Intersection of Two Checkpoints: Could Inhibiting the DNA Damage Response Checkpoint Rescue Immune Checkpoint-Refractory Cancer?

Metastatic cancers resistant to immunotherapy require novel management strategies. DNA damage response (DDR) proteins, including ATR (ataxia telangiectasia and Rad3-related), ATM (ataxia telangiectasia mutated) and DNA-PK (DNA-dependent protein kinase), have been promising therapeutic targets for decades. Specific, potent DDR inhibitors (DDRi) recently entered clinical trials. Surprisingly, preclinical studies have now indicated that DDRi may stimulate anti-tumor immunity to augment immunotherapy. The mechanisms governing how DDRi could promote anti-tumor immunity are not well understood; however, early evidence suggests that they can potentiate immunogenic cell death to recruit and activate antigen-presenting cells to prime an adaptive immune response. Merkel cell carcinoma (MCC) is well suited to test these concepts. It is inherently immunogenic as ~50% of patients with advanced MCC persistently benefit from immunotherapy, making MCC one of the most responsive solid tumors. As is typical of neuroendocrine cancers, dysfunction of p53 and Rb with upregulation of Myc leads to the very rapid growth of MCC. This suggests high replication stress and susceptibility to DDRi and DNA-damaging agents. Indeed, MCC tumors are particularly radiosensitive. Given its inherent immunogenicity, cell cycle checkpoint deficiencies and sensitivity to DNA damage, MCC may be ideal for testing whether targeting the intersection of the DDR checkpoint and the immune checkpoint could help patients with immunotherapy-refractory cancers.

Characterization of SPK 98, a Torin2 analog, as ATR and mTOR dual kinase inhibitor.

A series of Torin2, a second-generation ATP-competitive inhibitor, analogues were biologically characterized to identify their potential for ATR and mTOR kinase inhibition. Compound SPK 98 was observed to inhibit ATR/mTOR kinase selectively over ATM kinase in HCT 116 cell line. In addition to that, SPK 98 on 30 min incubation with human, mice and rat liver microsomes showed improved properties with an increased half-life (a maximum T ½ of 157 min) and internal clearance in mouse as compared to Torin2. Further, SPK 98 was also noticed to indulge in inducing premature chromatin condensation as a result of ATR/mTOR kinase inhibition at 50 nM. In a nutshell, our work presents the identification and characterization of SPK 98, a small molecule inhibitor, which exhibits improved specific inhibition for ATR at a lower concentration than Torin2.

MDC1 depletion promotes cisplatin induced cell death in cervical cancer cells.

OBJECTIVE: Cisplatin, the most common chemotherapeutic drug for the treatment of advanced stage cervical cancers has limitations in terms of drugs resistance observed in patients partly due to functional DNA damage repair (DDR) processes in the cell. Mediator of DNA damage checkpoint 1 (MDC1) is an important protein in the Ataxia telangiectasia mutated (ATM) mediated double stranded DNA break (DSB) repair pathway. In this regard, we investigated the effect of MDC1 change in expression on the cisplatin sensitivity in cervical cancer cells. RESULTS: Through modulation of MDC1 expression in the cervical cancer cell lines; Hela, SiHa and Caski, we found that all the three cell lines silenced for MDC1 exhibited higher sensitivity to cisplatin treatment with inefficiency in accumulation of p γH2AX, Ser 139 foci and increased accumulation of pChk2 Thr 68 at the damaged chromatin followed by enhanced apoptosis. Further, we observed the increased p53 Ser 15 phosphorylation in the MDC1 depleted cells. Our studies suggest that MDC1 expression could be a key determinant in cervical cancer prognosis and its depletion in combination with cisplatin has the potential to be explored for the sensitisation of chemo-resistant cervical cancer cells.

Novel Pyrazolo[4, 3-c]Quinolin-3-One Derivatives as PDE5A Inhibitors.

BACKGROUND: PDE5A is a phosphodiesterase which specifically hydrolyzes the cGMP to GMP. It takes part in several physiological and pathological pathways and is considered an important drug target. Currently, PDE5 inhibitors (ex; Sildenafil, Tadalafil) available in the market are not only being used for the treatment of erectile dysfunction but at the same time, they are also in clinical trials being investigated as anticancer agents. MATERIALS & METHODS: In this work, we have examined pyrazolo [4,3-c]quinolin-3-ones as PDE5A inhibitors. Pyrazolo [4,3-c]quinolin-3-ones are the class of tricyclic heterocyclic derivatives having a variety of therapeutically interesting drug candidates known for their anti-inflammatory, anti-viral, anti-anxiety and anti-cancer activity. Therefore, synthetic methods providing access to pyrazolo [4, 3-c] quinolin-3-ones are immensely valuable. Here, we are reporting a simple but efficient route for the synthesis of novel 8-morpholino-2-aryl - 2, 5-dihydro-3H-pyrazolo [4, 3-c] quinolin-3-one derivatives. RESULTS: Further, molecular docking studies of synthesized compounds with human PDE5A protein showed that all the compounds exhibited good docking score in comparison with known inhibitors. In addition, all the synthesized molecules were evaluated against HCT116 cell lines for their antitumor activity. CONCLUSION: Among all the synthesized compounds, compound 5a, 5d, and 6e showed better cytotoxicity. Thus, these derivatives can be studied as potential inhibitors of PDE5A.

Evaluation of Maltose Binding Protein-Tagged hATR Kinase Domain Catalytic Activity with p53 Ser-15 Phosphorylation.

DNA damage response (DDR) pathways form an integral part of the body's repair machinery, and ATR (ataxia-telangiectasia and Rad3-related kinase) protein is one of the key mediators in the DDR pathway that helps in maintaining genomic integrity. A growing body of evidence suggests that inhibition of ATR can help sensitize tumor cells to combinatorial treatment. However, specific ATR kinase inhibitors have largely remained elusive until now. Despite much interest in the protein for more than a decade, there has been little characterization of only the kinase domain, an essential target site for a variety of ATR inhibitors. Here, we report our findings for the bacterial expression, purification, and biological characterization of this potentially important recombinant kinase domain, which could further be considered for structure elucidation studies. Introduction of a solubility partner, i.e., maltose binding protein (MBP), at the N-terminus of the ATR kinase domain generated a soluble form of the protein, i.e., MBP-tagged hATR kinase domain (MBP-ATR-6X His), which was found to be catalytically active, as assessed by substrate p53 Ser-15 phosphorylation (EPPLSQEAFADLWKK). Our results also highlight the prospect of utilization of the overexpressed recombinant ATR kinase domain in characterization of kinase domain specific inhibitors.

Design, Synthesis, and Docking Studies of New Torin2 Analogs as Potential ATR/mTOR Kinase Inhibitors.

Targeting DNA damage and response (DDR) pathway has become an attractive approach in cancer therapy. The key mediators involved in this pathway are ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia-mutated, Rad3-related kinase (ATR). These kinases induce cell cycle arrest in response to chemo- and radio-therapy and facilitate DNA repair via their major downstream targets. Targeting ATP-binding site of these kinases is currently under study. Torin2 is a second generation ATP competitive mTOR kinase inhibitor (EC50 = 250 pmol/L) with better pharmacokinetic profile. Torin2 also exhibits potent biochemical and cellular activity against ATM (EC50 = 28 nmol/L) and ATR (EC50 = 35 nmol/L) kinases. In this study, eight new Torin2 analogs were designed and synthesized through multistep synthesis. All the synthesized compounds were characterized by NMR and mass analysis. The newly synthesized analogs were evaluated for their anti-cancer activity via CellTiter-Glo® assay. Additionally, compounds 13 and 14 also showed significant inhibition for ATR and mTOR substrates, i.e., p-Chk1 Ser 317 and p70 S6K Thr 389, respectively. Compounds 13 and 14 displayed promising anti-cancer activity with HCT-116 cell lines in the preliminary study. Further, a comparative model of ATR kinase was generated using the SWISS-MODEL server and validated using PROCHECK, ProSA analysis. Synthesized compounds were docked into the ATP-binding site to understand the binding modes and for the rational design of new inhibitors.

Learning of bimanual motor sequences in normal aging.

While it is well accepted that motor performance declines with age, the ability to learn simple procedural motor tasks appears to remain intact to some extent in normal aging. Here we examined the impact of aging on the acquisition of a simple sequence of bimanual actions. We further asked whether such learning results from an overall decrease in response time or is also associated with improved coordination between the hands. Healthy young and old individuals performed a bimanual version of the classic serial reaction time task. We found no learning deficit in older adults and noted that older subjects were able to learn as much as young participants. We also observed that learning in both groups was associated with an overall decrease in response time, but switch cost, the increase in response time when a switch in hands was required during sequence execution, did not decrease with learning. Surprisingly however, overall switch cost was lower in the older group compared to the younger subjects. These findings are discussed in the context of interactions between procedural and declarative memory, reduced interhemispheric inhibition and more symmetric cortical activation during motor performance in normal aging.