An Electrochemical Oxo-amination of 2H-Indazoles: Synthesis of Symmetrical and Unsymmetrical Indazolylindazolones.

We have established a supporting-electrolyte free electrochemical method for the synthesis of indazolylindazolones through oxygen reduction reaction (eORR) induced 1,3-oxo-amination of 2H-indazoles where 2H-indazole is used as both aminating agent as well as the precursor of indazolone. Moreover, we have merged indazolone and indazole to get unsymmetrical indazolylindazolones through direct electrochemical cross-dehydrogenative coupling (CDC). This exogenous metal-, oxidant- and catalyst-free protocol delivered a number of multi-functionalized products with high tolerance of diverse functional groups.

DNAJB8 facilitates autophagic-lysosomal degradation of viral Vif protein and restricts HIV-1 virion infectivity by rescuing APOBEC3G expression in host cells.

HSP40/DNAJ family of proteins is the most diverse chaperone family, comprising about 49 isoforms in humans. Several reports have demonstrated the functional role of a few of these isoforms in the pathogenesis of various viruses, including HIV-1. Our earlier study has shown that several isoforms of HSP40 get significantly modulated at the mRNA level during HIV-1 infection in T cells. To explore the biological role of these significantly modulated isoforms, we analyzed their effect on HIV-1 gene expression and virus production using knockdown and overexpression studies. Among these isoforms, DNAJA3, DNAJB1, DNAJB7, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC22, and DNAJC30 seem to positively regulate virus replication, whereas DNAJB3, DNAJB6, DNAJB8, and DNAJC5 negatively regulate virus replication. Further investigation on the infectivity of the progeny virion demonstrated that only DNAJB8 negatively regulates the progeny virion infectivity. It was further identified that DNAJB8 protein is involved in the downregulation of Vif protein, required for the infectivity of HIV-1 virions. DNAJB8 seems to direct Vif protein for autophagic-lysosomal degradation, leading to rescue of the cellular restriction factor APOBEC3G from Vif-mediated proteasomal degradation, resulting in enhanced packaging of APOBEC3G in budding virions and release of less infective progeny virion particles. Finally, our results also indicate that during the early stage of HIV-1 infection, enhanced expression of DNAJB8 promotes the production of less infective progeny virions, but at the later stage or at the peak of infection, reduced expression of DNJAB8 protein allows the HIV-1 to replicate and produce more infective progeny virion particles.

Tetrahydropalmatine from medicinal plants activates human glucokinase to regulate glucose homeostasis.

Many synthetic glucokinase activators (GKAs), modulating glucokinase (GK), an important therapeutic target in diabetes have failed to clear clinical trials. In this study, an in silico structural similarity search with differing scaffolds of reference GKAs have been used to identify derivatives from natural product databases. Ten molecules with good binding score and similar interactions to that in the co-crystallized GK as well good activation against recombinant human GK experimentally were identified. Tetrahydropalmatine, an alkaloid present in formulations and drugs from medicinal plants, has not been explored as an antidiabetic agent and no information regarding its mechanism of action or GK activation exists. Tetrahydropalmatine activates GK with EC50 value of 71.7 ± 17.9 µM while lowering the S0.5 (7.1 mM) and increasing Vmax (9.22 µM/min) as compared to control without activator (S0.5 = 10.37 mM; Vmax = 4.8 µM/min). Kinetic data (α and ß values) suggests it to act as mixed, nonessential type activator. Using microscale thermophoresis, Kd values of 3.8 µM suggests a good affinity for GK. In HepG2 cell line, the compound potentiated the uptake of glucose and maintained glucose homeostasis by increasing the expression of GK, glycogen synthase, and insulin receptor genes and lowering the expression of glucokinase regulatory protein (GKRP) and glucagon. Tetrahydropalmatine at low concentrations could elicit a good response by reducing expression of GKRP, increasing expression of GK while also activating it. Thus, it could be used alone or in combination as therapeutic drug as it could effectively modulate GK and alter glucose homeostasis.

Exploring binding mode assessment of novel kaempferol, resveratrol, and quercetin derivatives with PPAR-α as potent drug candidates against cancer.

Peroxisome proliferator-activated receptors (PPAR)-α, a ligand-activated transcription factor stands out to be a valuable protein target against cancer. Given that ligand binding is the crucial process for the activation of PPAR-α, fibrate class of synthetic compounds serves as potent agonist for the receptor. However, their serious side effects limit the long-term application in cancer. This emphasizes the dire need to identify new candidates that would exert desired activation by abrogating the adverse effects caused by synthetic agonists. Natural dietary products serve as an important source of drug discovery. Hence, the present study encompasses the investigation of the role of natural plant phenolic compounds: kaempferol, resveratrol, and quercetin and their 8708 derivatives by the means of computational pipeline comprising molecular docking and molecular dynamic (MD) simulation techniques. Docking calculations shortlisted potential candidates, namely 6-cinnamylchrysin (6-CC), resveratrol potassium-4-sulfate (RPS) and 6-[2-(3,4-Dihydroxyphenyl)-5-hydroxy-4-oxochromen-7-yl]oxyhexyl nitrate (DHOON), and derivatives of kaempferol, resveratrol, and quercetin, respectively. 6-CC, RPS, and DHOON manifested better affinities of - 32.83 kcal/mol (Ala333, Lys358, His440), - 27.22 kcal/mol (Tyr314, Met355), and - 30.18 kcal/mol (Ser280, Tyr314, Ala333), respectively, and were found to act as good stimulants for PPAR-α. Among these three compounds, 6-CC caused relatively least deviations and fluctuations analyzed through MD simulation which judiciously held responsible to attain most favorable interaction with PPAR-α. Followed by the binding free energy (ΔG) calculations using MM-GBSA confirmed the key role of 6-CC toward PPAR-α. The compound 6-CC also achieved high drug-likeness and pharmacokinetic properties. Thus, these findings stipulate new drug leads for PPAR-α receptor which abets a way to develop new anti-cancer drugs.

An in silico scientific basis for LL-37 as a therapeutic for Covid-19.

A multi-pronged approach with help in all forms possible is essential to completely overcome the Covid-19 pandemic. There is a requirement to research as many new and different types of approaches as possible to cater to the entire world population, complementing the vaccines with promising results. The need is also because SARS-CoV-2 has several unknown or variable facets which get revealed from time to time. In this work, in silico scientific findings are presented, which are indicative of the potential for the use of the LL-37 human anti-microbial peptide as a therapeutic against SARS-CoV-2. This indication is based on the high structural similarity of LL-37 to the N-terminal helix, with which the virus interacts, of the receptor for SARS-CoV-2, Angiotensin Converting Enzyme 2. Moreover, there is positive prediction of binding of LL-37 to the receptor-binding domain of SARS-CoV-2; this is the first study to have described this interaction. In silico data on the safety of LL-37 are also reported. As Vitamin D is known to upregulate the expression of LL-37, the vitamin is a candidate preventive molecule. This work provides the possible basis for an inverse correlation between Vitamin D levels in the body and the severity of or susceptibility to Covid-19, as widely reported in literature. With the scientific link put forth herein, Vitamin D could be used at an effective, medically prescribed, safe dose as a preventive. The information in this report would be valuable in bolstering the worldwide efforts to eliminate the pandemic as early as possible.

Novel B, C-ring truncated deguelin derivatives reveals as potential inhibitors of cyclin D1 and cyclin E using molecular docking and molecular dynamic simulation.

The overexpression of cyclin D1 and cyclin E due to their oncogenic potential and amplification has been associated with a higher mortality rate in many cancers. The deguelin is a natural compound, has shown promising anti-cancer activity by directly binding cyclin D1 and cyclin E and thus suppressing its function. The C7a atomic position of deguelin structure contains a proton that generates stabilized radical, as a result, decomposed deguelin reduces its structural stability and significantly decreases its biological activity. To design deguelin derivatives with the reduced potential side effect, series of B, C-ring truncated derivatives were investigated as cyclin D1 and cyclin E inhibitors. R-group-based enumeration was implemented in the deguelin scaffold using the R-group enumeration module of Schrödinger. Drug-Like filters like, REOS and PAINs series were applied to the enumerated compound library to remove compounds containing reactive functional groups. Further, screened compounds were docked within the ligand-binding cavity of cyclin D1 and cyclin E crystal structure, using Glide SP and XP protocol to obtain docking poses. Enrichment calculations were done using SchrÖdinger software, with 1000 decoy compounds (from DUD.E database) and 60 compounds (XP best poses) along with deguelin, to validate the docking protocol. The receiver operating characteristic (ROC) curve indicates R2 = 0.94 for cyclin D1 and R2 = 0.79 for cyclin E, suggesting that the docking protocol is valid. Besides, we explored molecular dynamics simulation to probe the binding stability of deguelin and its derivatives within the binding cavity of cyclin D1 and cyclin E structures which are associated with the cyclin D1 and cyclin E inhibitory mechanism.

Molecular engineering of an efficient four-domain DAF-MCP chimera reveals the presence of functional modularity in RCA proteins.

The complement system is highly efficient in targeting pathogens, but lack of its apposite regulation results in host-cell damage, which is linked to diseases. Thus, complement activation is tightly regulated by a series of proteins, which primarily belong to the regulators of complement activation (RCA) family. Structurally, these proteins are composed of repeating complement control protein (CCP) domains where two to four successive domains contribute to the regulatory functions termed decay-accelerating activity (DAA) and cofactor activity (CFA). However, the precise constitution of the functional units and whether these units can be joined to form a larger composition with dual function have not been demonstrated. Herein, we have parsed the functional units for DAA and CFA by constructing chimeras of the decay-accelerating factor (DAF) that exhibits DAA and membrane cofactor protein (MCP) that exhibits CFA. We show that in a four-CCP framework, a functional unit for each of the regulatory activities is formed by only two successive CCPs wherein each participates in the function, albeit CCP2 has a bipartite function. Additionally, optimal activity requires C-terminal domains that enhance the avidity of the molecule for C3b/C4b. Furthermore, by composing a four-CCP DAF-MCP chimera with robust CFA (for C3b and C4b) and DAA (for classical and alternative pathway C3 convertases), named decay cofactor protein, we show that CCP functional units can be linked to design a dual-activity regulator. These data indicate that the regulatory determinants for these two biological processes are distinct and modular in nature.

Fluorination of 2H-Indazoles Using N-Fluorobenzenesulfonimide.

An efficient, simple, and metal-free fluorination of 2H-indazoles has been developed using N-fluorobenzenesulfonimide (NFSI) in water under ambient air. This transformation provides direct access to fluorinated indazole derivatives with broad functionalities in satisfactory yields. The experimental results suggest a radical mechanistic pathway of this protocol.

Potassium Persulfate Mediated Chemodivergent C-3 Functionalization of 2H-Indazoles with DMSO as C1 Source.

A facile, efficient, and transition-metal-free chemodivergent C-3 functionalization of 2H-indazoles was developed under aerobic conditions using carboxylic acid and DMSO as the combined source of the carboxylic acid ester group and DMSO as the formylating agent. A series of formylated indazoles and carboxylic acid esters of indazole derivatives were produced in moderate to excellent yields. The mechanistic studies suggest that the reactions probably proceed through a radical pathway.

Reconstruction of transcriptional regulatory networks of Fis and H-NS in Escherichia coli from genome-wide data analysis.

Fis (Factor for inversion stimulation) and H-NS (Histone-like nucleoid-structuring protein) are two well-known nucleoid-associated proteins (NAPs) in proteobacteria, which play crucial roles in genome organization and transcriptional regulation. We performed RNA-sequencing to identify genes regulated by these NAPs. Study reveals that Fis and H-NS affect expression of 462 and 88 genes respectively in Escherichia coli at mid-exponential growth phase. By integrating available ChIP-seq data, we identified direct and indirect regulons of Fis and H-NS proteins. Functional analysis reveals that Fis controls expression of genes involved in translation, oxidative phosphorylation, sugar metabolism and transport, amino acid metabolism, bacteriocin transport, cell division, two-component system, biofilm formation, pilus organization and lipopolysaccharide biosynthesis pathways. However, H-NS represses expression of genes in cell adhesion, recombination, biofilm formation and lipopolysaccharide biosynthesis pathways under mid-exponential growth condition. The current regulatory networks thus provide a global glimpse of coordinated regulatory roles for these two important NAPs.

N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibits HIV-1 by suppressing the activity of viral protease.

Human Immunodeficiency Virus (HIV), the etiological agent for Acquired Immunodeficiency Syndrome (AIDS), continues to kill humans despite stupendous advances in antiviral research. With the presently available combination antiretroviral therapeutic arsenal, AIDS is now a manageable disease but with no cure available till date. The development of novel antivirals consumes an extensive amount of time and resources. Hence, repurposing of the established gold standard molecules for their anti-HIV application is enormously advantageous. In this study, we report that N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibits HIV-1 replication in a highly-conserved manner. Further, TPCK inhibits HIV-1 replication at the late stages of its life cycle by impeding viral protease (PR) enzyme activity. Additionally, our results demonstrate that the combination of TPCK with established HIV-1 PR inhibitors shows significant synergistic inhibitory potential, suggesting the potential use of TPCK in cART regimen. Collectively, we report the anti-HIV activity of TPCK, which should be further characterized for its translational applications.

Rhodium-Catalyzed Directed C(sp2)-H Bond Addition of 2-Arylindazoles to N-Sulfonylformaldimines and Activated Aldehydes.

Rhodium-catalyzed directed C-H functionalization of 2-arylindazoles with N-sulfonylformaldimines has been developed to provide a variety of N-benzylarylsulfonamide derivatives with good to excellent yields. Different activated aldehydes like ethyl glyoxalate and 2,2,2-trifluoroacetaldehyde also efficiently underwent nucleophilic addition with 2-arylindazoles. These selective transformations occur through the control of C3 nucleophilicity of indazole moiety. Mechanistic studies suggest that C-H activation step may be a rate-limiting step.

Rhodium(iii)-catalyzed ortho-C-H amidation of 2-arylindazoles with a dioxazolone as an amidating reagent.

A simple and efficient method for the directed amidation of a wide range of 2-arylindazoles has been established for the first time through a rhodium-catalyzed C-H activation reaction with alkyl, aryl and heteroaryl dioxazolones. A series of N-(2-(2H-indazol-2-yl)phenyl)acetamide derivatives were synthesized in excellent yields. A mechanistic study was conducted to describe C-H bond cleavage which is likely to be involved in the rate determining step.

Regioselective hydroarylation and arylation of maleimides with indazoles via a Rh(iii)-catalyzed C-H activation.

Switchable Rh(iii)-catalyzed highly regioselective hydroarylation and oxidative arylation of maleimides with 2-arylindazoles via C-H activation have been demonstrated. The reaction affords 3-(2-(2H-indazol-2-yl)phenyl)succinimide and 3-(2-(2H-indazol-2-yl)phenyl)maleimide derivatives in high yields with wide functional group tolerance. A mechanistic study was performed to depict C-H bond cleavage that might be involved in the turnover limiting step.

Genome analysis identifies a spontaneous nonsense mutation in ppsD leading to attenuation of virulence in laboratory-manipulated Mycobacterium tuberculosis.

BACKGROUND: A previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1. This phenotype was intriguing since the parental strain H37Rv, devR mutant (Mut1) and additional complemented strains, Comp9 and Comp11, were virulent in the guinea pig model. RESULTS: Towards deciphering the mechanism underlying the attenuation of Comp1, a whole genome sequencing approach was undertaken. Eight Single Nucleotide Polymorphisms (SNPs) unique to the Comp1 strain were identified. Of these, 5 SNPs were non-synonymous and included a G➞A mutation resulting in a W1591Stop mutation in ppsD gene of the phthiocerol dimycocerosate (PDIM) biosynthetic cluster. Targeted sequence analysis confirmed this mutation in only Comp1 strain and not in wild type (H37Rv), devR knockout (Mut1) or other complemented (Comp9 and Comp11) bacteria. Differential expression of the PDIM locus in Comp1 bacteria was observed which was associated with a partial deficiency of PDIM, an increased sensitivity to detergent and a compromised ability to infect human THP-1 cells. CONCLUSIONS: It is proposed that a spontaneous mutation in the ppsD gene of Comp1 underlies down-modulation of the PDIM locus which is associated with defects in permeability and infectivity as well as virulence attenuation in guinea pigs. Our study demonstrates the value of whole genome sequencing for resolving unexplainable bacterial phenotypes and recommends the assessment of PDIM status while assessing virulence properties of laboratory-manipulated strains of M. tuberculosis.

Comparison and storage study of ultra-filtered clarified jamun (Syzygium cumini) juice.

Ultrafiltration is one of the types of clarification process that was undertaken in this study together with examining and comparing the storage period of clarified juice by other methods (enzymatically treated, centrifugation, microfiltration). Centrifugation, ultrafiltration and microfiltration of jamun juice was carried out using a laboratory scale refrigerated centrifuge and filtration system. The juices obtained from various processes were evaluated on the basis of their physiochemical and microbial aspects over a period of 8 weeks. Enzyme treated and centrifuged juices were found to be degraded within 15-30 days while other juices had lesser changes in their properties. However microfiltered juice contained some yeasts and molds which increased with time. Ultrafiltration with 50 kDa pore size and 20 psi pressure was found to be the best method for clarification of jamun juice having a prolong shelf life with optimum qualities.

Cyclin F/FBXO1 Interacts with HIV-1 Viral Infectivity Factor (Vif) and Restricts Progeny Virion Infectivity by Ubiquitination and Proteasomal Degradation of Vif Protein through SCFcyclin F E3 Ligase Machinery.

Cyclin F protein, also known as FBXO1, is the largest among all cyclins and oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, cyclin F functions as the substrate-binding subunit of SCFcyclin F E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4+ T cells, we observed down-regulation of the cyclin F gene (CCNF). Later, using gene overexpression and knockdown studies, we identified cyclin F as negatively influencing HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that cyclin F negatively regulates the expression of viral protein Vif (viral infectivity factor) at the protein level. We also identified a novel host-pathogen interaction between cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a cyclin F-specific amino acid motif in the C-terminal region of Vif indicated rescue of the protein from cyclin F-mediated down-regulation. Subsequently, we showed that Vif is a novel substrate of the SCFcyclin F E3 ligase, where cyclin F mediates the ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we showed that cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate that cyclin F is a novel F-box protein that functions as an intrinsic cellular regulator of HIV-1 Vif and has a negative regulatory effect on the maintenance of viral infectivity by restoring APOBEC3G expression.

Metal-Free Trifluoromethylation of Indazoles.

A simple and efficient tert-butyl hydroperoxide-mediated direct trifluoromethylation of indazoles using sodium trifluoromethanesulfinate has been developed under metal-free conditions. A library of trifluoromethylated products with broad functionalities has been synthesized with moderate to good yields. A radical mechanistic pathway has been proposed for the present protocol.

Utility of Intermediate-Delay Washout CT Images for Differentiation of Malignant and Benign Adrenal Lesions: A Multivariate Analysis.

OBJECTIVE: The objective of this study was to identify features that impact the diagnostic performance of intermediate-delay washout CT for distinguishing malignant from benign adrenal lesions. MATERIALS AND METHODS: This retrospective study evaluated 127 pathologically proven adrenal lesions (82 malignant, 45 benign) in 126 patients who had undergone portal venous phase and intermediate-delay washout CT (1-3 minutes after portal venous phase) with or without unenhanced images. Unenhanced images were available for 103 lesions. Quantitatively, lesion CT attenuation on unenhanced (UA) and delayed (DL) images, absolute and relative percentage of enhancement washout (APEW and RPEW, respectively), descriptive CT features (lesion size, margin characteristics, heterogeneity or homogeneity, fat, calcification), patient demographics, and medical history were evaluated for association with lesion status using multiple logistic regression with stepwise model selection. Area under the ROC curve (Az) was calculated from both univariate and multivariate analyses. The predictive diagnostic performance of multivariate evaluations was ascertained through cross-validation. RESULTS: Az for DL, APEW, RPEW, and UA was 0.751, 0.795, 0.829, and 0.839, respectively. Multivariate analyses yielded the following significant CT quantitative features and associated Az when combined: RPEW and DL (Az = 0.861) when unenhanced images were not available and APEW and UA (Az = 0.889) when unenhanced images were available. Patient demographics and presence of a prior malignancy were additional significant factors, increasing Az to 0.903 and 0.927, respectively. The combined predictive classifier, without and with UA available, yielded 85.7% and 87.3% accuracies with cross-validation, respectively. CONCLUSION: When appropriately combined with other CT features, washout derived from intermediate-delay CT with or without additional clinical data has potential utility in differentiating malignant from benign adrenal lesions.

Differentiation of Malignant and Benign Adrenal Lesions With Delayed CT: Multivariate Analysis and Predictive Models.

OBJECTIVE: The purpose of this study is to identify imaging and patient parameters that affect the diagnostic performance of delayed contrast-enhanced CT for distinguishing malignant from benign adrenal lesions larger than 1 cm in adult patients and to derive predictive models. MATERIALS AND METHODS: This retrospective study assessed 97 pathologically proven adrenal lesions that had undergone unenhanced, portal venous, and 15-minute delayed CT. Quantitatively, single-parameter evaluations of lesion attenuation (in Hounsfield units) and absolute percentage enhancement washout (APEW) and relative percentage enhancement washout (RPEW) were performed. In addition, descriptive CT features (lesion size, margin definition, heterogeneity vs homogeneity, fat, and calcification) and patients' demographic characteristics and medical history of malignancy were evaluated for association with lesion status using multiple logistic regression with stepwise model selection. Areas under the ROC curve (Az) were determined for univariate and multivariate analyses. Leave-one-lesion-out cross-validation was applied to ascertain the predictive performance of single-parameter and multivariate evaluations. RESULTS: The Az values for unenhanced attenuation, portal venous attenuation, delayed attenuation, APEW, and RPEW were 0.835, 0.534, 0.847, 0.792, and 0.871, respectively. Multivariate analyses revealed that portal venous attenuation, delayed attenuation, and APEW were significant features, with an Az of 0.923 when combined. The addition of the descriptive CT features increased the Az to 0.938; patient age and a history of malignancy were additional significant factors, increasing the Az to 0.956 and 0.972, respectively. The combined predictive classifier yielded 89% accuracy under cross-validation, compared with the best commonly applied single-parameter evaluation (77% for RPEW < 40%). CONCLUSION: Multivariate imaging evaluation applied to delayed contrast-enhanced CT alone, with or without patient characteristics, improves diagnostic performance for characterizing adrenal lesions beyond those of single-parameter evaluations. Predictive formulas assessing the probabilities of lesion benignity or malignancy are provided.