threats: A systemic review.

During the catastrophic wave of Coronavirus disease 2019, health agencies started to report an infrequent but lethal mucormycosis or black fungal infection. Primarily, it causes sinusitis by affecting nasal, oral, lung, brain, ocular, and other body tissues. It becomes more fatal, especially in diabetic, cancer, and immune-compromised patients. Before 2020, the prevalence of mucormycosis was very rare but it has rapidly emerged globally from late 2020 to mid-2021. Recently, the mucormycosis got worse and epidemic with more than 30,000 cases reported across India. The etiology of infection can be diagnosed by molecular, serological, microscopic, and clinical methods. However, early diagnosis of this ailment is still a challenging task due to no standalone diagnostic tool available along with clinical manifestations of the ailment resembling other fungal diseases. The treatment of mucormycosis is also challenging and frequently requires long-term treatment. Amphotericin B was found to be an effective antifungal for preventing mucormycosis but it failed if infection disseminated to necrotizing tissues or adjacent organs. Removal of infected tissue/organ by surgery is an alternative treatment to control mucormycosis. In addition, reversal of underlying predisposing conditions based on therapy is also in practice for its prevention. This review highlights different aspects of mucormycosis such as pathogenesis, diagnosis, treatment, and their challenges and so on. We also emphasized the epidemiological shift during the recent outbreak and its influence on the different regions of India.

Green synthesis and characterization of silver nanoparticles using Pteris vittata extract and their therapeutic activities.

The bacterial infections have been substantially increasing with higher mortality and new regimens required for their management. The present work deals with the green synthesis of silver nanoparticles (AgNPs) using leaf extract of Pteris vittata at pH 9.0. The AgNPs showed a single absorption peak at 407 nm. The morphology of AgNPs was found to be spherical in shape analyzed by scanning electron micrographs. The X-ray diffraction studies revealed the face-centered cubic structure of AgNPs with a 17-nm average crystallite size. They showed the antimicrobial activity against Pseudomonas aeruginosa, and the cell growth was completely ceased at the minimum inhibitory concentration (MIC); 100 µg/mL, with rapidly decreased cell viability. This bactericidal effect was due to the enhancement of cell permeability caused by cell disruption. The AgNPs lead to show a promising antiquorum-sensing activity by inhibition of toxin protease and pyocyanin in P. aeruginosa by 88% and, 94% respectively, at the sub-MIC concentration (0.25× MIC). These results conclude that the green synthesis of AgNPs shows a promising antimicrobial and antivirulence activity against P. aeruginosa.

Information theoretic measures and mutagenesis identify a novel linchpin residue involved in substrate selection within the nucleotide-binding domain of an ABCG family exporter Cdr1p.

ABC transporters are membrane-bound pumps composed of two major domains: the transmembrane domain(s) (TMDs) and the nucleotide-binding domain(s) (NBDs). Sequence analyses of the NBDs of key ABC exporters revealed a residue position within the H-loop to be differentially conserved in the ABCG family, wherein there lies glutamine instead of positively charged arginine/lysine as in non-ABCG members. Consequently, contrasting NBD sequences of fungal Pleiotropic Drug Resistance transporters (PDR/ABCG) with that of Cholesterol/Phospholipid and Retinal (CPR/ABCA) Flippase family revealed a high Cumulative Relative Entropy (CRE) score of this residue position implying its family-specific functional significance. Further, substitution of the glutamine by arginine in both the NBDs of a representative PDR/ABCG member, (Candida drug resistance 1 protein) Cdr1p led to selective susceptibility of the Saccharomyces cerevisiae strains overexpressing the corresponding mutant proteins (Q362R and Q1060R) towards antifungal substrates without any impact on the ATPase activity. Consistent with the findings from previous studies on H-loop motif of fungal PDR transporters, the current report points towards a role of the glutamine residue within both canonical and divergent H-loop of Cdr1p in conferring substrate selection in a precisely identical manner.

Functional characterization of host toxic EcdB transcription factor protein of echinocandin B biosynthetic gene cluster.

The ecdB is a transcription factor, located in the echinocandin B biosynthetic gene cluster of Emericella rugulosa NRRL11440. Here, we validated the ecdB mRNA sequence for functional expression and to explore the role of EcdB protein in the echinocandin B regulation. The sequence alignment study revealed that the ecdB coding sequence was found 75 bp shorter than the reference mRNA sequence. This coding sequence encodes for EcdB protein and comprises three conserved domains; DNA binding domain (DBD), coiled-coil domain, and signature middle homology region. The full-length and DBD (truncated) DNA sequences were expressed in Escherichia coli BL21(DE3) under different tested conditions. The expression of EcdB protein was found to be toxic, which curbs the cell growth. In contrast to truncated protein (GST:EcdB1-54), the full-length (GST:EcdB) protein was expressed at very low titer and not detectable in SDS-PAGE under the varying isopropyl ß-d-1-thiogalactopyranoside (IPTG), temperature, and media conditions. However, GST:EcdB1-54 was successfully purified under standard conditions (0.5 mM IPTG at 0.5OD) with 33 kDa expected size. The functionality of GST:EcdB1-54 was attained by electrophoretic mobility shift assay study as a clear band shifting showed with ecdA promoter. Taken together, we conclude that EcdB interacts with the ecdA promoter that reflected to require for echinocandin B regulation.

Ouabain potentiates the antimicrobial activity of aminoglycosides against Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a notorious pathogen which often causes nosocomial and community attained infections. These infections steadily increased after evolving the resistance due to indecorous practice of antibiotics and now become a serious health issue. Ouabain is a Na+/K+-ATPase inhibitor that leads to increase the heart contraction in patients with congestive heart failure. METHODS: In the present study, in vitro antimicrobial effect of ouabain together with aminoglycosides was determined against clinical and non-clinical S. aureus strains. Using checkerboard, Gentamycin uptake and biofilm assays, we analysed he interactions of ouabain with aminoglycosides. RESULTS: Ouabain induced the staphylocidal potency of aminoglycosides by remarkably reducing the MIC of gentamycin (GEN) by 16 (0.25 µg/mL), 8 folds (0.5 µg/mL) amikacin (AMK); and 16 folds (1.0 µg/mL) with kanamycin (KAN), compared to their individual doses. OBN severely reduced cell viability within 60 min with GEN (1 µg/mL), KAN (2 µg/mL) and 90 min with AMK (1 µg/mL). This bactericidal effect was enhanced due to GEN uptake potentiated by 66% which led to increase the cell permeability as revealed by leakage of bacterial ATP and nitrocefin assay. The biofilm adherence disrupted by 80 and 50% at 5 mg/mL and 1.5 mg/mL OBN and 50 and 90% biofilm formation was inhibited at 5 mg/mL (MBIC50) and 10 mg/mL (MBIC90), respectively. Moreover, OBN with GEN further induced biofilm inhibition by 67 ± 5% at pH 7.0. CONCLUSIONS: Taken together, we established that OBN synergizes the antimicrobial activity of aminoglycosides that induces cell killing due to intracellular accumulation of GEN by disturbing cell homeostasis. It may be proven an effective approach for the treatment of staphylococcal infections.

A key structural domain of the Candida albicans Mdr1 protein.

A major multidrug transporter, MDR1 (multidrug resistance 1), a member of the MFS (major facilitator superfamily), invariably contributes to an increased efflux of commonly used azoles and thus corroborates their direct involvement in MDR in Candida albicans. The Mdr1 protein has two transmembrane domains, each comprising six transmembrane helices, interconnected with extracellular loops and ICLs (intracellular loops). The introduction of deletions and insertions through mutagenesis was used to address the role of the largest interdomain ICL3 of the MDR1 protein. Most of the progressive deletants, when overexpressed, eliminated the drug resistance. Notably, restoration of the length of the ICL3 by insertional mutagenesis did not restore the functionality of the protein. Interestingly, most of the insertion and deletion variants of ICL3 became amenable to trypsinization, yielding peptide fragments. The homology model of the Mdr1 protein showed that the molecular surface-charge distribution was perturbed in most of the ICL3 mutant variants. Taken together, these results provide the first evidence that the CCL (central cytoplasmic loop) of the fungal MFS transporter of the DHA1 (drug/proton antiporter) family is critical for the function of MDR. Unlike other homologous proteins, ICL3 has no apparent role in imparting substrate specificity or in the recruitment of the transporter protein.

2-Deoxy-D-Glucose: A Novel Pharmacological Agent for Killing Hypoxic Tumor Cells, Oxygen Dependence-Lowering in Covid-19, and Other Pharmacological Activities.

The nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) has shown promising pharmacological activities, including inhibition of cancerous cell growth and N-glycosylation. It has been used as a glycolysis inhibitor and as a potential energy restriction mimetic agent, inhibiting pathogen-associated molecular patterns. Radioisotope derivatives of 2-DG have applications as tracers. Recently, 2-DG has been used as an anti-COVID-19 drug to lower the need for supplemental oxygen. In the present review, various pharmaceutical properties of 2-DG are discussed.

Unlocking therapeutic potential: integration of drug repurposing and immunotherapy for various disease targeting.

Drug repurposing, also known as drug repositioning, entails the application of pre-approved or formerly assessed drugs having potentially functional therapeutic amalgams for curing various disorders or disease conditions distinctive from their original remedial indication. It has surfaced as a substitute for the development of drugs for treating cancer, cardiovascular diseases, neurodegenerative disorders, and various infectious diseases like Covid-19. Although the earlier lines of findings in this area were serendipitous, recent advancements are based on patient centered approaches following systematic, translational, drug targeting practices that explore pathophysiological ailment mechanisms. The presence of definite information and numerous records with respect to beneficial properties, harmfulness, and pharmacologic characteristics of repurposed drugs increase the chances of approval in the clinical trial stages. The last few years have showcased the successful emergence of repurposed drug immunotherapy in treating various diseases. In this light, the present review emphasises on incorporation of drug repositioning with Immunotherapy targeted for several disorders.

Divergent signature motifs of nucleotide binding domains of ABC multidrug transporter, CaCdr1p of pathogenic Candida albicans, are functionally asymmetric and noninterchangeable.

Nucleotide binding domains (NBDs) of the multidrug transporter of Candida albicans, CaCdr1p, possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved signature motifs, while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site-directed mutagenesis, we made three categories of mutant variants. These included mutants where all the signature motif residues were replaced with either alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where signature motifs were swapped to have variants with either both the conserved or degenerated entire signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2 and swapped signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogation in ATPase and R6G efflux activities. However, some of the mutants displayed a selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show not only that certain conserved residues of NBD1 signature sequence (S304, G306, and E307) are important in ATP hydrolysis and R6G efflux but also that a few divergent residues (N1002 and E1004) of NBD2 signature motif have evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the signature motifs of CaCdr1p, whether it is divergent or conserved, are nonexchangeable and are functionally critical for ATP hydrolysis.

1,2,3-Triazole ß-lactam conjugates as antimicrobial agents.

A convenient and efficient synthesis of new triazole ß-lactam conjugates using click chemistry is described. ß-lactam 15 and 16 were prepared using cycloaddition strategy and propargylated at N-1 to afford compounds 17 and 18. Cu-catalyzed click reaction of these ß-lactams 17 and 18 with different aryl azides provided 1,2,3-triazole conjugates 6 and 7, respectively. The products were fully characterized spectroscopically and tested against Gram-(+) and Gram-(-) bacteria. Compound 7a and 7c were found to be most active.

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans.

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg2+ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg2+ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg2+ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the gamma-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.

Structural elucidation of transmembrane domain zero (TMD0) of EcdL: A multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporter protein revealed by atomistic simulation.

ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 µs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.

Fungal-derived xenobiotic exhibits antibacterial and antibiofilm activity against Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen, responsible for superficial and invasive infections both in nosocomial and community-acquired settings. The incidences of infection have become more problematic attributable to emerging drug resistance and biofilm formation. These challenges suggest the need for new antimicrobial agents against S. aureus. In present work, we purified a fungal xenobiotic (FI3) which elicits a potent antimicrobial activity against a list of tested microbes including methicillin sensitive (MSSA) and methicillin resistance (MRSA) S. aureus. The cell growth of MSSA and MRSA were completely ceased with the 1× minimum inhibitory concentration (MIC); 32 µg/mL and 128 µg/mL, respectively. The cell viability severely decreased within 90 min, due to disturbance of membrane homeostasis. This bactericidal effect was enhanced at lower pH (pH 4) with a speculation to retain positive charge. The FI3 potently disrupts biofilm adherence at 64 µg/mL and found to be a safe with no toxic effect on mammalian tissue. FI3 also leads to increase the potency of tested antibiotics. Taken together, we established that FI3 has a potent antimicrobial activity against tested microbes and safer to human tissue. It may be proven a leading molecule for the treatment of bacterial infections.

Functional redundancy in Echinocandin B in-cluster transcription factor ecdB of Emericella rugulosa NRRL 11440.

Echinocandin B is a potent antifungal against the majority of fungal pathogens and its biosynthesis occurred by ecd and hty gene clusters in Emericella rugulosa NRRL 11440. We elucidated the functional necessity of in-clustered transcription factor; ecdB in the production of echinocandin B. We deleted the ecdB gene and found that ΔecdB mutant has no significant effect on echinocandin B production. The expression level of most of the ecd and hty cluster genes was not significantly altered except few of them up-regulated in knockout strain. The complete abrogation in ecdB gene expression was observed in ΔecdB strain. However, the interactions of purified EcdB protein with DNA sequence of ecdA, ecdH, ecdK and ecdI promoter was confirmed in-vitro. Our results conclude that EcdB protein in-vitro binds to the ecdA, ecdH, ecdK and ecdI promoter but in-vivo, it could not significantly affect the gene expression and echinocandin B production in Emericella rugulosa.