Toward Chemical Validation of Leishmania infantum Ribose 5-Phosphate Isomerase as a Drug Target.

Neglected tropical diseases caused by kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.) place a significant health and economic burden on developing nations worldwide. Current therapies are largely outdated, inadequate, and face mounting drug resistance from the causative parasites. Thus, there is an urgent need for drug discovery and development. Target-led drug discovery approaches have focused on the identification of parasite enzymes catalyzing essential biochemical processes, which significantly differ from equivalent proteins found in humans, thereby providing potentially exploitable therapeutic windows. One such target is ribose 5-phosphate isomerase B (RpiB), an enzyme involved in the nonoxidative branch of the pentose phosphate pathway, which catalyzes the interconversion of d-ribose 5-phosphate and d-ribulose 5-phosphate. Although protozoan RpiB has been the focus of numerous targeted studies, compounds capable of selectively inhibiting this parasite enzyme have not been identified. Here, we present the results of a fragment library screening against Leishmania infantum RpiB (LiRpiB), performed using thermal shift analysis. Hit fragments were shown to be effective inhibitors of LiRpiB in activity assays, and several fragments were capable of selectively inhibiting parasite growth in vitro. These results support the identification of LiRpiB as a validated therapeutic target. The X-ray crystal structure of apo LiRpiB was also solved, permitting docking studies to assess how hit fragments might interact with LiRpiB to inhibit its activity. Overall, this work will guide structure-based development of LiRpiB inhibitors as antileishmanial agents.

Apoptotic marker expression in the absence of cell death in staurosporine-treated Leishmania donovani.

The protozoan parasite Leishmania donovani undergoes several developmental transitions in its insect and vertebrate hosts that are induced by environmental changes. The roles of protein kinases in these adaptive differentiation steps and their potential as targets for antiparasitic intervention are only poorly characterized. Here, we used the generic protein kinase inhibitor staurosporine to gain insight into how interference with phosphotransferase activities affects the viability, growth, and motility of L. donovani promastigotes in vitro. Unlike the nonkinase drugs miltefosine and amphotericin B, staurosporine strongly reduced parasite biosynthetic activity and had a cytostatic rather than a cytotoxic effect. Despite the induction of a number of classical apoptotic markers, including caspase-like activity and surface binding of annexin V, we determined that, on the basis of cellular integrity, staurosporine did not cause cell death but caused cell cycle arrest and abrogated parasite motility. In contrast, targeted inhibition of the parasite casein kinase 1 (CK1) protein family by use of the CK1-specific inhibitor D4476 resulted in cell death. Thus, pleiotropic inhibition of L. donovani protein kinases and possibly other ATP-binding proteins by staurosporine dissociates apoptotic marker expression from cell death, which underscores the relevance of specific rather than broad kinase inhibitors for antiparasitic drug development.

The crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1: Implications to protein function and drug design.

The de novo crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1 (LiSir2rp1) has been solved at 1.99Å in complex with an acetyl-lysine peptide substrate. The structure is broadly commensurate with Hst2/SIRT2 proteins of yeast and human origin, reproducing many of the structural features common to these sirtuin deacetylases, including the characteristic small zinc-binding domain, and the larger Rossmann-fold domain involved in NAD+-binding interactions. The two domains are linked via a cofactor binding loop ordered in open conformation. The peptide substrate binds to the LiSir2rp1 protein via a cleft formed between the small and large domains, with the acetyl-lysine side chain inserting further into the resultant hydrophobic tunnel. Crystals were obtained only with recombinant LiSir2rp1 possessing an extensive internal deletion of a proteolytically-sensitive region unique to the sirtuins of kinetoplastid origin. Deletion of 51 internal amino acids (P253-E303) from LiSir2rp1 did not appear to alter peptide substrate interactions in deacetylation assays, but was indispensable to obtain crystals. Removal of this potentially flexible region, that otherwise extends from the classical structural elements of the Rossmann-fold, specifically the ß8-ß9 connector, appears to result in lower accumulation of the protein when expressed from episomal vectors in L. infantum SIR2rp1 single knockout promastigotes. The biological function of the large serine-rich insertion in kinetoplastid/trypanosomatid sirtuins, highlighted as a disordered region with strong potential for post-translational modification, remains unknown but may confer additional cellular functions that are distinct from their human counterparts. These unique molecular features, along with the resolution of the first kinetoplastid sirtuin deacetylase structure, present novel opportunities for drug design against a protein target previously established as essential to parasite survival and proliferation.

Inhibitors of Trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease.

Chagas disease remains one of the most neglected diseases in the world despite being the most important parasitic disease in Latin America. The characteristic chronic manifestation of chagasic cardiomyopathy is the region's leading cause of heart-related illness, causing significant mortality and morbidity. Due to the limited available therapeutic options, new drugs are urgently needed to control the disease. Sirtuins, also called Silent information regulator 2 (Sir2) proteins have long been suggested as interesting targets to treat different diseases, including parasitic infections. Recent studies on Trypanosoma cruzi sirtuins have hinted at the possibility to exploit these enzymes as a possible drug targets. In the present work, the T. cruzi Sir2 related protein 1 (TcSir2rp1) is genetically validated as a drug target and biochemically characterized for its NAD+-dependent deacetylase activity and its inhibition by the classic sirtuin inhibitor nicotinamide, as well as by bisnaphthalimidopropyl (BNIP) derivatives, a class of parasite sirtuin inhibitors. BNIPs ability to inhibit TcSir2rp1, and anti-parasitic activity against T. cruzi amastigotes in vitro were investigated. The compound BNIP Spermidine (BNIPSpd) (9), was found to be the most potent inhibitor of TcSir2rp1. Moreover, this compound showed altered trypanocidal activity against TcSir2rp1 overexpressing epimastigotes and anti-parasitic activity similar to the reference drug benznidazole against the medically important amastigotes, while having the highest selectivity index amongst the compounds tested. Unfortunately, BNIPSpd failed to treat a mouse model of Chagas disease, possibly due to its pharmacokinetic profile. Medicinal chemistry modifications of the compound, as well as alternative formulations may improve activity and pharmacokinetics in the future. Additionally, an initial TcSIR2rp1 model in complex with p53 peptide substrate was obtained from low resolution X-ray data (3.5 Å) to gain insight into the potential specificity of the interaction with the BNIP compounds. In conclusion, the search for TcSir2rp1 specific inhibitors may represent a valuable strategy for drug discovery against T. cruzi.

Validation of standard operating procedures in a multicenter retrospective study to identify -omics biomarkers for chronic low back pain.

Chronic low back pain (CLBP) is one of the most common medical conditions, ranking as the greatest contributor to global disability and accounting for huge societal costs based on the Global Burden of Disease 2010 study. Large genetic and -omics studies provide a promising avenue for the screening, development and validation of biomarkers useful for personalized diagnosis and treatment (precision medicine). Multicentre studies are needed for such an effort, and a standardized and homogeneous approach is vital for recruitment of large numbers of participants among different centres (clinical and laboratories) to obtain robust and reproducible results. To date, no validated standard operating procedures (SOPs) for genetic/-omics studies in chronic pain have been developed. In this study, we validated an SOP model that will be used in the multicentre (5 centres) retrospective "PainOmics" study, funded by the European Community in the 7th Framework Programme, which aims to develop new biomarkers for CLBP through three different -omics approaches: genomics, glycomics and activomics. The SOPs describe the specific procedures for (1) blood collection, (2) sample processing and storage, (3) shipping details and (4) cross-check testing and validation before assays that all the centres involved in the study have to follow. Multivariate analysis revealed the absolute specificity and homogeneity of the samples collected by the five centres for all genetics, glycomics and activomics analyses. The SOPs used in our multicenter study have been validated. Hence, they could represent an innovative tool for the correct management and collection of reliable samples in other large-omics-based multicenter studies.

'Omics' biomarkers associated with chronic low back pain: protocol of a retrospective longitudinal study.

INTRODUCTION: Chronic low back pain (CLBP) produces considerable direct costs as well as indirect burdens for society, industry and health systems. CLBP is characterised by heterogeneity, inclusion of several pain syndromes, different underlying molecular pathologies and interaction with psychosocial factors that leads to a range of clinical manifestations. There is still much to understand in the underlying pathological processes and the non-psychosocial factors which account for differences in outcomes. Biomarkers that may be objectively used for diagnosis and personalised, targeted and cost-effective treatment are still lacking. Therefore, any data that may be obtained at the '-omics' level (glycomics, Activomics and genome-wide association studies-GWAS) may be helpful to use as dynamic biomarkers for elucidating CLBP pathogenesis and may ultimately provide prognostic information too. By means of a retrospective, observational, case-cohort, multicentre study, we aim to investigate new promising biomarkers potentially able to solve some of the issues related to CLBP. METHODS AND ANALYSIS: The study follows a two-phase, 1:2 case-control model. A total of 12 000 individuals (4000 cases and 8000 controls) will be enrolled; clinical data will be registered, with particular attention to pain characteristics and outcomes of pain treatments. Blood samples will be collected to perform -omics studies. The primary objective is to recognise genetic variants associated with CLBP; secondary objectives are to study glycomics and Activomics profiles associated with CLBP. ETHICS AND DISSEMINATION: The study is part of the PainOMICS project funded by European Community in the Seventh Framework Programme. The study has been approved from competent ethical bodies and copies of approvals were provided to the European Commission before starting the study. Results of the study will be reviewed by the Scientific Board and Ethical Committee of the PainOMICS Consortium. The scientific results will be disseminated through peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT02037789; Pre-results.

FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter.

We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.

The role of glycosylation in IBD.

A number of genetic and immunological studies give impetus for investigating the role of glycosylation in IBD. Experimental mouse models have helped to delineate the role of glycosylation in intestinal mucins and to explore the putative pathogenic role of glycosylation in colitis. These experiments have been extended to human studies investigating the glycosylation patterns of intestinal mucins as well as levels of glycans of serum glycoproteins and expression of glycan receptors. These early human studies have generated interesting hypotheses regarding the pathogenic role of glycans in IBD, but have generally been restricted to fairly small underpowered studies. Decreased glycosylation has been observed in the intestinal mucus of patients with IBD, suggesting that a defective inner mucus layer might lead to increased bacterial contact with the epithelium, potentially triggering inflammation. In sera, decreased galactosylation of IgG has been suggested as a diagnostic marker for IBD. Advances in glycoprofiling technology make it technically feasible and affordable to perform high-throughput glycan pattern analyses and to build on previous work investigating a much wider range of glycan parameters in large numbers of patients.

Probing the dynamic nature of signalling pathways by IMAC and SELDI-tof MS.

One major obstacle to the analysis of signalling pathways is the dynamic nature of signalling response to environmental stimuli. To overcome this limitation we applied immobilized metal affinity chromatography (IMAC) in combination with SELDI-tof MS to investigate the temporal variation of protein phosphorylation. We analysed the phospho-proteome variations in our model organism, Leishmania donovani, in response to changes in pH and temperature, which induce differentiation from promastigotes to amastigotes. Investigation of total cell extracts did not allow promastigote and amastigote life cycle stages to be distinguished. However, using IMAC enriched samples, the pattern and intensity of phospho-proteins analysed distinguished both stages reproducibly. Approximately 61% of the phospho-proteins analysed were significantly different in abundance (p<0.02). Of these 61%, 73% showed an increased phosphorylation in promastigotes while 27% showed an increase phosphorylation in amastigotes. The workflow developed is currently being applied to the temporal analysis of environmental stimuli.