Supported-amine-catalyzed cascade synthesis of spiro-thiazolone-tetrahydrothiophenes: assessing HSA binding activity.

We have devised a supported-amine-catalyzed efficient synthesis of spiro-thiazolone-tetrahydrothiophenes via a sulfa-Michael/aldol cascade approach. The catalyst demonstrated sustained efficacy over 21 cycles. These derivatives were found to exhibit excellent binding abilities with purified human serum albumin as indicated by both in silico and in vitro-based experiments.

The rapid construction and biological evaluation of densely substituted pyrrolo[1,2-a]indoles via a BF3·OEt2-assisted cascade approach.

Lewis-acid cascade reactions promoted by BF3·OEt2 are reported for the synthesis of highly substituted pyrrolo[1,2-a]indoles and congeners of benzofuro[2,3-b]indoles. These reactions are highly regio- and diastereoselective towards generating up to five contiguous stereogenic centers, including two vicinal quaternary centers. Furthermore, an established cascade approach and the mechanism proposed herein are well supported by quantum chemistry calculations. In addition, a self-dimerization intermediate was trapped and isolated to establish a strategy for potential access to both pyrrolo and benzo indole derivatives, leaving sufficient freedom for broadening. Furthermore, in-silico molecular docking and all atomistic molecular dynamic (MD) simulation analysis suggests that the synthesized pyrrolo[1,2-a]indole derivatives stably bind at the active site of the mycobacterial secreted tyrosine phosphatase B (MptpB) enzyme, an emerging anti-mycobacterial drug target. Deep learning-based affinity predictions and MMPBGBSA-based energy calculations of the docked poses are presented herein.

Selective targeting of the inactive state of hematopoietic cell kinase (Hck) with a stable curcumin derivative.

Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID-19 patients. Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases. Curcumin has been previously identified as an excellent DYRK-2 inhibitor, but curcumin's fate is tainted by its instability in the cellular environment. Besides, small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity. Here, we show that functionalization of the 4-arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor (Kd = 50 ± 10 nM). The mustard curcumin derivative preferentially interacts with the inactive conformation of Hck, similar to type-II kinase inhibitors that are less promiscuous. Moreover, the lead compound showed no inhibitory effect on three other kinases (DYRK2, Src, and Abl). We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signaling pathway in triple-negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodeling its target affinity and cellular stability.

Stereoselective synthesis of tri-substituted tetrahydrothiophenes and their in silico binding against mycobacterial protein tyrosine phosphatase B.

A facile approach to tri-substituted tetrahydrothiophenes via thia-Michael/aldol has been developed. The cascade reaction was carried out in the presence of 5 mol% of DABCO in ethyl acetate to afford diversely functionalized tetrahydrothiophenes (THTs) with excellent diastereoselectivity. The present methodology has broad substrate tolerance. Gram-scale reaction proceeds with equal efficiency. Functional group transformations further highlight the synthetic potential of the THTs. An asymmetric version of the cascade reaction has also been investigated and a maximum of 72% ee was observed with cinchonidine derived squaramide. Moreover, in silico based molecular docking followed by deep learning based affinity prediction and molecular dynamics simulation analysis indicate the synthesized THT derivatives can act as potent competitive inhibitors of MptpB at low micromolar to nanomolar concentrations. In silico ADME analysis further suggests the plausibility of these compounds to act as future anti-mycobacterial therapeutic leads.

Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase.

The Gam protein of transposable phage Mu is an ortholog of eukaryotic and bacterial Ku proteins, which carry out nonhomologous DNA end joining (NHEJ) with the help of dedicated ATP-dependent ligases. Many bacteria carry Gam homologs associated with either complete or defective Mu-like prophages, but the role of Gam in the life cycle of Mu or in bacteria is unknown. Here, we show that MuGam is part of a two-component bacterial NHEJ DNA repair system. Ensemble and single-molecule experiments reveal that MuGam binds to DNA ends, slows the progress of RecBCD exonuclease, promotes binding of NAD+-dependent Escherichia coli ligase A, and stimulates ligation. In vivo, Gam equally promotes both precise and imprecise joining of restriction enzyme-digested linear plasmid DNA, as well as of a double-strand break (DSB) at an engineered I-SceI site in the chromosome. Cell survival after the induced DSB is specific to the stationary phase. In long-term growth competition experiments, particularly upon treatment with a clastogen, the presence of gam in a Mu lysogen confers a distinct fitness advantage. We also show that the role of Gam in the life of phage Mu is related not to transposition but to protection of genomic Mu copies from RecBCD when viral DNA packaging begins. Taken together, our data show that MuGam provides bacteria with an NHEJ system and suggest that the resulting fitness advantage is a reason that bacteria continue to retain the gam gene in the absence of an intact prophage.

Characterization of Caenorhabditis elegans Nucleosome Assembly Protein 1 Uncovers the Role of Acidic Tails in Histone Binding.

Nucleosome assembly proteins (Naps) influence chromatin dynamics by directly binding to histones. Here we provide a comprehensive structural and biochemical analysis of a Nap protein from Caenorhabditis elegans (CeNap1). CeNap1 naturally lacks the acidic N-terminal tail and has a short C-terminal tail compared to many other Nap proteins. Comparison of CeNap1 with full length and tail-less constructs of Saccharomyces cerevisiae Nap1 uncovers the role of these tails in self-association, histone binding, and Nap competition with DNA for H2A-H2B. We find that the presence of tails influences the stoichiometry of H2A-H2B binding and is required to complete the interactions between H2A-H2B and DNA. The absolute stoichiometry of the Nap protein and H2A-H2B complex is 2:1 or 2:2, with only a very small population of higher-order oligomers occurring at 150 mM NaCl. We also show that H3-H4 binds differently than H2A-H2B and that an (H3-H4)2 tetramer can simultaneously bind two Nap2 protein homodimers.

Design, synthesis and characterization of novel inhibitors against mycobacterial ß-ketoacyl CoA reductase FabG4.

We report the design and synthesis of triazole-polyphenol hybrid compounds 1 and 2 as inhibitors of the FabG4 (Rv0242c) enzyme of Mycobacterium tuberculosis for the first time. A major advance in this field occurred only a couple of years ago with the X-ray crystal structure of FabG4, which has helped us to design these inhibitors by the computational fragment-based drug design (FBDD) approach. Compound 1 has shown competitive inhibition with an inhibition constant (Ki) value of 3.97 ± 0.02 µM. On the other hand, compound 2 has been found to be a mixed type inhibitor with a Ki value of 0.88 ± 0.01 µM. Thermodynamic analysis using isothermal titration calorimetry (ITC) reveals that both inhibitors bind at the NADH co-factor binding domain. Their MIC values, as determined by resazurin assay against M. smegmatis, indicated their good anti-mycobacterial properties. A preliminary structure-activity relationship (SAR) study supports the design of these inhibitors. These compounds may be possible candidates as lead compounds for alternate anti-tubercular drugs. All of the reductase enzymes of the Mycobacterium family have a similar ketoacyl reductase (KAR) domain. Hence, this work may be extrapolated to find structure-based inhibitors of other reductase enzymes.

Crystal structure of hexanoyl-CoA bound to ß-ketoacyl reductase FabG4 of Mycobacterium tuberculosis.

FabGs, or ß-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of ß-oxoacyl-ACP (acyl-carrier protein) into ß-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal 'flavodoxintype' domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce ß-oxoacyl-CoA to ß-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4-NADH binary complex and the FabG4-NAD+-hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

Discovery of Quinazoline and Quinoline-Based Small Molecules as Utrophin Upregulators via AhR Antagonism for the Treatment of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease caused by the absence of a dystrophin protein. Elevating utrophin, a dystrophin paralogue, offers an alternative therapeutic strategy for treating DMD, irrespective of the mutation type. Herein, we report the design and synthesis of novel quinazoline and quinoline-based small molecules as potent utrophin modulators screened via high throughput In-Cell ELISA in C2C12 cells. Remarkably, lead molecule SG-02, identified from a library of 70 molecules, upregulates utrophin 2.7-fold at 800 nM in a dose-dependent manner, marking the highest upregulation within the nanomolar range. SG-02's efficacy was further validated through DMD patient-derived cells, demonstrating a significant 2.3-fold utrophin expression. Mechanistically, SG-02 functions as an AhR antagonist, with excellent binding affinity (Kd = 41.68 nM). SG-02 also enhances myogenesis, as indicated by an increased MyHC expression. ADME evaluation supports SG-02's oral bioavailability. Overall, SG-02 holds promise for addressing the global DMD population.

Molecular Mechanisms Driving and Regulating the AAA+ ATPase VCP/p97, an Important Therapeutic Target for Treating Cancer, Neurological and Infectious Diseases.

p97/VCP, a highly conserved type II ATPase associated with diverse cellular activities (AAA+ ATPase), is an important therapeutic target in the treatment of neurodegenerative diseases and cancer. p97 performs a variety of functions in the cell and facilitates virus replication. It is a mechanochemical enzyme that generates mechanical force from ATP-binding and hydrolysis to perform several functions, including unfolding of protein substrates. Several dozens of cofactors/adaptors interact with p97 and define the multifunctionality of p97. This review presents the current understanding of the molecular mechanism of p97 during the ATPase cycle and its regulation by cofactors and small-molecule inhibitors. We compare detailed structural information obtained in different nucleotide states in the presence and absence of substrates and inhibitors. We also review how pathogenic gain-of-function mutations modify the conformational changes of p97 during the ATPase cycle. Overall, the review highlights how the mechanistic knowledge of p97 helps in designing pathway-specific modulators and inhibitors.

Crystal structure of FadA2 thiolase from Mycobacterium tuberculosis and prediction of its substrate specificity and membrane-anchoring properties.

Tuberculosis (TB) is one of the leading causes of human death caused by Mycobacterium tuberculosis (Mtb). Mtb can enter into a long-lasting persistence where it can utilize fatty acids as the carbon source. Hence, fatty acid metabolism pathway enzymes are considered promising and pertinent mycobacterial drug targets. FadA2 (thiolase) is one of the enzymes involved in Mtb's fatty acid metabolism pathway. FadA2 deletion construct (ΔL136-S150) was designed to produce soluble protein. The crystal structure of FadA2 (ΔL136-S150) at 2.9 Å resolution was solved and analysed for membrane-anchoring region. The four catalytic residues of FadA2 are Cys99, His341, His390 and Cys427, and they belong to four loops with characteristic sequence motifs, i.e., CxT, HEAF, GHP and CxA. FadA2 is the only thiolase of Mtb which belongs to the CHH category containing the HEAF motif. Analysing the substrate-binding channel, it has been suggested that FadA2 is involved in the ß-oxidation pathway, i.e., the degradative pathway, as the long-chain fatty acid can be accommodated in the channel. The catalysed reaction is favoured by the presence of two oxyanion holes, i.e., OAH1 and OAH2. OAH1 formation is unique in FadA2, formed by the NE2 of His390 present in the GHP motif and NE2 of His341 present in the HEAF motif, whereas OAH2 formation is similar to CNH category thiolase. Sequence and structural comparison with the human trifunctional enzyme (HsTFE-ß) suggests the membrane-anchoring region in FadA2. Molecular dynamics simulations of FadA2 with a membrane containing POPE lipid were conducted to understand the role of a long insertion sequence of FadA2 in membrane anchoring.

A structurally novel hemopexin fold protein of rice plays role in chlorophyll degradation.

Proteins containing hemopexin fold domain are suggested to have diverse functions in various living organisms. In order to investigate the structure and function of this type of protein in rice plant (Oryza sativa), the gene encoding a hemopexin fold protein (OsHFP) was cloned, analyzed in silico and characterized. Molecular modeling revealed that the OsHFP is closely related to other hemopexin fold proteins, but is unique with a cylindrical central tunnel as well as extended N- and C-terminal domains. The recombinant OsHFP was found to bind hemin, the oxidized form of heme in vitro. The expression of the single copy OsHFP gene was detected in rice flower buds. Heterologous expression of OsHFP in green leaf tissues resulted in chlorophyll degradation; however, stable expression of OsHFP was observed in transgenic hairy roots, a non-green tissue. The possible role of OsHFP in regulating programmed cell death in anther green tissues of rice is proposed.

Crystallization and preliminary X-ray diffraction analysis of the high molecular weight ketoacyl reductase FabG4 complexed with NADH.

FabG4 from Mycobacterium tuberculosis belongs to the high molecular weight ketoacyl reductases (HMwFabGs). The enzyme requires NADH for ß-ketoacyl reductase activity. The protein was overexpressed, purified to homogeneity and crystallized as a FabG4-NADH complex. A mountable FabG4:NADH complex crystal diffracted to 2.59 Šresolution and belonged to space group P1, with unit-cell parameters a = 63.07, b = 71.03, c = 92.92 Å, α = 105.02, ß = 97.06, γ = 93.66°. The Matthews coefficient suggested the presence of four monomers in the unit cell. In addition, a self-rotation function revealed the presence of two twofold NCS axes and one fourfold NCS axis. At χ = 180° the highest peak corresponds to the twofold NCS between two monomers, whereas the second peak corresponds to the twofold NCS between two dimers.

Effective Synthesis and Biological Evaluation of Functionalized 2,3-Dihydrofuro[3,2-c]coumarins via an Imidazole-Catalyzed Green Multicomponent Approach.

For the first time, an eco-friendly and efficient one-pot green multicomponent approach has been described to synthesize functionalized trans-2,3-dihydrofuro[3,2-c]coumarins (DHFCs). In this synthesis, imidazole and water were used as the catalyst and solvent, respectively, under mild conditions. Applications of the developed catalytic process in a water medium revealed the outstanding activity, productivity, and broad functional group tolerance, affording a series of newly designed DHFC and derivatives in excellent yields (72-98%). Moreover, the human serum albumin (HSA) binding ability of the synthesized DHFC derivatives has been uncovered through the detailed in silico and in vitro-based structure-activity analysis. The ability to bind HSA, the most abundant serum protein, in the low micromolar ranges unequivocally reflects the suitable absorption, distribution, metabolism, and elimination profile of the synthesized compounds, which may further be envisaged for their therapeutic usage endeavors.

The C-terminal end of mycobacterial HadBC regulates AcpM interaction during the FAS-II pathway: a structural perspective.

Comprehending the molecular strategies employed by Mycobacterium tuberculosis (Mtb) in FAS-II regulation is of paramount significance for curbing tuberculosis progression. Mtb employs two sets of dehydratases, namely HadAB and HadBC (ß-hydroxyacyl acyl carrier protein dehydratase), for the regulation of the fatty acid synthase (FAS-II) pathway. We utilized a sequence similarity network to discern the basis for the presence of two copies of the dehydratase gene in Mtb. This analysis groups HadC and HadA in different clusters, which could be attributed to the variability in their physiological role with respect to the acyl chain uptake. Our study reveals structural details pertaining to the crystal structure of the last remaining enzyme of the FAS-II pathway. It also provides insights into the highly flexible hot-dog helix and substrate regulatory loop. Additionally, mutational studies assisted in establishing the role of the C-terminal end in HadC of HadBC in the regulation of acyl carrier protein from Mtb-mediated interactions. Complemented with surface plasmon resonance and molecular dynamics simulation studies, the present study provides the first evidence of the molecular mechanisms involved in the differential binding affinity of the acyl carrier protein from Mtb towards both mtbHadAB and mtbHadBC.

Effective Synthesis and Biological Evaluation of Natural and Designed Bis(indolyl)methanes via Taurine-Catalyzed Green Approach.

An ecofriendly, inexpensive, and efficient route for synthesizing 3,3'-bis(indolyl)methanes (BIMs) and their derivatives was carried out by an electrophilic substitution reaction of indole with structurally divergent aldehydes and ketones using taurine and water as a green catalyst and solvent, respectively, under sonication conditions. Using water as the only solvent, the catalytic process demonstrated outstanding activity, productivity, and broad functional group tolerance, affording the required BIM natural products and derivatives in excellent yields (59-90%). Furthermore, in silico based structure activity analysis of the synthesized BIM derivatives divulges their potential ability to bind antineoplastic drug target and spindle motor protein kinesin Eg5. The precise binding mode of BIM derivatives with the ATPase motor domain of Eg5 is structurally reminiscent with previously reported allosteric inhibitor Arry520, which is under phase III clinical trials. Nevertheless, detailed analysis of the binding poses indicates that BIM derivatives bind the allosteric pocket of the Eg5 motor domain more robustly than Arry520; moreover, unlike Arry520, BIM binding is found to be resistant to drug-resistant mutations of Eg5. Accordingly, a structure-guided mechanism of Eg5 inhibition by synthesized BIM derivatives is proposed.

Anti-SARS-CoV-2 potential of Cissampelos pareira L. identified by connectivity map-based analysis and in vitro studies.

BACKGROUND: Viral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present. Herbal medicines have been a source of various antiviral compounds such as Oseltamivir, extracted using shikimic acid from star anise (Illicium verum) and Acyclovir from Carissa edulis are FDA (Food and Drug Administration) approved antiviral drugs. In this study, we dissect the anti-coronavirus infection activity of Cissampelos pareira L (Cipa) extract using an integrative approach. METHODS: We analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map ( https://clue.io/ ). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. Molecular docking analyses of constituents of with key targets of SARS-CoV2 protein viz. spike protein, RNA­dependent RNA­polymerase (RdRp) and 3C­like proteinase. was also performed. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed. RESULTS: Several predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhibition of SARS-COV-2 replication in infected Vero cell cultures with the whole extract. Some of its prominent pure constituents e.g. pareirarine, cissamine, magnoflorine exhibited 40-80% inhibition. Comparison of genes between BALF and Cipa showed an enrichment of biological processes like transcription regulation and response to lipids, to be downregulated in Cipa while being upregulated in COVID-19. CMAP also showed that Triciribine, torin-1 and VU-0365114-2 had positive connectivity with BALF 1 and 2, and negative connectivity with Cipa. Amongst all the tested compounds, Magnoflorine and Salutaridine exhibited the most potent and consistent strong in silico binding profiles with SARS-CoV2 therapeutic targets.

Crystal structure of FabG4 from Mycobacterium tuberculosis reveals the importance of C-terminal residues in ketoreductase activity.

Rv0242c, also known as FabG4, is a beta-ketoacyl CoA reductase in Mycobacterium tuberculosis. The crystal structure of C-terminal truncated FabG4 is solved at 2.5Å resolution which shows the presence of two distinct domains, domain I and II. Domain I partially resembles "flavodoxin type domain" and the domain II is a typical "ketoacyl CoA reductase (KAR) domain". The enzyme exhibits ketoacyl CoA reductase activity by reducing acetoacyl CoA to 3-hydroxyacyl CoA in presence of NADH. Conserved catalytic triad Ser347, Tyr360, and Lys364 constitute the active site residues of the KAR domain. Presence of the Tyr and the Lys residues in the triad in a particular orientation is imperative for effective catalytic mechanism. The importance of loop I and II and the role of the C-terminal residues of KAR domain are highlighted. Comparative structural analyses clearly demonstrate that loop II is stabilized by hydrophobic interaction with C-terminal residues to sustain the orientation of Tyr360. Loop I interacts with loop II via H-bonding network to restrict the active site residue Lys364 in a catalytically favorable orientation.

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from Staphylococcus aureus MSSA476.

The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li(2)SO(4), 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 Šresolution using a Rigaku MicroMax-007 HF Cu Kα X-ray generator and a Rigaku R-AXIS IV(++) detector. The diffraction data were consistent with the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 Å, α = ß = γ = 90°, and the crystal contained two molecules in the asymmetric unit.

Comparison of vaginal misoprostol tablets and prostaglandin E2 gel for the induction of labor in premature rupture of membranes at term: a randomized comparative trial.

AIM: To compare immediate induction with vaginal misoprostol tablets and immediate induction with vaginal dinoprostone (naturally occurring prostaglandin E2 [PGE2]) gel in women with premature rupture of membranes (PROM) at term. METHODS: Two hundred and twelve women with PROM at term were assigned randomly to receive either an intravaginal 25 µg misoprostol tablet, 4-hourly, with a maximum of five doses, or 0.5 mg intravaginal PGE2 gel, 6-hourly, with a maximum of two doses. The primary outcome measures were the admission-to-delivery interval and the induction-to-delivery interval. Secondary outcomes included cesarean section rate, mode of delivery, and maternal and neonatal safety outcome. Results were calculated applying Fisher's exact test, χ2-test, t-test and calculating the P-value using an alpha level of 0.05 for Type I errors. RESULTS: The mean time from admission to delivery was 13.53 h in the misoprostol group and 12.30 h in the PGE2 group (P = 0.090). The induction-to-delivery interval was also comparable between the groups (10.75 h vs. 9.37 h), while the cesarean section rate did not differ significantly between them (7.61% vs. 15.30%). More women in the misoprostol group had an instrumental delivery (12.38% vs. 2.94%). The only significant difference in neonatal outcome was a greater number of babies born with Apgar score < 7 at 1 min in the misoprostol group. Maternal outcomes were not significantly different, except for a higher number of digital vaginal examinations in the misoprostol group. CONCLUSION: Vaginal misoprostol is equally efficacious in labor induction and demonstrates a similar fetal and maternal safety profile to PGE2 gel.