Discovery of a novel and highly selective JAK3 inhibitor as a potent hair growth promoter.

JAK-STAT signalling pathway inhibitors have emerged as promising therapeutic agents for the treatment of hair loss. Among different JAK isoforms, JAK3 has become an ideal target for drug discovery because it only regulates a narrow spectrum of γc cytokines. Here, we report the discovery of MJ04, a novel and highly selective 3-pyrimidinylazaindole based JAK3 inhibitor, as a potential hair growth promoter with an IC50 of 2.03 nM. During in vivo efficacy assays, topical application of MJ04 on DHT-challenged AGA and athymic nude mice resulted in early onset of hair regrowth. Furthermore, MJ04 significantly promoted the growth of human hair follicles under ex-vivo conditions. MJ04 exhibited a reasonably good pharmacokinetic profile and demonstrated a favourable safety profile under in vivo and in vitro conditions. Taken together, we report MJ04 as a highly potent and selective JAK3 inhibitor that exhibits overall properties suitable for topical drug development and advancement to human clinical trials.

The Expression of the RUVBL1 Component of the R2TP Complex Correlates with Poor Prognosis in DLBCL.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of non-Hodgkin's lymphoma (NHL) accounting for 30% of adult NHL worldwide and 50% in developing countries like India. DNA damage and Myc-induced transformation are well-known contributing factors towards development of DLBCL. A recently identified HSP90 co-chaperone complex R2TP has been shown to contribute towards DNA damage and Myc-induced transformation. This study aimed to analyse the immunohistochemical (IHC) expression of R2TP complex components RUVBL1, PIH1D1, and RPAP3 in DLBCL patients and correlate with prognosis. METHODS: DLBCL (n = 54) histological slides were retrieved from archives, and detailed histomorphological and clinical features were noted. IHC staining of R2TP complex components RUVBL1, PIH1D1, and RPAP3 was performed on 54 cases (FFPE) of DLBCL. Expression data were correlated with survival and clinical features. RESULTS: Out of the 54 DLBCL cases, 59.26% (n = 32) stained positive for RUVBL1. The RUVBL1 expression was associated with poor prognosis in both progression-free survival (PFS) (p = 0.0146) and overall survival (OS) (p = 0.0328). The expression was positively correlated with bone marrow involvement (p = 0.0525). The expression of PIH1D1 was observed in 68.51% (n = 32) of DLBCL cases, and positive correlation was observed with international prognostic index score (p = 0.0246); however, no correlation was observed with PFS or OS. Finally, RPAP3 was found immunopositive in only 1 case of DLBCL. CONCLUSIONS: Immunopositivity for RUVBL1 is associated with poor prognosis along with a higher relapse rate amongst the DLBCL patients. PIH1D1 immunopositivity correlated with a higher IPI score.

Metallothionein levels in gingival crevicular fluid, saliva, and serum of smokers and non-smokers with chronic periodontitis.

BACKGROUND: Metallothionein (MT), a cysteine rich protein is involved as a radical scavenger in several pathological conditions associated with oxidative stress; however, its role in periodontal disease still remains elusive. The aim of this cross-sectional study is to determine the serum, saliva and gingival crevicular fluid (GCF) levels of MT in smokers (S) and non-smokers (NS) with chronic periodontitis (CP), and compare them with those of periodontally healthy (PH) individuals. METHODS: A total of 85 participants were enrolled: 45 patients with CP (23 S [CP+S] and 22 NS [CP+NS]) and 40 PH individuals (20 S [PH+S] and 20 NS [PH+NS]). In all the study participants, clinical periodontal parameters (plaque index, gingival index, sulcus bleeding index, probing depth, and clinical attachment level) were recorded and samples of serum, saliva and GCF were collected. Enzyme-linked immunosorbent assay was used to determine the levels of MT in the samples. RESULTS: All periodontal clinical parameters were significantly higher in the CP groups as compared to PH groups (P < 0.05). MT levels in CP+S group were significantly raised in comparison to other three groups. There was no statistically significant difference in MT levels among CP+NS and PH+S groups (P > 0.05); however, relatively higher levels were observed in GCF and saliva in CP+NS group. When all the study groups were observed together, MT levels were positively correlated with clinical parameters. CONCLUSIONS: Results of present study suggest that smoking and CP can induce the synthesis of MT owing to increased oxidative stress and heavy metals intoxication. Further longitudinal studies with large sample size and an interventional arm are needed to substantiate the role of MT as a potential biomarker in periodontitis.

Βeta-catenin N-terminal domain: An enigmatic region prone to cancer causing mutations.

The Wnt/ß-catenin is a highly conserved signaling pathway involved in cell fate decisions during various stages of development. Dysregulation of canonical Wnt/ß-catenin signaling has been associated with various diseases including cancer. ß-Catenin, the central component of canonical Wnt signaling pathway, is a multi-functional protein playing both structural and signaling roles. ß-Catenin is composed of three distinct domains: N-terminal domain, C-terminal domain and a central armadillo repeat domain. N-terminal domain of ß-catenin harbours almost all of the cancer causing mutations, thus deciphering its critical structural and functional roles offers great potential in cancer detection and therapy. Here, in this review, we have collected information from pharmacological analysis, bio-physical and structural studies, molecular modeling, in-vivo and in-vitro assays, and transgenic animal experiments employing various N-terminal domain variants of ß-catenin to discuss the interaction of ß-catenin with its binding partners that specifically interact with this domain and the implications of these interactions on signaling, cell fate determination, and in tumorigenesis. A thorough understanding of interactions between ß-catenin and its binding partners will enable us to more effectively understand how ß-catenin switches between its multiple roles, and will lead to the development of specific assays for the identification of small molecules as chemotherapeutic agents to treat diseases, including cancer and neurological disorders, where Wnt/ß-catenin signaling is dysregulated.

Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response.

Mammalian Ecdysoneless (ECD) is a highly conserved ortholog of the DrosophilaEcd gene product whose mutations impair the synthesis of Ecdysone and produce cell-autonomous survival defects, but the mechanisms by which ECD functions are largely unknown. Here we present evidence that ECD regulates the endoplasmic reticulum (ER) stress response. ER stress induction led to a reduced ECD protein level, but this effect was not seen in PKR-like ER kinase knockout (PERK-KO) or phosphodeficient eukaryotic translation initiation factor 2α (eIF2α) mouse embryonic fibroblasts (MEFs); moreover, ECD mRNA levels were increased, suggesting impaired ECD translation as the mechanism for reduced protein levels. ECD colocalizes and coimmunoprecipitates with PERK and GRP78. ECD depletion increased the levels of both phospho-PERK (p-PERK) and p-eIF2α, and these effects were enhanced upon ER stress induction. Reciprocally, overexpression of ECD led to marked decreases in p-PERK, p-eIF2α, and ATF4 levels but robust increases in GRP78 protein levels. However, GRP78 mRNA levels were unchanged, suggesting a posttranscriptional event. Knockdown of GRP78 reversed the attenuating effect of ECD overexpression on PERK signaling. Significantly, overexpression of ECD provided a survival advantage to cells upon ER stress induction. Taken together, our data demonstrate that ECD promotes survival upon ER stress by increasing GRP78 protein levels to enhance the adaptive folding protein in the ER to attenuate PERK signaling.

Biophysical characterization and modeling of human Ecdysoneless (ECD) protein supports a scaffolding function.

The human homolog of Drosophila ecdysoneless protein (ECD) is a p53 binding protein that stabilizes and enhances p53 functions. Homozygous deletion of mouse Ecd is early embryonic lethal and Ecd deletion delays G1-S cell cycle progression. Importantly, ECD directly interacts with the Rb tumor suppressor and competes with the E2F transcription factor for binding to Rb. Further studies demonstrated ECD is overexpressed in breast and pancreatic cancers and its overexpression correlates with poor patient survival. ECD overexpression together with Ras induces cellular transformation through upregulation of autophagy. Recently we demonstrated that CK2 mediated phosphorylation of ECD and interaction with R2TP complex are important for its cell cycle regulatory function. Considering that ECD is a component of multiprotein complexes and its crystal structure is unknown, we characterized ECD structure by circular dichroism measurements and sequence analysis software. These analyses suggest that the majority of ECD is composed of α-helices. Furthermore, small angle X-ray scattering (SAXS) analysis showed that deletion fragments, ECD(1-432) and ECD(1-534), are both well-folded and reveals that the first 400 residues are globular and the next 100 residues are in an extended cylindrical structure. Taking all these results together, we speculate that ECD acts like a structural hub or scaffolding protein in its association with its protein partners. In the future, the hypothetical model presented here for ECD will need to be tested experimentally.

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function.

Experimental validation of predicted subcellular localizations of human proteins.

BACKGROUND: Computational methods have been widely used for the prediction of protein subcellular localization. However, these predictions are rarely validated experimentally and as a result remain questionable. Therefore, experimental validation of the predicted localizations is needed to assess the accuracy of predictions so that such methods can be confidently used to annotate the proteins of unknown localization. Previously, we published a method called ngLOC that predicts the localization of proteins targeted to ten different subcellular organelles. In this short report, we describe the accuracy of these predictions using experimental validations. FINDINGS: We have experimentally validated the predicted subcellular localizations of 114 human proteins corresponding to nine different organelles in normal breast and breast cancer cell lines using live cell imaging/confocal microscopy. Target genes were cloned into expression vectors as GFP fusions and cotransfected with RFP-tagged organelle-specific gene marker into normal breast epithelial and breast cancer cell lines. Subcellular localization of each target protein is confirmed by colocalization with a co-expressed organelle-specific protein marker. Our results showed that about 82.5% of the predicted subcellular localizations coincided with the experimentally validated localizations. The highest agreement was found in the endoplasmic reticulum proteins, while the cytoplasmic location showed the least concordance. With the exclusion of cytoplasmic location, the average prediction accuracy increased to 90.4%. In addition, there was no difference observed in the protein subcellular localization between normal and cancer breast cell lines. CONCLUSIONS: The experimentally validated accuracy of ngLOC method with (82.5%) or without cytoplasmic location (90.4%) nears the prediction accuracy of 89%. These results demonstrate that the ngLOC method can be very useful for large-scale annotation of the unknown subcellular localization of proteins.

Down regulation of a matrix degrading cysteine protease cathepsin L, by acetaldehyde: role of C/EBPα.

BACKGROUND: The imbalance between extra cellular matrix (ECM) synthesis and degradation is critical aspect of various hepatic pathologies including alcohol induced liver fibrosis. This study was carried out to investigate the effect of acetaldehyde on expression of an extra cellular matrix degrading protease cathepsin L (CTSL) in HepG2 cells. METHODOLOGY AND RESULTS: We measured the enzymatic activity, protein and, mRNA levels of CTSL in acetaldehyde treated and untreated cells. The binding of CAAT enhancer binding protein α (C/EBP α) to CTSL promoter and its key role in the transcription from this promoter and conferring responsiveness to acetaldehyde was established by site directed mutagenesis, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assays and siRNA technology. Acetaldehyde treatment significantly decreased CTSL activity and protein levels in HepG2 cells. A similar decrease in the mRNA levels and promoter activity was also observed. This decrease by acetaldehyde was attributed to the fall in the liver enriched transcription factor C/EBP α levels and it's binding to the CTSL promoter. Mutagenesis of C/EBP α binding motifs revealed the key role of this factor in CTSL transcription as well as conferring responsiveness to acetaldehyde. The siRNA mediated silencing of the C/EBP α expression mimicked the effect of acetaldehyde on CTSL levels and its promoter activity. It also abolished the responsiveness of this promoter to acetaldehyde. CONCLUSION: Acetaldehyde down regulates the C/EBP α mediated CTSL expression in hepatic cell lines. The decreased expression of CTSL may at least in part contribute to ECM deposition in liver which is a hallmark of alcoholic liver fibrosis.

Post-transcriptional regulation of human cathepsin L expression.

The expression of cathepsin L, a lysosomal protease, is known to be elevated in cancer and other pathologies. Multiple splice variants of human cathepsin L with variable 5'UTRs exist, which encode for the same protein. Previously we have observed that variant hCATL A (bearing the longest 5'UTR) was translated in vitro with significantly lower efficiency than variant hCATL AIII (bearing the shortest 5'UTR). Contrary to these findings, results of the present study reveal that in cancer cells, hCATL A mRNA exhibits higher translatability in spite of having lower stability than AIII. This is the first report demonstrating a highly contrasting trend in translation efficiencies of hCATL variants in rabbit reticulocytes and live cells. Expression from chimeric mRNAs containing 5'UTRs of A or AIII upstream to luciferase reporter cDNA established the A UTR to be the sole determinant for this effect. Transient transfections of bicistronic plasmids and mRNAs confirmed the presence of a functional Internal Ribosome Entry Site in this UTR. Our data suggest that differential stability and translation initiation modes mediated by the 5'UTRs of human cathepsin L variants are involved in regulating its expression.

Wild type p53-dependent transcriptional upregulation of cathepsin L expression is mediated by C/EBPα in human glioblastoma cells.

Mutations in the tumor suppressor gene p53 are frequent in human glioblastomas. Similarly cathepsin L, a lysosomal cysteine protease, is overexpressed and secreted by most human tumors including glioblastomas. However, hitherto there is no information on whether or not the mutation(s) in the p53 gene affect(s) expression of this protease. Using human glioblastoma cell lines harboring wild type and mutant p53, we demonstrate here for the first time that only the wild type but not the mutant p53 upregulates cathepsin L expression. By transfection of promoter reporter constructs, site-directed mutagenesis and chip assays we have established that wild type p53 elevates the levels of cathepsin L in these cells. It does so directly by binding to the cathepsin L promoter and also indirectly by inducing the expression of C/EBPα, which is crucial for the transcription of this protease. In view of its role in tumorigenesis, angiogenesis and tumor cell invasion, increased expression of cathepsin L in glioblastoma cells harboring wild type p53 might confer invasive ability and growth advantage to these cells. Therefore, use of cathepsin L inhibitors could prove useful in the management of these tumors.