Structure based designing of thiazolidinone-pyrimidine derivatives as ERK2 inhibitors: Synthesis and in vitro evaluation.

Breast cancer has been associated with an overexpression of various molecular targets; accordingly, various target-specific chemotherapeutic agents have been developed. Inhibition of ERK2, a member of MAPK pathway, is an important target involved in the treatment of both oestrogen receptor-positive and triple-negative breast cancer. Thus, in continuation of our previous work on the ERK2 target, we here report novel inhibitors of this kinase. Out of three lead molecules reported in our previous study, we selected the thiazolidinone-pyrimidine scaffold for further development of small molecule inhibitors of ERK2. Analogues of the lead molecule were docked in the target kinase, followed by molecular dynamic simulations and MM-GBSA calculations. Analogues maintaining key interactions with amino acid residues in the ATP-binding domain of ERK2 were selected and duly synthesized. In vitro biochemical evaluation of these molecules against ERK2 kinase disclosed that two molecules possess significant kinase inhibitory potential with IC50 values ≤ 0.5 µM.

Exploring RdRp-remdesivir interactions to screen RdRp inhibitors for the management of novel coronavirus 2019-nCoV.

A novel coronavirus recently identified in Wuhan, China (2019-nCoV) has resulted in an increasing number of patients globally, and has become a highly lethal pathogenic member of the coronavirus family affecting humans. 2019-nCoV has established itself as one of the most threatening pandemics that human beings have faced, and therefore analysis and evaluation of all possible responses against infection is required. One such strategy includes utilizing the knowledge gained from the SARS and MERS outbreaks regarding existing antivirals. Indicating a potential for success, one of the drugs, remdesivir, under repurposing studies, has shown positive results in initial clinical studies. Therefore, in the current work, the authors have attempted to utilize the remdesivir-RdRp complex - RdRp (RNA-dependent RNA polymerase) being the putative target for remdesivir - to screen a library of the already reported RdRp inhibitor database. Further clustering on the basis of structural features and scoring refinement was performed to filter out false positive hits. Finally, molecular dynamics simulation was carried out to validate the identification of hits as RdRp inhibitors against novel coronavirus 2019-nCoV. The results yielded two putative hits which can inhibit RdRp with better potency than remdesivir, subject to further biological evaluation.

Overexpression of BLM promotes DNA damage and increased sensitivity to platinum salts in triple-negative breast and serous ovarian cancers.

Background: Platinum-based therapy is an effective treatment for a subset of triple-negative breast cancer and ovarian cancer patients. In order to increase response rate and decrease unnecessary use, robust biomarkers that predict response to therapy are needed. Patients and methods: We performed an integrated genomic approach combining differential analysis of gene expression and DNA copy number in sensitive compared with resistant triple-negative breast cancers in two independent neoadjuvant cisplatin-treated cohorts. Functional relevance of significant hits was investigated in vitro by overexpression, knockdown and targeted inhibitor treatment. Results: We identified two genes, the Bloom helicase (BLM) and Fanconi anemia complementation group I (FANCI), that have both increased DNA copy number and gene expression in the platinum-sensitive cases. Increased level of expression of these two genes was also associated with platinum but not with taxane response in ovarian cancer. As a functional validation, we found that overexpression of BLM promotes DNA damage and induces sensitivity to cisplatin but has no effect on paclitaxel sensitivity. Conclusions: A biomarker based on the expression levels of the BLM and FANCI genes is a potential predictor of platinum sensitivity in triple-negative breast cancer and ovarian cancer.

Hypoalbuminaemia is an independent predictor of poor outcome in metastatic Ewing's sarcoma family of tumours: a single institutional experience of 150 cases treated with uniform chemotherapy protocol.

AIMS: Data on metastatic Ewing's sarcoma family of tumours (ESFT) with uniform chemotherapy protocol are minimal. MATERIALS AND METHODS: This was a single institutional patient review of patients treated between June 2003 and November 2011 and evaluated on an intent-to-treat analysis. All patients received uniform chemotherapy: neoadjuvant chemotherapy (NACT), surgery and/or radiotherapy as local treatment followed by adjuvant chemotherapy. Local treatment was offered if the patient achieved a complete response and/or a partial response at both the primary and the metastatic site. RESULTS: In total, 150/374 (40%) ESFT patients were metastatic, with a median age of 15 years (range: 2-50); a tumour diameter of 10 cm (range: 1.8-26). Most common metastatic sites were lung only (53; 35%), bone only (35; 23%) and combined bone/lung (25; 17%). Twenty patients underwent surgery; 55 patients received radical radiotherapy after NACT. After a median follow-up of 26.1 months (range: 1.6-101.6), 5 year event-free survival (EFS), overall survival and local control rate (LCR) were 9.1 ± 3.3%, 16.9 ± 5.2% and 31.8 ± 7.9%, respectively. Univariate analysis showed serum albumin ≤3.4 g/dl (P < 0.001) to predict inferior EFS. Tumour size >8 cm (P = 0.05), haemoglobin ≤10 g/dl (P = 0.04), hypoalbuminaemia (P = 0.003) and radical radiotherapy as local treatment (P = 0.03) predicted inferior overall survival. No factor significantly predicted LCR, although age ≤15 years (P = 0.08) and radical radiotherapy as local treatment (P = 0.09) had a trend towards inferior LCR. Hypoalbuminaemia was the only prognostic factor to predict EFS on multivariate analysis. CONCLUSION: This was the largest study of metastatic ESFT from Asia and identified a unique prognostic factor. In view of dismal prognosis with conventional chemotherapy in metastatic ESFT with hypoalbuminaemia, palliative intent therapy may be a potential therapeutic alternative for this subgroup of patients, especially in resource-challenged situations.

Review of spectrum and sensitivity of bacterial bloodstream isolates in children with malignancy: A retrospective analysis from a single center.

BACKGROUND: Febrile neutropenia is a life-threatening emergency in pediatric cancer patients. Its management is based on established guidelines that emphasize on prompt action. Consideration of local microbiologic spectrum and its susceptibility is pivotal in devising a rational protocol. AIMS: To study the spectrum of bacterial isolates and its antibiotic sensitivity profile in bloodstream infections (BSIs) of pediatric cancer patients. SETTINGS AND DESIGN: Retrospective study. MATERIALS AND METHODS: This study was conducted at a tertiary cancer center for pediatric cancer patients. Blood culture samples sent during the evaluation of patients with clinical diagnosis of febrile neutropenia during the year of 2013 were analyzed. The microbiological and antibiotic sensitivity patterns were studied. RESULTS: A total of 27 isolates represented BSIs out of 412 blood cultures sent (6.5%). These were predominantly Gram-negative (92%) with Klebsiella contributing to the majority of them. Extended spectrum beta-lactamase production was seen in 59% of all isolates. Multidrug resistance phenotype was seen in 48%, extreme drug resistance in 32% and pan drug resistance in 16% of Gram-negative isolates. Klebsiella predominated in all of these isolates. Mortality resulted in 15% isolates, majorly contributed by Klebsiella. Colistin was the most sensitive antibiotic (75% sensitivity) and in significant number of cases the only salvage option. CONCLUSION: Gram-negative bacteria are the most common etiologic agents. The emergence of drug resistant strains of Klebsiella and the poor sensitivity of most of these strains to common first choice empiric agents is alarming. Low prevalence of Gram-positive organisms questions the routine use of empiric vancomycin.

Detection and quantification of bovine papilloma virus type 2 (BPV-2) by real-time PCR in urine and urinary bladder lesions in enzootic bovine haematuria (EBH)-affected cows.

This study was conducted with the objectives of detecting bovine papillomavirus type 2 (BPV-2) in urine samples and urinary bladder lesions in bovines using polymerase chain reaction (PCR) and real-time PCR-based molecular diagnostic tests, and quantifying BPV-2 in urinary bladder lesions especially in enzootic bovine haematuria (EBH)-affected animals. BPV-2 viral DNA was detected in urine samples (50%) and urinary bladder tissue (68.6%). Cloning and sequencing results showed a close homology with other Indian BPV-2 sequences. Quantitative real-time PCR (SYBR Green assay) showed that the BPV-2 load was low and similar irrespective of inflammatory or neoplastic lesions in the bladder. It was concluded that BPV-2 DNA is frequently present in urine and urinary bladder lesions in cows in an EBH endemic region and virus load was low in urinary bladder lesions.

Preliminary assessment of binary ethylenimine inactivated and saponized cutaneous warts (BPV-2) therapeutic vaccine for enzootic bovine haematuria in hill cows.

A preliminary therapeutic vaccine trial was conducted in hill cows to evaluate the therapeutic potential of binary ethylenimine (BEI) inactivated and saponized bovine papillomavirus-2 (BPV-2) for enzootic bovine haematuria (EBH). Although the vaccine failed to show favorable clinical vaccine results in treatment of EBH affected cows at 120 days post-vaccination but immunopathological responses were encouraging. A significant difference was observed in humoral (against Brucella abortus strain 19S) and cell-mediated (in vivo phytohaemagglutination delayed type hypersensitivity (PHA DTH) test and CD4+/CD8+ T-cells ratio by FACS analysis) immune responses following vaccination. The vaccinated animals grossly failed to show regression of bladder tumours but microscopically engorgement and marked perivascular infiltration of mononuclear cells was observed which are indicative of the induction of initial stages of tumour regression. Overall results indicated that the therapeutic vaccine developed can have potentials for treating EBH in cows, for which further modifications in vaccine dose and field trial is required.

Ectopic expression of alkaline phosphatase in proximal tubular brush border membrane of human renal cell carcinoma.

The present study was conducted to find out any alteration in the expression and activity of alkaline phosphatase in the brush border membrane (BBM) from renal cell carcinoma (RCC) in comparison to normal renal BBM. The specific activity of alkaline phosphatase was drastically reduced in homogenate as well as BBM from RCC kidney when compared to ALP activity in BBM of normal kidney. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (V(max)) and increase in affinity constant (K(m)) of the enzyme. SDS-PAGE studies showed that the BBM proteins having molecular weights ranging from 95 to 170 kDa were poorly expressed in RCC BBM in relative to normal kidney BBM. Incubation of SDS-PAGE gel with BCIP/NBT dye clearly showed that the expression of ALP in tumor renal BBM was markedly reduced as compared to normal kidney. Further, Western blot analysis using anti-alkaline phosphatase antibody also confirmed the reduced expression of ALP in tumor renal BBM. Lipid composition in reference to phospholipids, glycolipids and cholesterol in tumor renal BBM was altered to that of normal renal BBM, indicating alteration in membrane fluidity of tumor renal BBM.

A general model for site-specific recombination by the integrase family recombinases.

We present here a general model for integrase family site-specific recombination using the geometric relationships of the cleavable phosphodiester bonds and the disposition of the recombinase monomers (defined by their binding planes) with respect to them. The 'oscillation model' is based largely on the conformations of the recombinase-bound DNA duplexes and their dynamics within Holliday junctions. The duplex substrate or the Holliday junction intermediate is capable of 'oscillating' between two cleavage-competent asymmetric states with respect to corres-ponding chemically inert 'equilibrium positions'. The model accommodates several features of the Flp system and predicts two modes of DNA cleavage during a normal recombination event. It is equally applicable to other systems that mediate recombination across 6, 7 or 8 bp long strand exchange regions (or spacers). The model is consistent with approximately 0-1, 1-2 and 2-3 bp of branch migration during recombination reactions involving 6, 7 and 8 bp spacers, respectively.

Flp ribonuclease activities. Mechanistic similarities and contrasts to site-specific DNA recombination.

The ribonuclease active site harbored by the Flp site-specific recombinase can act on two neighboring phosphodiester bonds to yield mechanistically distinct chain breakage reactions. One of the RNase reactions apparently proceeds via a covalent enzyme intermediate and targets the phosphodiester position involved in DNA recombination (Flp RNase I activity). The second activity (Flp RNase II) targets the phosphodiester immediately to the 3' side but appears not to involve an enzyme-linked intermediate. Flp RNase I is absolutely dependent upon Tyr-343 of Flp and is competitive with respect to the normal strand joining reaction. It can utilize the 2'-hydroxyl group from any one of the four ribonucleotides with comparable efficiencies in the cleavage reaction. On the other hand, the RNase II reaction mediated by Flp(Y343F) is specific for U and cannot utilize the 2'-hydroxyl group from ribo-A, -G, or -C under standard reaction conditions. The RNase II activity is also sensitive to the 3'-neighboring base. Although dT is functional, the activity is stimulated by U or U-2'-OMe. The Flp RNase II reaction effectively competes with the normal strand cleavage reaction mediated by Tyr-343, even though their phosphodiester targets are not the same.

Structural alterations and conformational dynamics in Holliday junctions induced by binding of a site-specific recombinase.

Binding of a cleavage-incompetent mutant of the Flp recombinase induces a roughly square-planar geometry in synthetic immobile Holliday junctions. The branch points, which are rigidly fixed in these junctions in their free forms, tend to be more flexible in their protein-bound forms. Our results (1) suggest a plausible mechanism for the switching of the recombination complex from the Holliday-forming mode to the Holliday-resolving mode, (2) provide a rationale for previous observations that Flp resolves preformed immobile Holliday structures in the parental or in the recombinant mode in a relatively unbiased manner, and (3) accommodate two modes of DNA cleavage by Flp (transhorizontal or transdiagonal) in Holliday substrates.