Polydispersity-mediated high efficacy of an in-situ aqueous nanosuspension of PPEF.3HCl in methicillin resistant Staphylococcus aureus sepsis model.

Recently, World Health Organization declared antimicrobial resistance as the third greatest threat to human health. Absence of known cross-resistance, new class, new target, and a new mode of action are few major strategies being undertaken by researches to combat multidrug resistant pathogen. PPEF.3HCl, a bisbenzimidazole was developed as highly potent antibacterial agent against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, targeting topoisomerase IA. The present work encompasses a radical on-site generation of In-situ nanosuspension of PPEF.3HCl with enhanced efficacy against methicillin resistant S. aureus in septicemia model. We have generated instantaneously a PPEF.3HCl nanosuspension (IsPPEF.3HCl-NS) by mixing optimized monophasic PPEF.3HCl preconcentrate in propylene glycol into an aqueous medium comprising tween 80 as stabilizer. The IsPPEF.3HCl-NS showed precipitation efficiency of > 90 %, average particle size < 500 nm, retained upto 5 h, a negative zeta potential and bi/trimodal particle size distribution. Differential scanning calorimetry, X-ray diffraction confirmed partial amorphization and transmission electron microscopy revealed spherical particles. IsPPEF.3HCl-NS was non-hemolytic and exhibited good stability in serum. More significantly, it exhibited a âˆ¼ 1.6-fold increase in macrophage uptake compared to free PPEF.3HCl in the RAW 264.7 macrophage cell line. Confocal microscopy revealed accumulation of IsPPEF.3HCl-NS within the lysosomal compartment and cell cytosol, proposing high efficacy. In terms of antimicrobial efficacy, IsPPEF.3HCl-NS outperforms free PPEF.3HCl against clinical methicillin sensitive and resistant S. aureus strains. In a pivotal experiment, IsPPEF.3HCl-NS exhibited over 83 % survival at 8 mg/kg.bw and an impressive reduction of âˆ¼ 4-5 log-fold in bacterial load, primarily in the kidney, liver and spleen of septicemia mice. IsPPEF.3HCl-NS prepared by the In-situ approach, coupled with enhanced intramacrophage delivery and superior efficacy, positions IsPPEF.3HCl-NS as a pioneering and highly promising formulation in the battle against antimicrobial resistance.

Design, synthesis, X-ray crystal structures, anticancer, DNA binding, and molecular modelling studies of pyrazole-pyrazoline hybrid derivatives.

We have designed and synthesized three pyrazole analogs (4, 5a, 5b), pyrazole-based chalcones (6a-6d) and (8a-8h), and N-formyl/acetyl 1,3,5-trisubstituted pyrazoline analogs (7a-7d), (9a-9d). FT-IR, 1H, 13C NMR, and mass spectrometry techniques were used to describe the structures of all the synthesized analogs. The single crystal X-ray method was used to identify the molecular structure of derivatives 4 and 5a. All synthesized analogs were screened by MTT assay on two cancer cell lines, the human lung cancer cell line (A549) and cervical cancer cell line (HeLa). Among all compounds, analog 9d demonstrates significant anticancer activity against HeLa (IC50 = 23.6 µM) and A549 (IC50 = 37.59 µM). The non-interactive interaction of active compound (9d) with Calf thymus DNA (Ct-DNA) has been investigated through various methods, such as UV-vis absorption, emission, cyclic voltammetry and circular dichroism. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical has been used to measure the antioxidant capacity of the pyrazoline derivative (9d). The outcomes showed that active analog has significant antioxidant activity. In addition, MD simulation of the EGFR tyrosine kinase protein-ligand complex was performed at a time scale of 100 ns. The MMGBSA data of ligand-protein complex are showed stable interactions up to 100 ns.

Bronsted Acid-Catalyzed Regioselective Carboxamidation of 2-Indolylmethanols with Isonitriles.

A regioselective direct carboxamidation reaction of 2-indolylmethanols with readily available isocyanoesters/isocyanides has been reported in this work. The reaction was catalyzed by Bronsted acid such as p-TsOH to deliver the benzylic regioselective amides in 67-86% yield under mild conditions. The developed methodology provides alternative access to traditional metal-free carboxamidation via C-C and C-O bond formation with high atom economy. Furthermore, the developed approach was diversified to synthesize chiral indole-2-carboxamide derivatives with a moderate enantiomeric excess (61-73% ee) using an (R)-chiral phosphoric acid.

Multistep Synthesis of Analogues of Remdesivir: Incorporating Heterocycles at the C-1' Position.

Studies suggest that the 1'ß-CN moiety in remdesivir sterically clashes with the Ser861 residue of the RNA-dependent-RNA polymerase (RdRp), causing a delayed chain termination in the RNA replication process. Replacing C1'ß-CN with 5-membered heterocycles such as tetrazoles, oxadiazoles, and triazoles can augment the inhibitory activity and pharmacokinetic profile of C-nucleotides. Synthesis of tetrazole-, triazole-, and oxadiazole-integrated C1' analogues of remdesivir was attempted using general synthetic routes. The final compounds 26, 28, and 29 did not inhibit viral replication; however, the synthetic intermediates, i.e., 27 and 50, exhibited an IC90 = 14.1 µM each. The trifluoromethyl-substituted 1,2,4-oxadiazole 59 showed an IC90 of 33.5 µM. This work adds to the growing evidence of the beneficial medicinal impact of C1,1'-disubstituted C-nucleotides.

Synthesis of C3,C6-Diaryl 7-Azaindoles via One-Pot Suzuki-Miyaura Cross-Coupling Reaction and Evaluation of Their HIV-1 Integrase Inhibitory Activity.

There is a continuing demand of new inhibitors of HIV-1 Integrase (HIV-1 IN) due to mutations of HIV-1. This study aims to develop the synthesis of 3,6-diaryl 7-azaindoles and introspect the role of aryl groups on the strand transfer (ST) inhibition of HIV-1 IN. An efficient and chemo-selective one-pot method is established for the synthesis of the unexplored diverse C3 → C6 diaryl 7-azaindoles starting from 6-chloro-3-iodo-N-protected 7-azaindoles. Here we report Pd2dba3/SPhos catalyzed synthesis of eight selective C3 monoaryl 7-azaindoles (10a-h) and eight C3,C6-diaryl 7-azaindoles (11a-f, 12a,b) with yields in the ranges of 67-93% and 43-88% respectively. The synthesized derivatives inhibit the strand transfer (ST) activity of HIV-1 IN enzyme at 10 µM dose with 11d and 11f exhibiting %ST inhibitions of 72% and 71%, respectively. SAR studies indicate the para-substitution on the C3 aryl ring and C6 aryl is essential for enhanced %ST inhibition. 11b,c, 11e-f, and 12b showed lower cytotoxicity (IC50 > 200 µM) against TZM-bl cells. Molecular docking of the diaryl 7-azaindoles and Raltegravir (RAL), to the PFV-integrase revealed favorable binding interactions.

Unraveling topoisomerase IA gate dynamics in presence of PPEF and its preclinical evaluation against multidrug-resistant pathogens.

Type IA topoisomerases maintain DNA topology by cleaving ssDNA and relaxing negative supercoils. The inhibition of its activity in bacteria prevents the relaxation of negative supercoils, which in turn impedes DNA metabolic processes leading to cell death. Using this hypothesis, two bisbenzimidazoles, PPEF and BPVF are synthesized, selectively inhibiting bacterial TopoIA and TopoIII. PPEF stabilizes the topoisomerase and topoisomerase-ssDNA complex, acts as an interfacial inhibitor. PPEF display high efficacy against ~455 multi-drug resistant gram positive and negative bacteria. To understand molecular mechanism of inhibition of TopoIA and PPEF, accelerated MD simulation is carried out, and results suggested that PPEF binds, stabilizes the closed conformation of TopoIA with -6Kcal/mol binding energy and destabilizes the binding of ssDNA. The TopoIA gate dynamics model can be used as a tool to screen TopoIA inhibitors as therapeutic candidates. PPEF and BPVF cause cellular filamentation and DNA fragmentation leading to bacterial cell death. PPEF and BPVF show potent efficacy against systemic and neutropenic mouse models harboring E. coli, VRSA, and MRSA infection without cellular toxicity.

Moonlighting proteins: beacon of hope in era of drug resistance in bacteria.

Moonlighting proteins (MLPs) are ubiquitous and provide a unique advantage to bacteria performing multiple functions using the same genomic content. Targeting MLPs can be considered as a futuristic approach in fighting drug resistance problem. This review follows the MLP trail from its inception to the present-day state, describing a few bacterial MLPs, viz., glyceraldehyde 3'-phosphate dehydrogenase, phosphoglucose isomerase glutamate racemase (GR), and DNA gyrase. Here, we carve out that targeting MLPs are the beacon of hope in an era of increasing drug resistance in bacteria. Evolutionary stability, structure-functional relationships, protein diversity, possible drug targets, and identification of new drugs against bacterial MLP are given due consideration. Before the final curtain calls, we provide a comprehensive list of small molecules that inhibit the biochemical activity of MLPs, which can aid the development of novel molecules to target MLPs for therapeutic applications.

Short designed peptide unfolding human telomeric G-quadruplex: mimicking the helicase function.

Human telomeric DNA can fold into G-quadruplex structures involving the interaction of four guanine bases in a square planar arrangement. The highly distinctive nature of quadruplex topologies suggests that they can act as novel therapeutic targets. In this study, we provide the evidence of human telomeric G4 destabilization in dilute and cell-mimicking molecular crowing conditions upon peptide binding. We have used three human telomeric sequences of different lengths. CD data showed that these sequences folded into anti-parallel G-quadruplex and CD intensity decreased significantly on increasing the peptide concentration. UV-thermal melting results showed significant decrease in hypochromicity due to formation of G4-peptide complex at 295 nm. Fluorescence data showed the quenching on titrating the peptide with human telomere G4. Electrophoretic mobility shift assay confirmed the unfolding of G4 structure. Cell viability was significantly reduced in the presence of QW5 peptide with IC50 values as 8.78 µM and 7.72 µM after 72 and 96 hours of incubation respectively. These results confirmed that QW5 peptide has an ability to bind and unfold to human telomeric G-quadruplex and hence might be the key modulator for targeting diseases having over-representation of G4 motifs and their destabilization will be helpful in increasing the efficiency of DNA replication, transcription or duplex reannealing.Communicated by Ramaswamy H. Sarma.

DMA, a Small Molecule, Increases Median Survival and Reduces Radiation-Induced Xerostomia via the Activation of the ERK1/2 Pathway in Oral Squamous Cell Carcinoma.

Survival, recurrence, and xerostomia are considerable problems in the treatment of oral squamous carcinoma patients. In this study, we investigated the role of DMA (5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)5″benzimidazoyl]benzimidazole) as a salivary gland cytoprotectant in a patient-derived xenograft mouse model. A significant increase in saliva secretion was observed in the DMA-treated xenograft compared to radiation alone. Repeated doses of DMA with a high dose of radiation showed a synergistic effect on mice survival and reduced tumor growth. The mean survival rate of tumor-bearing mice was significantly enhanced. The increased number of Ki-67-stained cells in the spleen, intestine, and lungs compared to the tumor suggests DMA ablates the tumor but protects other organs. The expression of aquaporin-5 was restored in tumor-bearing mice injected with DMA before irradiation. The reduced expression of αvß3 integrin and CD44 in DMA alone and DMA with radiation-treated mice suggests a reduced migration of cells and stemness of cancer cells. DMA along with radiation treatment results in the activation of the Ras/Raf/MEK/ERK pathway in the tumor, leading to apoptosis through caspase upregulation. In conclusion, DMA has strong potential for use as an adjuvant in radiotherapy in OSCC patients.

Ag NCs as a potent antibiofilm agent against pathogenic Pseudomonas aeruginosa and Acinetobacter baumannii and drug-resistant Bacillus subtilis by affecting chemotaxis and flagellar assembly pathway genes.

Biofilm infections are highly resistant to commercial antibiotics. Therefore, developing a potent agent against such drug-resistant bacterial infections is highly desirable. Here, we synthesized positively charged silver nanoclusters (Ag NCs) with a diameter of <2 nm, which were found to be very effective antibacterial and antibiofilm agents against tetracycline-resistant Bacillus subtilis and most importantly multidrug-resistant pathogenic strains of Pseudomonas aeruginosa and Acinetobacter baumannii. Ag NCs were able to both prevent and eradicate the biofilm formation very effectively. The antibiofilm activity can be significantly increased with α-amylase and/or DNase which degrade the structural components of biofilms. The antibiofilm activity of antibiotics gets considerably lowered due to poor penetration and the acidic microenvironment of biofilms. However, the potency of antibiotics gets significantly increased when applied with Ag NCs. Finally, RNA seq-based analysis has demonstrated that the biofilm degradation was likely due to the regulation of bacterial chemotaxis and flagellar assembly pathway genes by Ag NCs.

Pixantrone confers radiosensitization in KRAS mutated cancer cells by suppression of radiation-induced prosurvival pathways.

Radioresistance towards radiation therapy has generated the need for the development of radiosensitizers as a potential drug. KRAS mutation brings radioresistance in tumor cells. The present work proves sensitization of cancer cells towards radiotherapy through inhibition of KRAS activation. Acquiring a drug repurposing approach, the in-silico screening revealed that pixantrone, an antineoplastic drug, possesses a high affinity towards KRAS G12C and G12D subtypes. The SPR study suggests that maximum affinity of pixantrone was observed with KRAS G12C>WT>G12D and G12S. Pixantrone potentially inhibited the KRAS activation in stable transfectants G12C and G12D cell lines and radiosensitized distinct KRAS mutant subtype cells. The combination of pixantrone with radiation causes enhanced dsDNA breaks along with enhanced ATM expression, and increased late apoptosis. The preclinical studies on NCr-fox1nu xenograft mice showed potent inhibition of tumor progression and prolonged survival of mcie due to the radiosensitizing effect of pixantrone. Radiation-induced activation of key effector proteins of RAS downstream pathways, like MAPK and PI3K/Akt/mTOR pathways, were downregulated in tumor cells upon combination treatment. Interestingly, a robust upregulation of senescence marker p21 was observed in the tumor cells in combination treatment. These findings reveal a convergence between KRAS signaling, pixantrone treatment, and radiation conferring tumor cell death.

Unraveling molecular mechanisms of head and neck cancer.

Malignancies that develop from mucosal epithelium of the upper aerodigestive tract are known as head and neck squamous cell carcinomas (HNSCC). Heterogeneity, late stage diagnosis and high recurrence rate are big hurdles in head and neck treatment regimen. Presently, the biomarkers available for diagnosis and prognosis of HNSCC are based on smoking as the major risk habit. This review shed light on the differential environment of HNSCC in smokeless tobacco consuming Indian patients. Frequent mutation in genes involved in DNA repair pathway (p53), cell proliferation (PIK3CA, HRAS) and cell death (CASP8, FADD) are common in western population. On the contrary, the genes involved in metastasis (MMPs, YAP1), lymphocyte proliferation (TNFRSF4, CD80), cell-cell adhesion (DCC, EDNRB), miRNA processing (DROSHA) and inflammatory responses (TLR9, IL-9) are mutated in Indian HNSCC patients. Gene ontology enrichment analysis highlighted that responses to chemical stimulus, immune pathways and stress pathways are highly enriched in Indian patients.

Development of Azaindole-Based Frameworks as Potential Antiviral Agents and Their Future Perspectives.

The azaindole (AI) framework continues to play a significant role in the design of new antiviral agents. Modulating the position and isosteric replacement of the nitrogen atom of AI analogs notably influences the intrinsic physicochemical properties of lead compounds. The intra- and intermolecular interactions of AI derivatives with host receptors or viral proteins can also be fine tuned by carefully placing the nitrogen atom in the heterocyclic core. This wide-ranging perspective article focuses on AIs that have considerable utility in drug discovery programs against RNA viruses. The inhibition of influenza A, human immunodeficiency, respiratory syncytial, neurotropic alpha, dengue, ebola, and hepatitis C viruses by AI analogs is extensively reviewed to assess their plausible future potential in antiviral drug discovery. The binding interaction of AIs with the target protein is examined to derive a structural basis for designing new antiviral agents.

Synthesis and Biological Evaluation of Novel 1H-Benzo[d]imidazole Derivatives as Potential Anticancer Agents Targeting Human Topoisomerase I.

Small molecules that modulate biological functions are targets of modern-day drug discovery efforts. A new series of novel 1H-benzo[d]imidazoles (BBZs) were designed and synthesized with different functional groups at the phenyl ring and variable lengths of the alkyl chain at the piperazine end as anticancer agents. We identified human topoisomerase I (Hu Topo I) as a probable target of these molecules through a computational study and DNA relaxation assay, a functional assay of the Hu Topo I enzyme. UV absorption, fluorescence, and circular dichroism spectroscopy were used to study interactions between BBZ and DNA. Out of 16 compounds, 11a, 12a, and 12b showed strong binding affinity and thermal stabilization of AT sequence-specific DNA. BBZs were screened against a panel of 60 human cancer cell lines at National Cancer Institute, USA. Most potent molecules 11a, 12a, and 12b showed 50% growth inhibition (GI50) in a concentration range from 0.16 to 3.6 µM cancer cells. Moreover, 12b showed 50% inhibition of the relaxation of DNA by Hu Topo I at 16 µM. Furthermore, flow cytometry revealed that 11a, 12a, and 12b cause prominent G2M arrest of cancer cells. In view of the above, we propose that 12b deserves to be further evaluated for its therapeutic use as an anticancer agent.

Prochlorperazine enhances radiosensitivity of non-small cell lung carcinoma by stabilizing GDP-bound mutant KRAS conformation.

Lung cancer is considered as leading cancer with the highest mortality. The KRAS-oncogenic mutations are dominant in lung carcinoma leading to poor prognosis and radioresistance, which is a major impediment to radiotherapy. Thus, KRAS mutant inhibitors that synergistically sensitize tumours to radiation are urgently needed. In pursuance of the search for a novel radiosensitizer, high-throughput screening of FDA-approved drugs was performed at active site of K-Ras. Prochlorperazine (PCZ), an antipsychotic drug, showed good binding affinity with KRAS-mutant proteins. PCZ binds to the GTP-binding pocket of KRAS-mutant protein and inhibits its constitutive activation by stabilizing the GDP-bound conformation of K-Ras mutants by 9 kcal/mol compared to WT. PCZ alongwith radiation decreased the clonogenic survival of KRAS-mutant NSCLC but not KRAS-WT cells. The combination treatment activates p-ATM, p53, and p21 proteins, leading to cell cycle arrest. PCZ with increasing radiation caused a linear increase in γH2AX foci, suggesting enhanced DSBs-associated apoptosis in radioresistant A549 cells. Pharmacokinetics study showed Cmax = 526 ng/ml at 30min, 4.6h half-life in plasma, and highest accumulation in tumours. PCZ and 10Gy irradiation synergistically radiosensitize mice xenografts via downregulation of Ras/Raf/MEK/ERK pathway. Our efforts have led to the discovery of PCZ as a lead compound. In preclinical analyses, treatment with PCZ alone and in combination with radiation led to regression of KRAS-G12S tumours.

Current Insights and Advancements in Head and Neck Cancer: Emerging Biomarkers and Therapeutics with Cues from Single Cell and 3D Model Omics Profiling.

Head and neck cancer (HNC) is among the ten leading malignancies worldwide, with India solely contributing one-third of global oral cancer cases. The current focus of all cutting-edge strategies against this global malignancy are directed towards the heterogeneous tumor microenvironment that obstructs most treatment blueprints. Subsequent to the portrayal of established information, the review details the application of single cell technology, organoids and spheroid technology in relevance to head and neck cancer and the tumor microenvironment acknowledging the resistance pattern of the heterogeneous cell population in HNC. Bioinformatic tools are used for study of differentially expressed genes and further omics data analysis. However, these tools have several challenges and limitations when analyzing single-cell gene expression data that are discussed briefly. The review further examines the omics of HNC, through comprehensive analyses of genomics, transcriptomics, proteomics, metabolomics, and epigenomics profiles. Patterns of alterations vary between patients, thus heterogeneity and molecular alterations between patients have driven the clinical significance of molecular targeted therapies. The analyses of potential molecular targets in HNC are discussed with connotation to the alteration of key pathways in HNC followed by a comprehensive study of protein kinases as novel drug targets including its ATPase and additional binding pockets, non-catalytic domains and single residues. We herein review, the therapeutic agents targeting the potential biomarkers in light of new molecular targeted therapies. In the final analysis, this review suggests that the development of improved target-specific personalized therapies can combat HNC's global plight.

Recent approaches to the synthesis of tetrahydrocarbazoles.

The tetrahydrocarbazole (THC) motif is ubiquitous in natural products and biologically active compounds. THCs can serve as favorable synthetic intermediates or precursors en-route to desired complex natural products. Despite a considerable number of strategies for the synthesis of THCs that have emerged in the last two decades, only a handful of reviews have been published on the subject. Herein, we have summarized synthetic methodologies published in the last ten years for the benefit of organic chemists. The review focuses on non-enantioselective syntheses of THCs, and encompasses ring opening reactions of donor-acceptor cyclopropanes, metal-catalyzed C-C/C-N bond formation, cycloaddition, conjugate addition, and miscellaneous reactions employed for accessing the THC framework.

Strategic Advances in Sequential C-Arylations of Heteroarenes.

Sequence-specific C-arylation strategies have important applications in medicinal and material research. These strategies allow C-C bond formations in a regioselective manner to synthesize large molecular libraries for studying structure-activity profiles. The past decade has seen the development of single C-C bond forming reactions using various transition-metal catalysts, cryogenic metalation strategies, and metal-free methods. Sequential arylations of heterocycles allow for the formation of multiaryl derivatives and are a preferred choice over de novo synthetic routes. This perspective sheds light on recent strategic advances to develop various sequential synthetic routes for the multiarylation of heteroarenes. This perspective addresses many challenges in optimizing sequential routes with respect to catalysts, reaction parameters, and various strategies adopted to obtain diversely arylated products.

Oxygen-releasing manganese clay hybrid complex triggers p53-mediated cancer cell death in hypoxia.

Hypoxia in tumor microenvironment is responsible for resistance to conventional modes of cancer therapeutics. A manganese-clay hybrid compound MHC was shown to generate molecular oxygen in aqueous solution. In this study we have shown that MHC, in hypoxia, causes cancer cell death, through release of molecular oxygen and via p53-dependent apoptosis. MHC treatment of cells results in depletion of mitochondrial membrane potential and inhibition of ROS production, in a cell-specific manner. In hypoxia, the oxygen from MHC releases cells from S-phase arrest thus causing p53-dependent apoptosis. The induction of apoptosis by MHC is higher in p53 Wt/Wt cells when it is compared with p53 Mt/Mt cells. The released oxygen from MHC triggers apoptosis via p53 activation through its enhanced homo-oligomerization, post-translational modifications and nuclear localization. Thus MHC as a cellular oxygen-releasing compound has high potential as a drug for hypoxic tumor regression.