Polyphosphate kinase-1 regulates bacterial and host metabolic pathways involved in pathogenesis of Mycobacterium tuberculosis.

Inorganic polyphosphate (polyP) is primarily synthesized by Polyphosphate Kinase-1 (PPK-1) and regulates numerous cellular processes, including energy metabolism, stress adaptation, drug tolerance, and microbial pathogenesis. Here, we report that polyP interacts with acyl CoA carboxylases, enzymes involved in lipid biosynthesis in Mycobacterium tuberculosis. We show that deletion of ppk-1 in M. tuberculosis results in transcriptional and metabolic reprogramming. In comparison to the parental strain, the Δppk-1 mutant strain had reduced levels of virulence-associated lipids such as PDIMs and TDM. We also observed that polyP deficiency in M. tuberculosis is associated with enhanced phagosome-lysosome fusion in infected macrophages and attenuated growth in mice. Host RNA-seq analysis revealed decreased levels of transcripts encoding for proteins involved in either type I interferon signaling or formation of foamy macrophages in the lungs of Δppk-1 mutant-infected mice relative to parental strain-infected animals. Using target-based screening and molecular docking, we have identified raloxifene hydrochloride as a broad-spectrum PPK-1 inhibitor. We show that raloxifene hydrochloride significantly enhanced the activity of isoniazid, bedaquiline, and pretomanid against M. tuberculosis in macrophages. Additionally, raloxifene inhibited the growth of M. tuberculosis in mice. This is an in-depth study that provides mechanistic insights into the regulation of mycobacterial pathogenesis by polyP deficiency.

Molecular modeling reveals binding interface of γ-tubulin with GCP4 and interactions with noscapinoids.

The initiation of microtubule assembly within cells is guided by a cone shaped multi-protein complex, γ-tubulin ring complex (γTuRC) containing γ-tubulin and atleast five other γ-tubulin-complex proteins (GCPs), i.e., GCP2, GCP3, GCP4, GCP5, and GCP6. The rim of γTuRC is a ring of γ-tubulin molecules that interacts, via one of its longitudinal interfaces, with GCP2, GCP3, or GCP4 and, via other interface, with α/ß-tubulin dimers recruited for the microtubule lattice formation. These interactions however, are not well understood in the absence of crystal structure of functional reconstitution of γTuRC subunits. In this study, we elucidate the atomic interactions between γ-tubulin and GCP4 through computational techniques. We simulated two complexes of γ-tubulin-GCP4 complex (we called dimer1 and dimer2) for 25 ns to obtain a stable complex and calculated the ensemble average of binding free energies of -158.82 and -170.19 kcal/mol for dimer1 and -79.53 and -101.50 kcal/mol for dimer2 using MM-PBSA and MM-GBSA methods, respectively. These highly favourable binding free energy values points to very robust interactions between GCP4 and γ-tubulin. From the results of the free-energy decomposition and the computational alanine scanning calculation, we identified the amino acids crucial for the interaction of γ-tubulin with GCP4, called hotspots. Furthermore, in the endeavour to identify chemical leads that might interact at the interface of γ-tubulin-GCP4 complex; we found a class of compounds based on the plant alkaloid, noscapine that binds with high affinity in a cavity close to γ-tubulin-GCP4 interface compared with previously reported compounds. All noscapinoids displayed stable interaction throughout the simulation, however, most robust interaction was observed for bromo-noscapine followed by noscapine and amino-noscapine. This offers a novel chemical scaffold for γ-tubulin binding drugs near γ-tubulin-GCP4 interface.

Rational design of biaryl pharmacophore inserted noscapine derivatives as potent tubulin binding anticancer agents.

We have strategically designed a series of noscapine derivatives by inserting biaryl pharmacophore (a major structural constituent of many of the microtubule-targeting natural anticancer compounds) onto the scaffold structure of noscapine. Molecular interaction of these derivatives with α,ß-tubulin heterodimer was investigated by molecular docking, molecular dynamics simulation, and binding free energy calculation. The predictive binding affinity indicates that the newly designed noscapinoids bind to tubulin with a greater affinity. The predictive binding free energy (ΔG(bind, pred)) of these derivatives (ranging from -5.568 to -5.970 kcal/mol) based on linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model showed improved binding affinity with tubulin compared to the lead compound, natural α-noscapine (-5.505 kcal/mol). Guided by the computational findings, these new biaryl type α-noscapine congeners were synthesized from 9-bromo-α-noscapine using optimized Suzuki reaction conditions for further experimental evaluation. The derivatives showed improved inhibition of the proliferation of human breast cancer cells (MCF-7), human cervical cancer cells (HeLa) and human lung adenocarcinoma cells (A549), compared to natural noscapine. The cell cycle analysis in MCF-7 further revealed that these compounds alter the cell cycle profile and cause mitotic arrest at G2/M phase more strongly than noscapine. Tubulin binding assay revealed higher binding affinity to tubulin, as suggested by dissociation constant (Kd) of 126 ± 5.0 µM for 5a, 107 ± 5.0 µM for 5c, 70 ± 4.0 µM for 5d, and 68 ± 6.0 µM for 5e compared to noscapine (Kd of 152 ± 1.0 µM). In fact, the experimentally determined value of ΔG(bind, expt) (calculated from the Kd value) are consistent with the predicted value of ΔG(bind, pred) calculated based on LIE-SGB. Based on these results, one of the derivative 5e of this series was used for further toxicological evaluation. Treatment of mice with a daily dose of 300 mg/kg and a single dose of 600 mg/kg indicates that the compound does not induce detectable pathological abnormalities in normal tissues. Also there were no significant differences in hematological parameters between the treated and untreated groups. Hence, the newly designed noscapinoid, 5e is an orally bioavailable, safe and effective anticancer agent with a potential for the treatment of cancer and might be a candidate for clinical evaluation.

Molecular insight into γ-γ tubulin lateral interactions within the γ-tubulin ring complex (γ-TuRC).

γ-Tubulin is essential for the nucleation and organization of mitotic microtubules in dividing cells. It is localized at the microtubule organizing centers and mitotic spindle fibres. The most well accepted hypothesis for the initiation of microtubule polymerization is that α/ß-tubulin dimers add onto a γ-tubulin ring complex (γTuRC), in which adjacent γ-tubulin subunits bind to the underlying non-tubulin components of the γTuRC. This template thus determines the resulting microtubule lattice. In this study we use molecular modelling and molecular dynamics simulations, combined with computational MM-PBSA/MM-GBSA methods, to determine the extent of the lateral atomic interaction between two adjacent γ-tubulins within the γTuRC. To do this we simulated a γ-γ homodimer for 10 ns and calculated the ensemble average of binding free energies of -107.76 kcal/mol by the MM-PBSA method and of -87.12 kcal/mol by the MM-GBSA method. These highly favourable binding free energy values imply robust lateral interactions between adjacent γ-tubulin subunits in addition to their end-interactions longitudinally with other proteins of γTuRC. Although the functional reconstitution of γ-TuRC subunits and their stepwise in vitro assembly from purified components is not yet feasible, we nevertheless wanted to recognize hotspot amino acids responsible for key γ-γ interactions. Our free energy decomposition data from converting a compendium of amino acid residues identified an array of hotspot amino acids. A subset of such mutants can be expressed in vivo in living yeast. Because γTuRC is important for the growth of yeast, we could test whether this subset of the hotspot mutations support growth of yeast. Consistent with our model, γ-tubulin mutants that fall into our identified hotspot do not support yeast growth.

Crystallographic landscape provides molecular insights into the modes of action of diverse ROR-γt modulators.

ROR-γt, the master regulator of Th-17 cells, is activated by the binding of small molecules at its orthosteric site, followed by the recruitment of co-activators or co-repressors in the ligand binding domain (LBD). Th-17 cells provide immune-dependent protection against cancers and pathogens. Their dysregulation causes inflammation and is therefore implicated in various autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. Consequently, there is enormous interest in the development of ROR-γt modulators, both agonist and inverse-agonists. Here, we review advances in the development of ROR-γt modulators that have been made over the past decade, focusing on the rich crystallography landscape for ROR-γt co-crystals that has delineated the relationship between the binding patterns of modulators and the resulting biological activities.

Rational design of 9-vinyl-phenyl noscapine as potent tubulin binding anticancer agent and evaluation of the effects of its combination on Docetaxel.

Docetaxel (DOX) based combination therapy is a novel therapeutic strategy that attracts great interest in breast cancer treatment but its clinical utility got limited due to side effects. In contrast, noscapine, an antitussive drug showed antitumor activity against many cancers without any side effects that targets microtubules and attenuates its dynamic instability. In the quest for an increase in the anticancer activity of noscapine, we strategically designed a novel derivative, 9-vinyl phenyl noscapine (VPN), based on our in silico molecular docking and molecular dynamics simulation effort. Molecular docking of VPN and DOX onto microtubule revealed a docking score of -4.82 kcal/mol and -6.67 kcal/mol respectively, while the docking score of VPN was changed to -3.23 kcal/mol when it was docked onto the co-complex of tubulin-DOX. Further, the binding free energy (ΔGbind,PBSA) of VPN and DOX with tubulin showed -24.04 and -18.65 kcal/mol respectively, while the binding free energy of DOX was increased further in combination with VPN (ΔGbind, PBSA was reduced to -21.41 kcal/mol), denoting combination effect of both ligands. The IC50 value amounted to 30.17 µM and 19.92 µM for VPN and 0.621 µM and 0.193 µM for DOX, respectively for 48 h and 72 h. The dose dependent cytotoxicity of DOX has been reduced considerably with the combination dose regimen of VPN. Further, the combine effect of both the agents improved the apoptotic cell death 28.5% compared to single agent treatment 5.71% and 10.5% for VPN and DOX, respectively. Both agents bind effectively to tubulin in single and in combination to interfere with cell cycle progression in G2/M transition. This study provides novel concept of combination treatment of DOX and VPN to amend efficiency in breast cancer treatment.Communicated by Ramaswamy H. Sarma.

Insights into structural dynamics of allosteric binding sites in HCV RNA-dependent RNA polymerase.

Inhibition of the viral RNA-dependent RNA polymerase (RdRp) to resolve chronic infection is a useful therapeutic strategy against Hepatitis C virus (HCV). Non-nucleoside inhibitors (NNIs) of RdRp are small molecules that bind tightly with allosteric sites on the enzyme, thereby inhibiting polymerase activity. A large number of crystal structures (176) were studied to establish the structure-activity relationship along with the mechanism of inhibition and resistance between HCV RdRp and NNIs at different allosteric sites. The structure and the associated dynamics are the blueprint to understand the function of the protein. We have implemented the ligand-based pharmacophore and molecular dynamic simulations to extract the possible local and global characteristics of RdRp upon NNI binding and the structural-dynamical features possessed by the known actives. Our results suggest that the NNI binding induces significant fluctuations at the atomic level which are critical for enzymatic activity, with minimal global structural alterations. Residue-wise mapping of interactions of NNIs at different sites exhibited some conserved interaction patterns of key amino acids and water molecules. Here, the structural insights are explored to understand the correlation between the dynamics of protein's subdomains and function at the molecular level, useful for genotype-specific rational designing of NNIs.Communicated by Ramaswamy H. Sarma.

Molecular dynamics simulation reveals the possible druggable hot-spots of USP7.

The plasticity in Ubiquitin Specific Proteases (USP7) inducing conformational changes at important areas has highlighted an intricate mechanism, by which USP7 is regulated. Given the importance of USP7 in oncogenic pathways and immune-oncology, identification of USP7 inhibitors has attracted considerable interest. Despite substantial efforts, the discovery of deubiquitinases (DUBs) inhibitors, knowledge of their binding site and understanding the possible mechanism of action has proven particularly challenging. We disclose the most likely binding site of P5091 (a potent USP7 inhibitor), which reveal a cryptic allosteric site through extensive computational studies in an inhibitor dependent and independent manner. Overall, these findings demonstrate the tractability and druggability of USP7. Through a series of molecular dynamics simulations and detailed quantitative analysis, a dynamically stable allosteric binding site near catalytic center of the inactive state of USP7 (site partially absent in active state), along with two newly identified sites have been revealed, which opens the avenue for rational structure-guided inhibitor designing in USP7 specific-manner.

Elucidation of the anticancer potential and tubulin isotype-specific interactions of ß-sitosterol.

Beta-sitosterol (ß-SITO), a phytosterol present in many edible vegetables, has been reported to possess antineoplastic properties and cancer treatment potential. We have shown previously that it binds at a unique site (the 'SITO-site') compared to the colchicine binding site at the interface of α- and ß-tubulin. In this study, we investigated the anticancer efficacy of ß-SITO against invasive breast carcinoma using MCF-7 cells. Since 'isotypes' of ß-tubulin show tissue-specific expression and many are associated with cancer drug resistance, using computer-assisted docking and atomistic molecular dynamic simulations, we also examined its binding interactions to all known isotypes of ß-tubulin in αß-tubulin dimer. ß-SITO inhibited MCF-7 cell viability by up to 50%, compared to vehicle-treated control cells. Indicating its antimetastatic potential, the phytosterol strongly inhibited cell migration. Immunofluorescence imaging of ß-SITO-treated MCF-7 cells exhibited disruption of the microtubules and chromosome organization. Far-UV circular dichroism spectra indicated loss of helical stability in tubulin when bound to ß-SITO. Docking and MD simulation studies, combined with MM-PBSA and MM-GBSA calculations revealed that ß-SITO preferentially binds with specific ß-tubulin isotypes (ßII and ßIII) in the αß-tubulin dimer. Both these ß-tubulin isotypes have been implicated in drug resistance against tubulin-targeted chemotherapeutics. Our data show the tubulin-targeted anticancer potential of ß-SITO, and its potential clinical utility against ßII and ßIII isotype-overexpressing neoplasms.

Nature of Hard Tissues in Oral Pathological Lesions -Using Modified Gallego's Stain.

INTRODUCTION: Histological stains are dyes that bind to various tissues. Special stains form an integral part of routine histopathology as an adjunct to Haematoxylin and Eosin (H&E), and give meaningful diagnostic information of the tissues available. By using routine histological procedure like H&E alone, it is difficult to differentiate the various hard tissues present in the oral pathological lesions. Modified Gallego's stain can be used as one of the differential stain for these hard tissues. AIM: To differentiate various hard tissues of teeth and to identify the presence of hard tissue components in different oral pathological lesions using Modified Gallego's stain under light microscope. MATERIALS AND METHODS: A total sample of 20 cases, amongst which 10 were human extracted teeth and 10 oral pathological lesions were included. From 10 human extracted teeth, five ground sections and five decalcified sections were prepared. From pathological lesions, two slides of each lesion were prepared. All the sections were stained with Modified Gallego's stain and viewed under light microscopy. RESULTS: In properly stained slides, cementum stained red, dentin and bone stained green and enamel stained pink in colour. CONCLUSION: Modified Gallego's stain can be used as a differential stain for various hard tissues in oral pathological lesions and also for ground sections for which histochemical stains are very rare.

Biochemical characterization and molecular dynamic simulation of ß-sitosterol as a tubulin-binding anticancer agent.

Βeta-sitosterol (ß-SITO), a phytosterol present in pomegranate, peanut, corn oil, almond, and avocado, has been recognized to offer health benefits and potential clinical uses. ß-SITO is orally bioavailable and, as a constituent of edible natural products, is considered to have no undesired side effects. It has also been considered as a potent anticancer agent. However, the molecular mechanism of action of ß-SITO as a tubulin-binding anticancer agent and its binding site on tubulin are poorly understood. Using a combination of biochemical analyses and molecular dynamic simulation, we investigated the molecular details of the binding interactions of ß-SITO with tubulin. A polymer mass assay comparing the effects of ß-SITO and of taxol and vinblastine on tubulin assembly showed that this phytosterol stabilized microtubule assembly in a manner similar to taxol. An 8-anilino-1-naphthalenesulfonic acid assay confirmed the direct interaction of ß-SITO with tubulin. Although ß-SITO did not show direct binding to the colchicine site on tubulin, it stabilized the colchicine binding. Interestingly, no sulfhydryl groups of tubulin were involved in the binding interaction of ß-SITO with tubulin. Based on the results from the biochemical assays, we computationally modeled the binding of ß-SITO with tubulin. Using molecular docking followed by molecular dynamic simulations, we found that ß-SITO binds tubulin at a novel site (which we call the 'SITO site') adjacent to the colchicine and noscapine sites. Our data suggest that ß-SITO is a potent anticancer compound that interferes with microtubule assembly dynamics by binding to a novel site on tubulin.

Myofibroma of the gingiva: a rare case report and literature review.

Myofibromas are benign uncommon fibroblastic tumors of the soft tissue, bone, or internal organs affecting all ages. These lesions histopathologically may mimic many other soft tissue tumors of the oral cavity such as spindle cell tumors of neurogenic and smooth muscle cell origin, thus leading to misdiagnosis and mistreatment. This case report describes a rare benign tumor, which presented as a soft tissue swelling on posterior gingiva. Surgical excision of the lesion was carried out under local anaesthesia. Histopathologic and immunohistochemical examination confirmed the diagnosis of myofibroma. Myofibroma should be included in the clinical differential diagnosis of masses of the oral soft tissues; however immunohistochemical examination is essential to establish an accurate diagnosis.

Comparision of Immunohistochemical Expression of CD10 in Odontogenic Cysts.

BACKGROUND: Expression of CD10 has been documented in various tumors like nasopharyngeal carcinoma, gastric carcinoma, squamous cell carcinoma, odontogenic tumors. AIM: To evaluate and compare CD10 expression in odontogenic cysts like radicular cyst, dentigerous cyst and odontogenic keratocyst (OKC). MATERIALS AND METHODS: Total 60 cases were included in the study, comprising 20 cases each of radicular, dentigerous and odontogenic keratocyst. Each case was evaluated and compared for immunohistochemical expression of CD10. RESULTS obtained were statistically analysed using ANOVA test followed by post hoc test Tukey-Kramer Multiple Comparisons Test for continuous variable and Chi-square test for discrete variable. RESULTS: More number of cases showing sub-epithelial stromal CD10 expression were found in OKC among the cysts. CONCLUSION: CD10 expression was more in OKC compared to radicular and dentigerous cysts.

Plasma fibrinogen degradation products in betel nut chewers - with and without oral submucous fibrosis.

CONTEXT: Oral submucous fibrosis (OSMF) has a multifactorial etiology. Recent studies have shown that there is an increased level of fibrinogen degradation products (FDP) in plasma of OSMF patients suggesting its possible role in etiopathogenesis of OSMF. AIMS: To detect the presence of FDP in the plasma of betel nut chewers with and without OSMF and in normal subjects without any habits, to correlate these levels with respect to the clinical and histological grading of OSMF and whether it can be used as a nonsurgical diagnostic aid in detection of suspected OSMF cases. MATERIALS AND METHODS: Study comprised of 35 cases of betel nut chewers with OSMF, 10 patients with betel nut chewing habit but having apparently normal oral mucosa, and 10 normal patients without any habits. The patients were evaluated for plasma FDP levels. RESULTS: All the betel nut chewers with OSMF showed the presence of plasma FDP. However, controls and subjects with habit, but without OSMF did not show FDP in the plasma. Spearman's rank correlation was used to find the association between the clinical and histological grades and it was not statistically significant (P = 0.910) and the correlation being 0.020. CONCLUSION: Since only those patients with OSMF have showed the presence of FDP in plasma, we suggest that our test can be utilized as a nonsurgical diagnostic aid in suspected OSMF patients.

A Non-Invasive Technique which Demonstrates the Iron in the Buccal Mucosa of Sickle Cell Anaemia and Thalassaemia Patients who Undergo Repeated Blood Transfusions.

BACKGROUND: Iron is vital for all the living organisms. However, excess iron is hazardous because it produces free radical formation. Therefore, the iron absorption is carefully regulated to maintain an equilibrium between the absorption and the body loss of iron. Considering the lack of specific excretory pathways for iron in humans, an iron overload in the tissues is frequently encountered. It can be precipitated by a variety of conditions such as increased iron absorption, as is seen in haemochromatosis or a frequent parenteral iron administration, as is seen in thalassaemia and sickle cell anaemia patients (a transfusional overload). OBJECTIVES: To demonstrate the iron overload at an early stage by oral exfoliative cytology in the oral mucosal cells of thalassaemia and sickle cell anaemia patients and to compare the presence of iron in the exfoliated oral epithelial cells with that of the serum ferritin levels in those patients. MATERIALS AND METHODS: The present study comprised of 40 ß- thalassaemia major and 20 sickle cell anaemia patients who were undergoing repeated blood transfusions of a minimum of 15/more, along with 60 clinically healthy individuals. Scrapings were obtained from the buccal mucosa and they were smeared onto glass slides. Then the slides were stained with a Perl's Prussian staining kit and they were examined under a light microscope. RESULTS: 72.5% of the thalassaemia patients and 35% of the sickle cell anaemia patients revealed a positivity for the Perl's Prussian blue reaction and none of the controls showed this positivity. It was also observed that as the serum ferritin levels increased, the iron overload in the oral mucosal cells of the thalassaemia patients also increased, which was not statistically significant, whereas it was statistically significant in case of the sickle cell anemia patients. CONCLUSION: Since the exfoliative cytology is a simple, painless, non-invasive and a quick procedure to perform, a lot of research should be carried out on the correlation of the Perl's Prussian blue reaction to the serum ferritin levels.

Periodontal findings in patients with oral submucous fibrosis and comet assay of affected gingival epithelial cells.

BACKGROUND: Oral submucous fibrosis (OSF) involves large sections of population and is related to certain chewing practices that involve direct exposure of the gingiva to noxious chemicals and additives. The impact of such practices, although studied extensively in relation to the cheek mucosa, is not elucidated as far as gingival tissues are concerned. The possible DNA damage on affected gingiva is also one of the objectives of the present study. METHODS: One hundred cases of individuals with OSF and 89 without OSF were examined for periodontal parameters and compared. Biopsies of gingival tissue from both categories were examined with conventional hematoxylin and eosin staining for epithelial, connective tissue, and inflammatory changes. Comet assay of epithelial cells obtained from scraping the gingival surface of both patients with OSF and control individuals was performed. RESULTS: Patients with OSF had poorer oral hygiene and greater loss of attachment, probing depth, and recession compared with controls. Significant histopathologic changes in the form of loss of rete ridges with gingival epithelium reduced to a flattened surface was observed. All the patients with OSF showed comet cells in larger numbers. The controls also showed comet cells but in fewer numbers, and the number of individuals showing comet cells was significantly less. CONCLUSION: OSF showed significant changes in affected gingiva, and the presence of comet cells in all the patients with OSF is a significant indicator of possible pathognomonic developments.