Cladosporium halotolerans: Exploring an Unheeded Human Pathogen.

BACKGROUND: Cladosporium halotolerans is a saprobic fungus, rarely implicated in human infections. The identification is challenging due to non-specific phenotypic features. OBJECTIVE: To decipher clinical spectrum, microbiological and susceptibility profile of clinical and environmental isolates of Cladosporium halotolerans. METHOD: All the isolates identified as Cladosporium halotolerans deposited in National Culture Collection for Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research, Chandigarh, India were revived. Phenotypic and molecular characterization targeting internal transcribed spacer (ITS) region of ribosomal DNA, large subunit of ribosomal DNA (LSU; NL1 and NL4), actin (ACT) and beta-tubulin (TUB) was done. Scanning electron microscopy (SEM) was performed to determine any phenotypic variations. Antifungal susceptibility testing (AFST) was carried out for eight antifungal agents as per CLSI M38 Ed3 guidelines. We also performed systematic literature review of all the cases of Cladosporium halotolerans reported till date. RESULTS: A total of four isolates (clinical, n = 3; soil, n = 1) identified as Cladosporium halotolerans were included in the study. The clinical sites were skin, maxillary tissue and nail. All patients were apparently immunocompetent, and history of trauma was recorded in one patient. All patients improved on antifungal therapy. The cultures revealed growth of black mycelial fungus and microscopic examination demonstrated dematiaceous septate hyphae with erect conidiophores and conidia in branched acropetal chains. Based on molecular methods, all the four isolates were identified as C. halotolerans. SEM revealed no variation in length and width of the conidia, conidiophores, ramoconidium and hyphae among the isolates. All molecular targets, such as ITS region, LSU (partially sequenced), ACT and TUB were able to differentiate the isolates. Minimum inhibitory concentrations for antifungals were: triazoles (0.12-2 µg/ml), amphotericin B (4 µg/ml) and echinocandins (2-8 µg/ml). CONCLUSION: We report role of the rarely isolated dematiaceous fungus, C. halotolerans, in causing human infections. The study emphasizes the role of molecular methods in precisely identifying these species. Triazoles are more active against these black fungi compared to polyenes or echinocandins.

Synthesis of Thieno[3,2-d]pyrimidine Derivatives through Sequential SN Ar and Suzuki Reactions as Selective h-NTPDase Inhibitors.

In this study various of thieno[3,2-d]pyrimidine derivatives have been synthesized by treating different secondary amines through aromatic nucleophilic substitution reaction (SN Ar) followed by Suzuki reaction with aryl and heteroaryl boronic acids. A bis-Suzuki coupling was also performed to generate bis-aryl thienopyrimidine derivatives. The synthesized compounds were screened for the hydrolytic activity of h-NTPdase1, h-NTPdase2, h-NTPdase3, and h-NTPdase8. The compound N-benzyl-N-methyl-7-phenylthieno[3,2-d]pyrimidin-4-amine 3 j selectively inhibits the activity of h-NTPdase1 with IC50 value of 0.62±0.02 µM whereas, the compound 4 d was the most potent inhibitor of h-NTPdase2 with sub-micromolar IC50 value of 0.33±0.09 µM. Similarly, compounds 4 c and 3 b were found to be selective inhibitors for isozymes h-NTPdase3 (IC50 =0.13±0.06 µM) and h-NTPdase8 (IC50 =0.32±0.10 µM), respectively. The molecular docking study of the compounds with the highest potency and selectivity revealed the interactions with the important amino acid residues.

A patent review of pharmaceutical and therapeutic applications of oxadiazole derivatives for the treatment of chronic diseases (2013-2021).

INTRODUCTION: Oxadiazole is a unique class of heterocycle, possessing numerous important biomedical and therapeutic applications, such as anti-bacterial, anti-cancer, anti-inflammatory, inhibitors for diverse enzymes, receptor modulators, and neuroprotective properties. The rapid development in the field of oxadiazole-containing structures is confirmed by the development of numerous clinical drugs, such as doxazosin, nesapidil, pleconaril, fasiplon, ataluren, zibotentan, and prenoxdiazine as selected examples. AREAS COVERED: This review provides a comprehensive overview of the range of biological applications of oxadiazole-containing drugs in a range of patents from 2013 to 2021. The information was collected from available data sources including SciFinder, Reaxys, MedLine, and Chemical Abstracts. EXPERT OPINION: Oxadiazole is an established class of compounds with fascinating biological properties. The importance of oxadiazoles can be recognized by their enormous application in a wide spectrum of medicinal chemistry from anticancer, lantibiotics, and antidiabetics to their use in agriculture and neuroprotection. For instance, the oxadiazole-based compounds have shown the ability to modulate a variety of receptors including the M4 receptor agonists, S1P1 receptor modulators, SSTR5 antagonists, orexin type-2 receptor agonists, liver X receptor agonists, and many more. This testifies to the special features associated with the oxadiazole scaffold, making it a significant pharmacophore.

De novo pathway is an active metabolic pathway of cysteine synthesis in Haemonchus contortus.

Haemonchus contortus, commonly known as Barber's pole worm, is an economically important gastrointestinal nematode of sheep and goats especially in tropical and sub-tropical regions of the world. Cysteine synthesis is a very important metabolic pathway for the parasite, however the functional aspects of cysteine synthesis in parasite are largely unknown. The key question which we have investigated in the study is; whether the parasite uses a de novo pathway of cysteine synthesis, which is unknown in multicellular organisms of the animal kingdom and known to be absent in mammals. Directional cloning of the cysteine synthase (CS) gene was done in pET303 champion vector using restriction sites XbaI and XhoI. The CS gene of the H.contortus was closely related to CS-A protein of Oesophagostomum dentatum and a hypothetical protein of Ancylostoma ceylanicum. Recombinant protein of the H contortus CS (rHC-CS) gene was expressed using pET303 vector in pLysS BL21 strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. Western blot using anti-His tag antibody confirmed the presence of rHC-CS. Biochemical assay, FTIR and enzyme kinetics studies revealed that rHC-CS used O-acetyl serine as substrate to produce cysteine using de novo pathway and CS activity was also confirmed with the homogenate of H.contortus. Upregulation of CS transcripts in the adult and its downregulation in the L3 larval stage suggests that de novo pathway contributes to the cysteine requirement of mature H.contortus. It is concluded that de novo pathway is an active metabolic pathway in H.contortus.

A combine approach of chemical synthesis, biological evaluation and structural dynamics studies revealed thiazole substituted arylamine derivatives as potent FabH enzyme inhibitors.

Bacterial FabH enzyme is a broad-spectrum antimicrobial target and can be used in the design of novel antibiotics. This study reports chemical synthesis of thiazole based amine compounds as FabH inhibitors, followed by biological evaluation, and computational drug designing analysis with ultimate objective to guide further biological optimization of the identified hits. The compounds were synthesized through Pd-PEPPSI catalyzed cross coupling strategy for the Buchwald-Hartwig amination of thiazole-substituted aryl bromide. Pd-PEPPSI pre-catalysts were utilized for the cross couple with the diverse range of functionalized electron-deficient and electron-rich anilines and aliphatic amines. The thiazole based heteroaryl bromide coupling was found to be challenging and only specialized Pd-PEPPSI-IPr and Pd-PEPPSI-IPent catalysts were found to be effective providing the coupling product yield in the range of 78% to 99%. Biological investigation depicted compound 3f to be effective against Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermis, and Escherichia coli with mean + standard deviation value of 9.6 ± 0.4, 11.6 ± 0.4, 15.6 ± 0.4, and 11.6 ± 0.4, respectively. This compound is also active against free radicals with EC90 value of 39.45 µg/ml. Comparative docking predictions unravel the 3f binding mode at FabH active tunnel as such to block complete access for the natural substrate and involved balanced hydrogen and hydrophobic interactions. FabH-3f complex dynamics in solution found the docked conformation between the protein and compound of higher stability with mean carbon alpha deviation of 1.87 Å and mean residual deviation of 0.88 Å. Intermolecular interactions analysis depicted Asn274 from FabH active pocket to be significant in compound holding and strengthening of interaction as the simulation progresses. This was supported further by radial distribution function (RDF) and axial frequency distribution (AFD) that demonstrated the high distribution of compound atoms in close proximity of Asn274 residue and decrease in interaction distance. Further, the docking and simulation findings were validated through MMPB/GBSA methods that complements the compound affinity for the said target. In a nutshell, the identified hit could be subjected to structure, biological and pharmacokinetic optimization for development of effective FabH inhibitors.

Synthesis of biphenyl oxazole derivatives via Suzuki coupling and biological evaluations as nucleotide pyrophosphatase/phosphodiesterase-1 and -3 inhibitors.

Oxazole derivatives are important medicinal compounds which are inhibitors of various enzymes such as NPP1, NPP2, NPP3, tyrosine kinase, dipeptidyl-peptidase IV, cyclooxygenase-2, and protein tyrosine phosphatase. In this study, an extensive range of new biologically active biphenyl oxazole derivatives was synthesized in high to excellent yields (57-93%) through Suzuki-Miyaura cross-coupling of bromophenyloxazole with different boronic acids. The reaction was carried out in wet toluene under mild conditions. Overexpression of nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and NPP3 has been associated with various health disorders including chondrocalcinosis, cancer, osteoarthritis, and type 2 diabetes. We evaluated the inhibitory potential and selectivity of the synthesized compounds (3a-3q) towards NPP1 and NPP3 at 100 µM concentrations. We found two compounds that were selective and potent inhibitors of these two enzymes on the artificial substrate thymidine 5'-monophosphate para-nitrophenyl ester: compound 3n inhibited NPP1 with an IC50 of 0.15 µM, and compound 3f inhibited NPP3 with an IC50 value of 0.17 µM. The compounds with promising inhibitory potential were docked inside the proteins of NPP1 and NPP3 isozymes to get insight into the plausible binding interactions with active site residues.

In silico homology modelling and prediction of novel epitopic peptides from P24 protein of Haemonchus contortus.

Haemonchus contortus (HC) causes Haemonchosis in sheep and goats, with high mortality and morbidity due to lack of effective vaccine and increasing resistance to anthelmintic drugs. The present study was aimed at developing the 3D model of HCP24 protein and to identify the candidate epitopic peptides for effective humoral and cell-mediated immune-response. The HCP24 protein was homology modelled using the Swiss server and developed model was validated by ERRAT, VERIFY3D, PROQ, RAMPAGE and PROCHECK servers. Linear and prominent antigenic epitopes were predicted by SVMTrip and Immuno-medicine group tool. Conformational B-cell epitopes were predicted by Ellipro. MHC-I and MHC-II binding peptides were predicted by MHCPRED2, MHC2PRED and Propred I server. Proteosomal cleavage sites were predicted by Netchop server, to assess the stability of peptides. Reverse and three frame translation was done by EMBOSS tool. Bepipred and IEDB analysis also confirmed that both the predicted peptides (pep-1 and pep-2) were important antigenic region but pep-1 should have better hydrophobicity and stability. The degree of confidence achieved on scientific validation of the generated 3D model of the protein allows us to prescribe its use for research purpose. We could determine the peptide Pep-1(EDCKCTNCVCSRDEAL) should be a conformational B cell epitope with high antigenic potential and should demonstrate good binding affinity with host MHC-II and MHC-I alleles as well as stability inside host. Thus, it could be an ideal vaccine candidate for developing sub-unit vaccine against the parasite and should be assessed for protective immune response by in vitro and in-vivo studies.

Design, synthesis and bioevaluation of tricyclic fused ring system as dual binding site acetylcholinesterase inhibitors.

Due to recently discovered non-classical acetylcholinesterase (AChE) function, dual binding-site AChE inhibitors have acquired a paramount attention of drug designing researchers. The unique structural arrangements of AChE peripheral anionic site (PAS) and catalytic site (CAS) joined by a narrow gorge, prompted us to design the inhibitors that can interact with dual binding sites of AChE. Eighteen homo- and heterodimers of desloratadine and carbazole (already available tricyclic building blocks) were synthesized and tested for their inhibition potential against electric eel acetylcholinesterase (eeAChE) and equine serum butyrylcholinesterase (eqBChE). We identified a six-carbon tether heterodimer of desloratadine and indanedione based tricyclic dihydropyrimidine (4c) as potent and selective inhibitor of eeAChE with IC50 value of 0.09 ±â€¯0.003 µM and 1.04 ±â€¯0.08 µM (for eqBChE) with selectivity index of 11.1. Binding pose analysis of potent inhibitors suggest that tricyclic ring is well accommodated into the AChE active site through hydrophobic interactions with Trp84 and Trp279. The indanone ring of most active heterodimer 4b is stabilized into the bottom of the gorge and forms hydrogen bonding interactions with the important catalytic triad residue Ser200.