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 EC50 of 250 pM with approximately 800-fold selectivity for cellular mTOR versus PI3K. Torin2 also exhibited potent biochemical and cellular activity against PIKK family kinases including ATM (EC50 28 nM), ATR (EC50 35 nM) and DNA-PK (EC50 118 nM) (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 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 oncological settings where mTOR signaling has a pathogenic role.

Background: Several new ATP-competitive mTOR inhibitors have been described, but their kinome-wide selectivity profiles have not been disclosed. Results: Four different profiling technologies revealed a different spectrum of targets for four recently described mTOR inhibitors. Conclusion: Diverse heterocyclic mTOR inhibitors have unique pharmacology.Significance: Profiling data guide choices of mTOR inhibitors for particular applications and provide new potential targets for medicinal chemistry efforts.

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 10(3) selectivity for the parasite enzyme over human IMPDH2.

Summary Through in vitro kinase assays and docking studies, we report the synthesis and biological evaluation of a phenothiazine analog J54 with potent TLK1 inhibitory activity for prostate cancer (PCa) therapy. Most PCa deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa. Treatments that can suppress the conversion to mCRPC have high potential to be rapidly implemented in the clinics. ADT results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DNA damage response that typically results in temporary cell-cycle arrest of androgen-responsive PCa cells, whereas its abrogation leads to apoptosis. We studied J54 as a potent inhibitor of this axis and as a mediator of apoptosis in vitro and in LNCaP xenografts, which has potential for clinical investigation in combination with ADT. J54 has low affinity for the dopamine receptor in modeling and competition studies and weak detrimental behavioral effects in mice and C. elegans.

Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The C. parvum and C. 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.

Helicobacter pylori (H. pylori), the major cause of several gastric disorders has been recognied as a type I carcinogen. By virtue of resistance developed by H. pylori strains, currently used antibiotic based treatments rather demonstrate high failure rates. Hence, there is an emerging need for identification of new targets to treat H. pylori infection. Inosine-5′-monophosphate dehydrogenase (IMPDH) has been studied as a potential target to treat H. pylori infection. Here, a detailed enzyme kinetic study of recombinant expressed H. pylori inosine-5′-monophosphate dehydrogenase (HpIMPDH) is presented. A new in-house synthesized indole-based scaffold is identified as an inhibitor for HpIMPDH. These indole-based compounds showed non-competitive inhibition against IMP and NAD+ whereas the benzimidazole compounds were found be uncompetitive inhibitors. The new indole scaffold ensures specificity due to its high selectivity for bacterial IMPDH over human IMPDH II. Our work aims to overcome the drawback of existing inhibitors by introducing new indole scaffold for targeting bacterial IMPDH.

Human Tousled-like kinases (TLKs) are highly conserved serine/threonine protein kinases responsible for cell proliferation, DNA repair, and genome surveillance. Their possible involvement in cancer via efficient DNA repair mechanisms have made them clinically relevant molecular targets for anticancer therapy. Innovative approaches in chemical biology have played a key role in validating the importance of kinases as molecular targets. However, the detailed understanding of the protein structure and the mechanisms of protein–drug interaction through biochemical and biophysical techniques demands a method for the production of an active protein of exceptional stability and purity on a large scale. We have designed a bacterial expression system to express and purify biologically active, wild-type Human Tousled-like Kinase 1B (hTLK1B) by co-expression with the protein phosphatase from bacteriophage λ. We have obtained remarkably high amounts of the soluble and homogeneously dephosphorylated form of biologically active hTLK1B with our unique, custom-built vector design strategy. The recombinant hTLK1B can be used for the structural studies and may further facilitate the development of new TLK inhibitors for anti-cancer therapy using a structure-based drug design approach.

Inosine 5′-monophosphate dehydrogenase (IMPDH) is a vital enzyme involved in thede novosynthesis of guanine nucleotides. Inhibitors of bacterial IMPDH hold great potential as new generation antimicrobial agents.