Triazole Inhibitors of Cryptosporidium parvum Inosine 5?-Monophosphate Dehydrogenase

Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5?-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Because C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series of 1,2,3-triazole containing ether CpIMPDH inhibitors are described. A structure?activity relationship study revealed that a small alkyl group on the ?-position of the ether was required, with the (R)-enantiomer significantly more active than the (S)-enantiomer. Electron-withdrawing groups in the 3- and/or 4-positions of the pendent phenyl ring were best, and conversion of the quinoline containing inhibitors to quinoline-N-oxides retained inhibitory activity both in the presence and absence of bovine serum albumin. The 1,2,3-triazole CpIMPDH inhibitors provide new tools for elucidating the role of IMPDH in C. parvum and may serve as potential therapeutics for treating cryptosporidiosis

The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5?-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 103 selectivity for the parasite enzyme over human IMPDH2

Structure–activity relationship study of selective benzimidazole-based inhibitors of Cryptosporidium parvum IMPDH

Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The Cryptosporidium parvum and Cryptosporidium hominis genomes indicate that the only route to guanine nucleotides is via inosine 5?-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazole-based inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure–activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro

Characterization of Torin2, an ATP-Competitive Inhibitor of mTOR, ATM, and ATR

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC[subscript 50] of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase–like kinase (PIKK) family kinases including ATM (EC[subscript 50], 28 nmol/L), ATR (EC[subscript 50], 35 nmol/L), and DNA-PK (EC[subscript 50], 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role

Diverse applications of Nanotechnology in Biomedicine, Chemistry, and Engineering

This chapter introduces the basic concepts of nano science to readers. Some novel methodologies for synthesizing nano particles are discussed briefly. Since the book title suggests diverse applications of nano materials, this chapter also summarizes the applications of nano technology in medicine (nano medicine), where tissue engineering and regenerative medicine are discussed. Other applications DNA nanotechnology in living organisms are discussed briefly. Overall, this chapter introduces the readers to broad sections of nano science and its applications in chemistry, engineering, and medicine.by Sivapriya Kirubakaran and Vijay Thiruvenkata

Development of potent and selective inhibitors for ATR: an adjuvant for DNA damage based chemotherapeutics

Inhibition of DNA damage checkpoint and repair function has been a challenging as well a promising approach in cancer therapy. ATR kinase is one of the key mediator of DNA damage response which induces cell cycle arrest and DNA repair via its downstream proteins. Blocking ATR has proved to prevent the Chk1 pathway from stalled replication fork and enhances the replication stress and premature mitotic entry. In addition inhibition of ATR can selectively sensitize the cancer cell to radio and chemotherapy, due to defective DNA damage signaling through the loss of ATM or p53 mutation in cancer cell. Due to its inherent role in DDR, ATR has been explored as a potential target in enhancing the effect of radiation and chemo-therapy in addition enables highly selective targeting the cancer cell through synthetic lethality. Despite the attractiveness of ATR inhibition in the cancer therapy, specific ATR inhibitors have remained indefinable. Further, there is a limited proof of concept data for ATR inhibition. Considering these facts and challenges we will be presenting some preliminary results with respect to In-silico design, synthesis and evolution of potential and selective inhibitors for ATR.by Sivapriya Kirubakaran, Vijay Thiruvenkatam and Althaf Shai

Molecular cloning and expression analysis of Human TLK1B-Kinase Domain construct in Escherichia coli

by Shashank Raman, Siddhant Bhoir and Sivapriya Kirubakara

Crystalline polymorphs of Diethyl 2-(((4-bromophenyl) amino) methylene) malonate: a detailed study

Althaf Shaik, Sivapriya Kirubakaran and Vijay Thiruvenkata

High MDC1 expression in cervical cancer cells can affect the chemo- and radiotherapeutic response as its depletion leads to increased cell death

Cervical cancer is the third most frequent cancer and common cause of death in women worldwide. The work presented identifies mediator of DNA damage checkpoint 1 (MDC1) as an important molecular target to increase sensitivity of cervical cancer cells to chemo or radiotherapy. MDC1 functions as an adaptor protein for recruitment and retention of many other DNA damage repair proteins in ataxia telangiectasia mutated (ATM) pathway for double-stranded DNA damage repair. It is reported to be highly expressed in cervical cancer cells. Also, its expression tends to increase with increase in malignancy. We have studied in detail MDC1 mRNA expression in three cervical cancer cell lines, HeLa, SiHa and CasKi, in response to various genotoxic stresses including some known inhibitors, UV exposure or gamma irradiation through quantitative PCR. The cellular response to the DNA damage resulted in increase in MDC1 expression, which declined with increase in treatment time period. Protein expression and activation by Western blotting with anti-MDC1 and anti-phosphoMDC1 antibody indicated a higher level of phosphorylated as compared to unphosphorylated MDC1. The significance of this increase in MDC1 expression was studied by generating stable cell lines knocked down for MDC1 expression. The modified cell lines were assessed for apoptosis through various assays, including flow cytometry, and showed greater cell death in response to DNA damage. In summary, high MDC1 expression can significantly affect chemo or radiotherapeutic response and its inhibition can improve sensitivity to these treatments.by Neeru Singh, Rashmi Bhakuni and Sivapriya Kirubakara