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.

Purification, functional characterization and enhanced production of serratiopeptidase from Serratia marcescens MES-4: An endophyte isolated from Morus rubra.

Serratiopeptidase, a proteolytic enzyme serves as an important anti-inflammatory and analgesic medication. Present study reports the production and purification of extracellular serratiopeptidase from an endophyte, Serratia marcescens MES-4, isolated from Morus rubra. Purification of the enzyme by Ion exchange chromatography led to the specific activity of 13,030 U/mg protein of serratiopeptidase, showcasing about 3.1 fold enhanced activity. The catalytic domain of the purified serratiopeptidase, composed of Zn coordinated with three histidine residues (His 209, His 213, and His 219), along with glutamate (Glu 210) and tyrosine (Tyr 249). The molecular mass, as determined by SDS-PAGE was ∼51 kDa. The purified serratiopeptidase displayed optimal activity at pH 9.0, temperature 50°C. Kinetic studies revealed Vmax and Km values of 33,333 U/mL and 1.66 mg/mL, respectively. Further, optimized conditions for the production of serratiopeptidase by Taguchi design led to the productivity of 87 U/mL/h with 87.9 fold enhanced production as compared to the previous conditions.

Orally bioavailable styryl derivative of rohitukine-N-oxide inhibits CDK9/T1 and the growth of pancreatic cancer cells.

The chromone alkaloid is one of the classical pharmacophores for cyclin-dependent kinases (CDKs) and represents the first CDK inhibitor to reach clinical trials. Rohitukine (1), a chromone alkaloid isolated from Dysoxylum binectariferum inspired the discovery of several clinical candidates. The N-oxide derivative of rohitukine occurs naturally, with no reports on its biological activity. Herein, we report isolation, biological evaluation, and synthetic modification of rohitukine N-oxide for CDK9/T1 inhibition and antiproliferative activity in cancer cells. Rohitukine N-oxide (2) inhibits CDK9/T1 (IC50 7.6 µM) and shows antiproliferative activity in the colon and pancreatic cancer cells. The chloro-substituted styryl derivatives, 2b, and 2l, inhibit CDK9/T1 with IC50 values of 0.17 and 0.15 µM, respectively. These derivatives display cellular antiproliferative activity in HCT 116 (colon) and MIA PaCa-2 (pancreatic) cancer cells with GI50 values of 2.5-9.7 µM with excellent selectivity over HEK293 (embryonic kidney) cells. Both analogs induce cell death in MIA PaCa-2 cells via inducing intracellular ROS production, reducing mitochondrial membrane potential, and inducing apoptosis. These analogs are metabolically stable in liver microsomes and have a decent oral pharmacokinetics in BALB/c mice. The molecular modeling studies indicated their strong binding at the ATP-binding site of CDK7/H and CDK9/T1.

Clinical relevance of biomarkers, new therapeutic approaches, and role of post-translational modifications in the pathogenesis of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive loss of cognitive functions like thinking, memory, reasoning, behavioral abilities, and social skills thus affecting the ability of a person to perform normal daily functions independently. There is no definitive cure for this disease, and treatment options available for the management of the disease are not very effective as well. Based on histopathology, AD is characterized by the accumulation of insoluble deposits of amyloid beta (Aß) plaques and neurofibrillary tangles (NFTs). Although several molecular events contribute to the formation of these insoluble deposits, the aberrant post-translational modifications (PTMs) of AD-related proteins (like APP, Aß, tau, and BACE1) are also known to be involved in the onset and progression of this disease. However, early diagnosis of the disease as well as the development of effective therapeutic approaches is impeded by lack of proper clinical biomarkers. In this review, we summarized the current status and clinical relevance of biomarkers from cerebrospinal fluid (CSF), blood and extracellular vesicles involved in onset and progression of AD. Moreover, we highlight the effects of several PTMs on the AD-related proteins, and provide an insight how these modifications impact the structure and function of proteins leading to AD pathology. Finally, for disease-modifying therapeutics, novel approaches, and targets are discussed for the successful treatment and management of AD.

Identification of a stretch of four discontinuous amino acids involved in regulating kinase activity of IGF1R.

IGF1R is pursued as a therapeutic target because of its abnormal expression in various cancers. Recently, we reported the presence of a putative allosteric inhibitor binding pocket in IGF1R that could be exploited for developing novel anti-cancer agents. In this study, we examined the role of nine highly conserved residues surrounding this binding pocket, with the aim of screening compound libraries in order to develop small-molecule allosteric inhibitors of IGF1R. We generated GFP fusion constructs of these mutants to analyze their impact on subcellular localization, kinase activity and downstream signaling of IGF1R. K1055H and E1056G were seen to completely abrogate the kinase activity of IGF1R, whereas R1064K and L1065A were seen to significantly reduce IGF1R kinase activity. During molecular dynamics analysis, various structural and conformational changes were observed in different conserved regions of mutant proteins, particularly in the activation loop, compromising the kinase activity of IGF1R. These results show that a stretch of four discontinuous residues within this newly identified binding pocket is critical for the kinase activity and structural integrity of IGF1R. This article has an associated First Person interview with the first author of the paper.

Purification and characterization of thermoactive serratiopeptidase from Serratia marcescens AD-W2.

Serratiopeptidase is a proteolytic enzyme extensively used as an anti-inflammatory and analgesic drug. Present work reports a thermoactive serratiopeptidase from Serratia marcescens AD-W2, a soil isolate from the North-Western Himalayan region of India. The extracellular metalloprotease has been purified by a simple two-step procedure resulting in a specific activity of 20,492 Units/mg protein with 5.28-fold purification. The molecular mass of the metalloprotease, as determined by SDS-PAGE was ~ 51 kDa. The purified serratiopeptidase presented optimum activity at pH 9.0, temperature 50 °C and stability in wide pH and temperature range. Critical temperature of 50 °C confirmed the thermoactivity of the purified serratiopeptidase. The kinetic studies of the purified serratiopeptidase revealed Vmax and Km of 57,256 Units/mL and 1.57 mg/mL, respectively, for casein. The purified serratiopeptidase from S. marcescens AD-W2 was found to be 100% identical to serralysin from Serratia marcescens ATCC 21074/E-15. The catalytic domain comprising of Zn coordinated with three histidine residues (His192, His196, His202), along with glutamate (Glu193) and tyrosine (Tyr232) residues, further confirmed that the purified protein is identical to serralysin.

Transcriptome-wide identification of squalene epoxidase genes from Glycyrrhiza glabra L.: expression analysis and heterologous expression of GgSQE1 suggest important role in terpenoid biosynthesis.

Squalene epoxidase (SQE) is a crucial regulatory enzyme for the biosynthesis of several important classes of compounds including sterols and triterpenoids. The present paper identified and characterised five SQE genes (GgSQE1 to GgSQE5) from Glycyrrhiza glabra through transcriptome data mining and homology-based cloning, for the first time. The phylogenetic analysis implied their functional divergence. The ORF corresponding to one of the five SQEs, namely, GgSQE1, was cloned and studied for its function in a heterologous system, following transient and stable expressions. The transient expression followed by GgSQE1 encoding protein purification suggested approximately 58.0-kDa protein following the predicted molecular mass of the deduced protein. The gene expression profiling based on qRT-PCR indicated its highest expression (6.4-folds) in the 10-month-old roots. Furthermore, ABA (12.4-folds) and GA3 (2.47) treatments upregulated the expression of GgSQE1 in the shoots after 10 and 12 hours, respectively, which was also reflected in glycyrrhizin accumulation. The inductive effects of ABA and GA3 over GgSQE1 expression were also confirmed through functional analysis of GgSQE1 promoters using GUS fusion construct. Stable constitutive expression of GgSQE1 in Nicotiana tabacum modulated the sterol contents. The study could pave the way for understanding the metabolic flux regulation concerning biosynthesis of related sterols and triterpenoids.

Identification of a cross-talk between EGFR and Wnt/beta-catenin signaling pathways in HepG2 liver cancer cells.

EGFRis a transmembrane receptor tyrosine kinase involved in regulating cell proliferation, differentiation and survival. EGFR is actively pursued as a therapeutic target because its aberrant expression or activity has been reported in several cancers. Several studies have reported the nuclear localization of the EGFR in various cell types, however, its exact nuclear functions are not clear yet. In this study, we have generated GFP fusion constructs of EGFR and its mutants to analyze their subcellular localizationin normal and cancer cells and impact of its sub-cellular location on its various activities using immunoblotting, confocal microscopy, reporter assays, loss-of-function EGFR mutants, and EGFR specific small molecule inhibitors. We show that EGFR is involved in modulating TCF dependent ß-catenin transcriptional activity in HepG2 cells in a similar fashion as IGF1R tyrosine kinase. Moreover, we show that cytoplasmic and nuclear functions are two independent activities of EGFR.

Analyzing structural differences between insulin receptor (IR) and IGF1R for designing small molecule allosteric inhibitors of IGF1R as novel anti-cancer agents.

IR and insulin-like growth factor-1 receptor (IGF-1R) share high degree of sequence and structural similarity that hinders the development of anticancer drugs targeting IGF1R, which is dysregulated in many cancers. Although IR and IGF1R mediate their activities through similar signalling pathways, yet they show different physiological effects. The exact molecular mechanism(s) how IR and IGF1R exert their distinct functions remain largely unknown. Here, we performed in silico analysis and generated GFP-fusion proteins of wild type IR and its K1079R mutant to analyze their subcellular localization, cytoplasmic and nuclear activities in comparison to IGF1R and its K1055R mutant. We showed that, like K1055R mutation in IGF1R, K1079R mutation does not impede the subcellular localization and nuclear activities of IR. Although K1079R mutation significantly decreases the kinase activity of IR but not as much as K1055R mutation, which was seen to drastically reduce the kinase activity of IGF1R. Moreover, K1079 residue in IR is seen to be sitting in a pocket which is different than the allosteric inhibitor binding pocket present in its homologue (IGF1R). This is for the first time such a study has been conducted to identify structural differences between these receptors that could be exploited for designing small molecule allosteric inhibitor(s) of IGF1R as novel anti-cancer drugs.

Identification of dinactin, a macrolide antibiotic, as a natural product-based small molecule targeting Wnt/ß-catenin signaling pathway in cancer cells.

PURPOSE: Despite the fact that hyper-activation of Wnt/ß-catenin signaling pathway has been seen in many cancers, including liver, colorectal and lung carcinoma, no small molecule inhibitors are available that specifically target this pathway. In this study, we analyzed the impact of dinactin (DA), an antibiotic ionophore produced by Streptomyces species, as an effective small molecule targeting Wnt/ß-catenin signaling pathway in cancer cells. METHODS: We performed MTT assays to investigate cell viability and proliferation after exposure to small molecules. Protein expression analysis was carried out by western blotting. Top-Flash reporter assays were used to score for ß-catenin signaling and cell cycle analysis was carried out by flow cytometry. RESULTS: In the first set of experiments, DA was seen to selectively inhibit the proliferation of HCT-116 and HepG2 cancer cells, unlike HEK-293 cells (a low tumorigenic cell line), in apoptosis-independent manner. Further, DA was seen to block the G1/S progression and decrease the expression of cyclin D1 in cancer cells. Since cyclin D1 is the downstream target gene of Wnt/ß-catenin signaling, we examined the impact of DA on TCF-dependent ß-catenin activity using Top-Flash reporter assay. Interestingly, DA significantly decreased Top-Flash activity at lower nano-molar concentrations when compared with salinomycin in HCT-116 and HepG2 cells. CONCLUSION: We report the identification of dinactin as a natural product-based small molecule that effectively blocks the Wnt/ß-catenin signaling pathway in cancer cells at nano-molar concentration. We anticipate that DA could be developed as a novel drug for anti-cancer therapy and for the management of neuropathic pain.

Analyzing the role of cannabinoids as modulators of Wnt/ß-catenin signaling pathway for their use in the management of neuropathic pain.

Neuropathic pain is a debilitating form of treatment-resistant chronic pain caused by damage to the nervous system. Cannabinoids have been known for suppressing neuropathic pain by modulating the endo cannabinoid system. Since the canonical Wnt/ß-catenin signaling has recently been implicated in pain sensation, we investigated the impact of major cannabinoids (1-6) from the leaves of Cannabis sativa and an epoxy derivative of compound 2, here upon referred to as 2a, on modulating Wnt/ß-catenin signaling pathway. The results presented in this study show that compound 1, 2 and 2a exhibited potent inhibitory activity against Wnt/ß-catenin pathway in a dose-dependent manner. Compound 2a was seen to inhibit this pathway at slightly lower concentrations than its parent molecule 2, under similar conditions. Taken together, compound 1, 2 and 2a, by virtue of their inhibition of Wnt/ß-catenin signaling pathway, could be developed as effective neuroprotective agents for the management of neuropathic pain.

p110α and p110ß isoforms of PI3K are involved in protection against H2O2 induced oxidative stress in cancer cells.

PURPOSE: Phosphatidylinositol-3 kinases (PI3Ks) are involved in regulating cell growth, proliferation, differentiation, apoptosis and survival. p110α and p110ß, two ubiquitously expressed isoforms of PI3K signalling, are involved in growth factor mediated signaling and survival by generating second messengers. Earlier, we have generated GFP-fusion proteins of p110α and p110ß and expressed them in normal and cancer cell-lines to investigate their subcellular localization and their role in various activities. Here, we sought to examine the role of p110α and p110ß isoforms in protecting MCF-7 breast cancer cells against oxidative stress. MATERIAL METHODS: We performed cytotoxicity assays, DNA transfection, Plasmid DNA preparation, western blotting, flourscence microscopy and statistical analysis. RESULTS: To know whether p110α and p110ß are involved in protecting MCF-7 breast cancer cells against oxidative stress, we subjected MCF-7 cells to H2O2 treatment and observed a dose dependent decrease in cell viability and a marked increase in the levels of pro-apoptotic markers which include PARP, Bcl-2, Bax and procaspase-9. We then over-expressed recombinant GFP-fusion p110α and p110ß proteins in MCF-7 cells and observed a significant decrease in apoptosis and a concomitant increase in pAkt levels. CONCLUSION: We report the involvement of p110α and p110ß isoforms of Class 1A PI3K signalling in rescue from oxidative stress-induced apoptosis in MCF-7 cells in Akt dependent manner.

Identification of a unique loss-of-function mutation in IGF1R and a crosstalk between IGF1R and Wnt/ß-catenin signaling pathways.

IGF1R is a ubiquitous receptor tyrosine kinase that plays critical roles in cell proliferation, growth and survival. Clinical studies have demonstrated upregulation of IGF1R mediated signaling in a number of malignancies including colon, breast, and lung cancers. Overexpression of the IGF1R in these malignancies is associated with a poor prognosis and overall survival. IGF1R specific kinase inhibitors have failed in multiple clinical trials partly because of the complex nature of IGF1R signaling. Thus identifying new binding partners and allosteric sites on IGF1R are emerging areas of research. More recently, IGF1R has been shown to translocate into the nucleus and perform many functions. In this study, we generated a library of IGF1R deletion and point mutants to examine IGF1R subcellular localization and activation of downstream signaling pathways. We show that the nuclear localization of IGF1R is primarily defined by its cytoplasmic domain. We identified a cross-talk between IGF1R and Wnt/ß-catenin signaling pathways and showed, for the first time, that IGF1R is associated with upregulation of TCF-mediated ß-catenin transcriptional activity. Using loss-of-function mutants, deletion analysis and IGF1R specific inhibitor(s), we show that cytoplasmic and nuclear activities are two independent functions of IGF1R. Furthermore, we identified a unique loss-of-function mutation in IGF1R. This unique loss-of-function mutant retains only nuclear functions and sits in a pocket, outside ATP and substrate binding region, that is suited for designing allosteric inhibitors of IGF1R.

Streptomyces puniceus strain AS13., Production, characterization and evaluation of bioactive metabolites: A new face of dinactin as an antitumor antibiotic.

A highly active actinobacterial strain isolated from untapped areas of Northwestern Himalayas and characterised as Streptomyces puniceus strain AS13 by 16S rRNA gene sequencing was selected for production of bioactive metabolites. The bioassay-guided fractionation of microbial cultured ethyl acetate extract of the strain, led to isolation of macrotetrolide compound 1 (Dinactin) and compound 2 (1-(2,4-dihydroxy-6-methylphenyl)-ethanone). Structures of the isolated compounds were elucidated by [corrected] interpretation of NMR and other spectroscopic data including HR-ESI-MS, FT-IR. These compounds are reported for first time from Streptomyces Puniceus. Compound 1 exhibited strong anti-microbial activity against all tested bacterial pathogens including Mycobacterium tuberculosis. The MIC values of compound 1 against Gram negative and Gram positive bacterial pathogens ranged between 0.019 - 0.156µgml-1 and 1µgml-1 against Mycobacterium tuberculosis H37Rv. Dinactin exhibited marked anti-tumor potential with IC50 of 1.1- 9.7µM in various human cancerous cell lines and showed least cytotoxicity (IC50∼80µM) in normal cells (HEK-293). Dinactin inhabited cellular proliferation in cancer cells, reduced their clonogenic survival as validated by clonogenic assay and also inhabited cell migration and invasion characteristics in colon cancer (HCT-116) cells. Our results expressed the antimicrobial potential of dinactin and also spotted its prospective as an antitumor antibiotic.

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.

The synthesis, biological evaluation and structure-activity relationship of 2-phenylaminomethylene-cyclohexane-1,3-diones as specific anti-tuberculosis agents.

The present study utilised whole cell based phenotypic screening of thousands of diverse small molecules against Mycobacterium tuberculosis H37Rv (M. tuberculosis) and identified the cyclohexane-1,3-dione-based structures 5 and 6 as hits. The selected hit molecules were used for further synthesis and a library of 37 compounds under four families was synthesized for lead generation. Evaluation of the library against M. tuberculosis lead to the identification of three lead antituberculosis agents (37, 39 and 41). The most potential compound, 2-(((2-hydroxyphenyl)amino)methylene)-5,5-dimethylcyclohexane-1,3-dione (39) showed an MIC of 2.5 µg mL-1, which falls in the range of MICs values found for the known antituberculosis drugs ethambutol, streptomycin and levofloxacin. Additionally, this compound proved to be non-toxic (<20% inhibition at 50 µM concentration) against four human cell lines. Like first line antituberculosis drugs (isoniazid, rifampicin and pyrazinamide) this compound lacks activity against general Gram positive and Gram negative bacteria and even against M. smegmatis; thereby reflecting its highly specific antituberculosis activity.

p110α and p110ß isoforms of PI3K signaling: are they two sides of the same coin?

Class-1 phosphatidylinositol-3-kinases (PI3Ks) are activated by a variety of extracellular stimuli and have been implicated in a wide range of cellular processes. p110α and p110ß are the two most studied isoforms of the class-1A PI3K signaling pathway. Although these two isoforms are ubiquitously expressed and play multiple redundant roles, they also have distinct functions within the cell. More recently, p110α and p110ß isoforms have been shown to translocate into the nucleus and play a role in DNA replication and repair, and in cell cycle progression. In the following Review article, we discuss the overlapping and unique roles of p110α and p110ß isoforms with a particular focus on their structure, expression analysis, subcellular localization, and signaling contributions in various cell types and model organisms.

Terminal regions of ß-catenin are critical for regulating its adhesion and transcription functions.

ß-Catenin, the central molecule of canonical Wnt signaling pathway, has multiple binding partners and performs many roles in the cell. Apart from being a transcriptional activator, ß-catenin acts as a crucial effector component of cadherin/catenin complex to physically interact with actin cytoskeleton along with α-catenin and E-cadherin for regulating cell-cell adhesion. Here, we have generated a library of ß-catenin point and deletion mutants to delineate regions within ß-catenin that are important for α-catenin-ß-catenin interaction, nuclear localization, and transcriptional activity of ß-catenin. We observed a unique mechanism for nuclear localization of ß-catenin and its mutants and show that N-terminal exon-3 region and C-terminal domain of ß-catenin are critical for this activity of ß-catenin. Furthermore, we show HepG2 cells have high ß-catenin mediated transcriptional activity due to the presence of an interstitial deletion at the N-terminal region of ß-catenin. Due to this deletion mutant (hereupon called TM), GSK3ß and HDAC inhibitors failed to show any impact whereas curcumin significantly inhibited ß-catenin mediated transcriptional activity reiterating that TM is primarily responsible for the high transcriptional activity of HepG2 cells. Moreover, we show the recombinant TM does not physically interact with α-catenin, localizes predominantly in the nucleus, and has nearly two-fold higher transcriptional activity than the wildtype ß-catenin.