Cyclopentyl-pyrimidine based analogues as novel and potent IGF-1R inhibitor.

A series of novel 2-amino-4-pyrazolecyclopentylpyrimidines have been prepared and evaluated as IGF-1R tyrosin kinase inhibitors. The in vitro activity was found to depend strongly on the substitution pattern in the 2- amino ring, 4-pyrazolo moieties and size of fused saturated ring with the central pyrimidine core. A stepwise optimization by combination of active fragments led to discovery of compound 6f and 6k, two structures with IGF-1R IC50 of 20 nM and 10 nM, respectively. 6f was further profiled for its anti cancer activity across various cell lines and pharmacokinetic studies in Sprague Dawley rats.

Lead optimization of isocytosine-derived xanthine oxidase inhibitors.

We report our attempts at improving the oral efficacy of low-nanomolar inhibitors of xanthine oxidase from isocytosine series through chemical modifications. Our lead compound had earlier shown good in vivo efficacy when administered intraperitoneally but not orally. Several modifications are reported here which achieved more than twofold improvement in exposure. A compound with significant improvement in oral efficacy was also obtained.

Synthesis of N-(6-(4-(Piperazin-1-yl)phenoxy)pyridin-3-yl)benzenesulfonamide Derivatives for the Treatment of Metabolic Syndrome.

Metabolic syndrome is a widely prevalent multifactorial disorder associated with an increased risk of cardiovascular disease and type 2 diabetes mellitus. High plasma levels of insulin and glucose due to insulin resistance are a major component of the metabolic disorder. Thiazolidinediones (TZDs) are potent PPARγ ligand and used as insulin sensitizers in the treatment of type 2 diabetes mellitus. They are potent insulin-sensitizing agents but due to adverse effects like hepatotoxicity, a safer alternative of TZDs is highly demanded. Here we report synthesis of N-(6-(4-(piperazin-1-yl)phenoxy)pyridin-3-yl)benzenesulfonamide derivatives as an alternate remedy for insulin resistance.

Isocytosine-based inhibitors of xanthine oxidase: design, synthesis, SAR, PK and in vivo efficacy in rat model of hyperuricemia.

Structure-activity relationship studies were carried out for lead generation following structure-guided design approach from an isocytosine scaffold identified earlier for xanthine oxidase inhibition. A 470-fold improvement in in vitro IC(50) was obtained in the process. Five most potent compounds with nanomolar IC(50) values were selected for pharmacokinetics and in vivo experiments. The best compound showed good in vivo activity when administered intraperitoneally but was not active by oral route. The results suggest that improvement in oral exposure could improve the in vivo efficacy of this series.

Identification of novel isocytosine derivatives as xanthine oxidase inhibitors from a set of virtual screening hits.

In recent years, xanthine oxidase has emerged as an important target not only for gout but also for cardiovascular and metabolic disorders involving hyperuricemia. Contrary to popular belief, recent clinical trials with uricosurics have demonstrated that enhanced excretion of uric acid is, by itself, not adequate to treat hyperuricemia; simultaneous inhibition of production of uric acid by inhibition of xanthine oxidase is also important. Virtual screening of in-house synthetic library followed by in vitro and in vivo testing led to the identification of a novel scaffold for xanthine oxidase inhibition. In vitro activity results corroborated the results from molecular docking studies of the virtual screening hits. The isocytosine scaffold maintains key hydrogen bonding and pi-stacking interactions in the deep end of the xanthine-binding pocket, which anchors it in an appropriate pose to inhibit binding of xanthine and shows promise for further lead optimization using structure-based drug design approach.

Managing protein flexibility in docking and its applications.

Docking, virtual screening and structure-based drug design are routinely used in modern drug discovery programs. Although current docking methods deal with flexible ligands, managing receptor flexibility has proved to be challenging. In this brief review, we present the current state-of-the-art for computationally handling receptor flexibility, including a novel statistical computational approach published recently. We conclude, from a comparison of the different approaches, that a combination of methods is likely to provide the most reliable solution to the problem of finding the right protein conformation for a given ligand.

A fluorinated analog of ISO-1 blocks the recognition and biological function of MIF and is orally efficacious in a murine model of colitis.

A promising therapeutic approach to diminish pathological inflammation is to inhibit the synthesis and/or biological activity of macrophage migration inhibitory factor (MIF). Prior studies have shown that intraperitoneal administration of small-molecule inhibitors targeting the catalytic pocket of MIF (e.g., ISO-1) elicits a therapeutic effect in mouse inflammation models. However, it remains to be elucidated whether these tautomerase activity inhibitors block the synthesis and/or biological activity of MIF. In this study, we investigated and compared the activity of representative MIF inhibitors from isoxazole series (fluorinated analog of ISO-1; ISO-F) and substituted quinoline series (compound 7E; 7E). Our results demonstrate that ISO-F is a more potent MIF inhibitor than 7E. Both ISO-F and 7E do not inhibit MIF synthesis but "bind-onto" MIF thereby blocking its recognition. However, in contrast to 7E, ISO-F docks well in the active site of MIF and also has a stronger binding affinity towards MIF. In line with these observations, ISO-F, but not 7E, robustly inhibits the biological function of MIF. Most importantly, ISO-F, when administered orally in a therapeutic regimen, significantly suppresses dextran sulphate sodium (DSS)-induced murine colitis. This study, which provides mechanistic insights into the anti-inflammatory efficacy of ISO-F, is the first documented report of in vivo anti-inflammatory efficacy of a MIF inhibitor upon oral administration. Moreover, the findings from this study reinforce the potential of catalytic site of MIF as a target for eliciting therapeutic effect in inflammatory disorders. Compounds (e.g., ISO-F) that block not only the recognition but also the biological function of MIF are potentially attractive for reducing pathological inflammation.

Modeling and selection of flexible proteins for structure-based drug design: backbone and side chain movements in p38 MAPK.

Receptor rearrangement upon ligand binding (induced fit) is a major stumbling block in docking and virtual screening. Even though numerous studies have stressed the importance of including protein flexibility in ligand docking, currently available methods provide only a partial solution to the problem. Most of these methods, being computer intensive, are often impractical to use in actual drug discovery settings. We had earlier shown that ligand-induced receptor side-chain conformational changes could be modeled statistically using data on known receptor-ligand complexes. In this paper, we show that a similar approach can be used to model more complex changes like backbone flips and loop movements. We have used p38 MAPK as a test case and have shown that a few simple structural features of ligands are sufficient to predict the induced variation in receptor conformations. Rigorous validation, both by internal resampling methods and on an external test set, corroborates this finding and demonstrates the robustness of the models. We have also compared our results with those from an earlier molecular dynamics simulation study on DFG loop conformations of p38 MAPK, and found that the results matched in the two cases. Our statistical approach enables one to predict the final ligand-induced conformation of the active site of a protein, based on a few ligand properties, prior to docking the ligand. We can do this without having to trace the step-by-step process by which this state is arrived at (as in molecular dynamics simulations), thereby drastically reducing computational effort.

A novel computational analysis of ligand-induced conformational changes in the ATP binding sites of cyclin dependent kinases.

Protein kinases in general are known to be very flexible macromolecules. In this article, the conformational plasticity of the ATP binding site in cyclin dependent kinases is analyzed. Movement of the two lysine residues lining the ATP binding site are shown to play a major role in the conformational variability of the site. Linear models are developed to identify and quantify ligand properties that maximally influence the lysine side chain conformations. A few simple properties of the ligands are shown to account for more than 70% of the variation in the lysine conformations. The results are validated using test data and molecular simulation studies. Illustrative applications of the results of this analysis to finding the appropriate crystal structure for molecular docking and binding mode predictions of novel ligands are provided. This work provides a new approach to quantify ligand-induced conformational changes in the active sites of flexible proteins and to find the appropriate crystal structure for docking novel ligands.

Beta(2)-adrenergic receptor polymorphisms and response to salbutamol among Indian asthmatics*.

INTRODUCTION: The beta(2)-adrenergic receptor (beta(2)AR or ADRbeta(2)) is the target for beta(2)-agonist drugs used for bronchodilation in asthma and other respiratory diseases. The aim of this study was to identify common single nucleotide polymorphisms (SNPs) and haplotypes in asthmatics and healthy individuals from an Indian population, and determine the influence of beta(2)AR SNPs in responsiveness to beta(2)-agonist therapy in asthma patients. METHODS: Ten variable SNP sites within a span of 2.193 kb were identified in the beta(2)AR gene by sequencing and genotyping 374 bronchial asthma patients and healthy individuals from an Indian population. Spirometry tests were performed on 80 unrelated patients before and after administration of 200 microg of salbutamol. A post-bronchodilator forced expiratory volume in one second (FEV(1)) change of >or= 15.3% was considered a good response, and a change of<15.3% was defined as a poor response, to salbutamol. RESULTS: The pattern of linkage disequilibrium between the ten SNPs showed a single, linked SNP block consisting of sites -468, -367, -47, -20, and 79 having strong linkage disequilibrium, while the SNPs at sites -1023, -654, 46, 252, and 523 showed very low linkage with one another and with the linked region. The SNPs were found to be organized into 16 haplotypes in the studied population. We found that patients with a homozygous Arg-16 form at nucleotide position 46 are poor responders with probability of 0.81, and patients with a homozygous Gly-16 form are good responders with a probability of 0.73. The responder status to salbutamol treatment and the genotype at nucleotide position 46 in beta(2)AR gene of an asthmatic patient are significantly associated in the studied Indian population (chi2=9.98, df=2, p=0.0068). Most importantly, this association for responsiveness to salbutamol at nucleotide position 46 is independent of other SNPs in the beta(2)AR gene. CONCLUSION: This study suggests that the SNP at nucleotide position 46 has particular relevance to pharmacogenetics in the Indian population studied.

MLC1 gene is associated with schizophrenia and bipolar disorder in Southern India.

BACKGROUND: Chromosome 22q13 has shown linkage with schizophrenia (SCZ) and bipolar affective disorder (BPAD). A missense mutation in MLC1 (putative cation-channel gene on 22q13) co-segregating with periodic catatonic schizophrenia has been reported. We have investigated the relationship of MLC1 with SCZ and BPAD in Southern India. METHODS: All exons and flanking intronic sequences of MLC1 were screened for novel variations. Case-control (216 BPAD, 193 SCZ, 116 control subjects) and family-based analyses (113 BPAD, 107 SCZ families) were performed to evaluate association of MLC1 with these disorders. RESULTS: We found 33 MLC1 sequence variations, including three novel mutations: Val210Ile, Leu308Gln, and Arg328His in six BPAD cases and Val210Ile in one control individual. Minor allele of a common variation, ss16339182 (in approximately 6 Kb Linkage-Disequilibrium [LD]-block) was associated with BPAD in case-control (p = .03) and family-based analyses (transmitted/nontransmitted [T/NT]-44/20; p = .003). Association was observed for rs2235349 and rs2076137 with SCZ and ss16339163 with BPAD in case-control study. Using Block 2 haplotype tagging single nucleotide polymorphisms (htSNPs), GC haplotype revealed association (p = .02) and excess transmission (p = .002) with BPAD. CONCLUSIONS: Association of MLC1 with SCZ and BPAD suggests involvement of a common pathway. Rare missense mutations and common variants associated with BPAD favors hypothesis about likely involvement of both rare and common polymorphisms in etiology of this complex disorder.

TGFbeta1 haplotypes and asthma in Indian populations.

BACKGROUND: Asthma is a complex disorder of the airways of the lungs. TGF-beta1 plays a key role in airway remodeling and asthma by having both proinflammatory and anti-inflammatory activities, making TGFbeta1 an important candidate gene to study. OBJECTIVE: To investigate the association of TGFbeta1 gene polymorphisms with asthma. METHODS: A case-control study was designed for identifying polymorphisms and haplotypes associated with asthma and associated phenotypes. We have verified our results in 2 independent cohorts collected from northern (number of patients, 187; number of controls, 187) and western India (number of patients, 209; number of controls, 190). We measured the serum TGF-beta1 levels of selected individuals and correlated them with genotypes and haplotypes. RESULTS: A novel (CT)n(CA)m repeat polymorphism (BV209662) 24.9 kb upstream of TGFbeta1 was identified. A significant association was seen at the level of alleles and genotypes with asthma in the 2 cohorts studied independently (P < .05). Interestingly, a novel 3-locus haplotype, 23_G_T, was found to be significantly associated with asthma (P = .00001 in cohorts A and B) as well as with higher serum TGF-beta1 level (P = .01). On the other hand, a novel haplotype, 22_G_C, was negatively associated with asthma (P = .00001 for cohorts A and B) and with lower serum TGF-beta1 level (P = .0019). CONCLUSION: This is the first study identifying novel risk and protective haplotypes--23_G_T and 22_G_C, respectively--in the TGFbeta1 gene that are associated with asthma. We also demonstrate the functional significance of these haplotypes with serum TGF-beta1 levels. These results would be valuable in elucidating the role of TGF-beta1 in asthma pathogenesis.

Signal transducer and activator of transcription 6 haplotypes and asthma in the Indian population.

In this paper, we report for the first time the results of an investigation on the association of signal transducer and activator of transcription 6 (STAT6) with asthma in the Indian population. A novel polymorphic CA-repeat in the proximal promoter region [R1] and a previously identified CA-repeat in the 5'-untranslated region [R3] were genotyped, and haplotypes [R1_R3] were generated using PHASE software. The 16 repeat allele at the R1 locus was positively associated (P = 0.01) with asthma. The 15 and 16 repeat alleles at the R3 locus were positively (P < 10(-4)) and negatively (P < 10(-5)) associated with asthma, respectively. Further, the 17_15 (P = 0.0031) and 16_15 (P = 0.001) haplotypes were found to be positively associated with asthma, whereas 17_14, 24_16, and 23_16 were negatively associated (P < 10(-5)). It appears that the R3 and R1 loci together play a bigger role in asthma than either of them alone, and the R3 locus has a larger effect than the R1 locus. Although alleles at the R1 locus appeared to be associated with total serum immunoglobulin E level, the genotypes showed no association, and the R3 locus showed no effect. As no exonic variants of STAT6 are known as yet, repeat polymorphisms in the regulatory regions and their haplotypes could be important in deciphering the genetic role of STAT6 in asthma and atopy.

(TG:CA)(n) repeats in human housekeeping genes.

The unravelling of human genome sequence gives a new opportunity to investigate the role of repetitive sequences in gene regulation. Among the various types of repetitive sequences, the dinucleotide (TG:CA)(n) repeats are one of the most abundant in human genome and exhibit polymorphism. Early on, it was observed that the (TG:CA)(n) repeats could modulate gene expression and has the propensity to undergo conformational transitions in in vivo conditions. Recent reports describe the role of polymorphic (TG:CA)(n) repeats in gene regulation in several genes. In this work, we have analysed the distribution of (TG:CA)(n) (n >or= 6) repeats in human 'housekeeping genes' on which recently released Gene Chip data is available. Our results indicate that (i). The number of short intragenic (TG:CA)(n) repeats is significantly higher than the number of long repeats (ii). the proportion of genes with (TG:CA)(n) repeats (n >or= 12 units) had lower mean expression levels compared to those without these repeats, (iii). the genes belonging to the functional class of 'signalling and communication' had a positive association with repeats in contrast to the genes belonging to the 'information' class that were negatively associated with repeats.

Nonrandom distribution of alu elements in genes of various functional categories: insight from analysis of human chromosomes 21 and 22.

The first draft of the human genome has revealed enormous variability in the global distribution of Alu repeat elements. There are regions such as the four homeobox gene clusters, which are nearly devoid of these repeats that contrast with repeat dense regions in other transcriptionally active regions of the genome. Our analysis of the completely sequenced chromosomes 21 and 22 revealed a striking bias in Alu distribution. These elements are more clustered in genes which are involved in metabolism, transport, and signaling processes. In contrast, they are significantly fewer in genes coding for information pathway components as well as structural proteins. This bias in Alu distribution is independent of the effect of Alu density of the flanking genomic region and is also not affected by the GC content of the gene and its upstream and downstream regions. The relative proportions of Alu subfamilies (Alu J, Alu S, and Alu Y) are not significantly different in genes with high Alu density belonging to the functional categories of transport, metabolism, and signaling. However, in the structural proteins and information genes, these proportions are lower than the other three categories. We suggest that Alu elements might be involved in regulatory mechanisms and are therefore differentially selected in primate genomes.

A novel complexity measure for comparative analysis of protein sequences from complete genomes.

Analysis of sequence complexities of proteins is an important step in the characterization and classification of new genomes. A new measure has been proposed to compute sequence complexity in protein sequences based on linguistic complexity. The algorithm requires a single parameter, is computationally simple and provides a framework for comparative genomic analysis. Protein sequences were classified into groups of high or low complexity based on a quantitative measure termed F(c), which is proportional to the fraction of low complexity sequence present in the protein. The algorithm was tested on sequences of 196 non-homologous proteins whose crystal structures are available at

Association of IFNG gene polymorphism with asthma in the Indian population.

Epidemiologic studies in India show that the prevalence of asthma is increasing, but no genetic studies have been reported on the Indian population thus far. We selected the IFNG locus on 12q21 as a candidate gene for asthma on the basis of its role in pathophysiology and positive linkage demonstrated in other populations. The aim of this study was to investigate association of a CA-repeat marker in this gene with asthma and total serum IgE levels in the North Indian population. The repeat region was PCR-amplified from patients and control subjects and analyzed through use of GeneScan. The distributions of allele sizes were found to be significantly different between patients and control subjects (Kolmogorov-Smirnov test, P < 10(-6)). Alleles 10 and 11 were found to be overrepresented in individuals with asthma, whereas alleles 13 and 15 were less likely in asthmatic individuals. We found that the CA-repeat polymorphism in the IFNG gene was significantly associated with total serum IgE levels (ANOVA, P < 10(-4) for control subjects and P =.0036 for patients). Furthermore, a previously reported promoter polymorphism at the -333 base pair position was not detected in our population. This is the first report on the association of a candidate gene with asthma from the Indian subcontinent.

Comparative genomics using data mining tools.

We have analysed the genomes of representatives of three kingdoms of life, namely, archaea, eubacteria and eukaryota using data mining tools based on compositional analyses of the protein sequences. The representatives chosen in this analysis were Methanococcus jannaschii, Haemophilus influenzae and Saccharomyces cerevisiae. We have identified the common and different features between the three genomes in the protein evolution patterns. M. jannaschii has been seen to have a greater number of proteins with more charged amino acids whereas S. cerevisiae has been observed to have a greater number of hydrophilic proteins. Despite the differences in intrinsic compositional characteristics between the proteins from the different genomes we have also identified certain common characteristics. We have carried out exploratory Principal Component Analysis of the multivariate data on the proteins of each organism in an effort to classify the proteins into clusters. Interestingly, we found that most of the proteins in each organism cluster closely together, but there are a few 'outliers'. We focus on the outliers for the functional investigations, which may aid in revealing any unique features of the biology of the respective organisms

Study of the single nucleotide polymorphism (SNP) at the palindromic sequence of hypersensitive site (HS)4 of the human beta-globin locus control region (LCR) in Indian population.

LCR, a genetic regulatory element, was examined in beta-thalassemia patients who do not show any mutation in the beta-globin genes. We sequenced LCR-HS2, HS3, and HS4 in samples from 16 such patients from the Indian population and found only one SNP A-G in the inverted repeat in HS4. A significant association was observed between the G allele and occurrence of beta-thalassemia by Fisher's exact test. The AG and GG genotypes showed higher relative risk as compared to the AA genotype. We also observed linkage disequilibrium between the A/G polymorphism and the AT-rich motif of the LCR HS2 region, suggesting that the G allele could be an evolutionarily new mutation in the study population.