Studies on Methylpyrazole-Substituted Benzimidazoles to Target Helicobacter pylori Infection through HpIMPDH Inhibition.

The prevalence of Helicobacter pylori infection has been increasing rapidly due to the genetic heterogeneity and antibacterial resistance shown by the bacteria, affecting over 50% of the world population and over 80% of the Indian population, in particular. In this regard, novel drug targets are currently being explored, one of which is the crucial metabolic enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) involved in the de novo nucleotide biosynthesis pathway, in order to combat the infection and devise efficient therapeutic strategies. The present study reports the development of methylpyrazole-substituted benzimidazoles as small molecule inhibitors of H. pylori IMPDH with a nanomolar range of enzyme inhibition. A set of 19 small molecules have been designed, synthesized, and further evaluated for their inhibitory potential against H. pylori IMPDH using in silico, in vitro, biochemical, and biophysical techniques. Compound 7j was found to inhibit H. pylori IMPDH with an IC50 value of 0.095 ± 0.023 µM, which is close to 1.5-fold increase in the inhibitory activity, in comparison to the previously reported benzimidazole-based hit C91. Moreover, kinetic characterization has provided significant insights into the uncompetitive inhibition shown by these small molecules on H. pylori IMPDH, thus providing details about the enzyme inhibition mechanism. In conclusion, methylpyrazole-based small molecules indicate a promising path to develop cheap and bioavailable drugs to efficiently treat H. pylori infection in the coming years, in comparison to the currently available therapy.

In silico Design, Synthesis and Biological Evaluation of Novel Thieno[3,2-d]pyrimidine Derivatives for Cancer Therapy - A Preliminary Study on the Inhibitory Potential towards ATR Kinase Domain and PIKK Family.

Continuing our studies in the field of new heterocyclic compounds with biological interest, herein we report the synthesis and anticancer activity of new N- and S-substituted derivatives of tetracyclic pyrido[3',2' : 4,5]thieno[3,2-d]pyrimidines. In this regard, starting from the thieno[2,3-b]pyridine-2-carboxylates, the corresponding 8(9)-aminopyrido[3',2' : 4,5]thieno[3,2-d]pyrimidin-7(8)-ones, as well as chloro derivatives were obtained. Based on the latter, amino, hydrazino and S-alkyl derivatives of pyrido[3',2' : 4,5]thieno[3,2-d]pyrimidines were synthesized subsequently. The current study focuses on identifying the potential of thieno[3,2-d]pyrimidine derivatives primarily towards ATR kinase inhibition, through computational predictions, followed by synthesis and cancer cell viability studies, along with an aim to develop the core as PIKK inhibitors for cancer therapy.

Targeting ATM and ATR for cancer therapeutics: Inhibitors in clinic.

The DNA Damage and Response (DDR) pathway ensures accurate information transfer from one generation to the next. Alterations in DDR functions have been connected to cancer predisposition, progression, and response to therapy. DNA double-strand break (DSB) is one of the most detrimental DNA defects, causing major chromosomal abnormalities such as translocations and deletions. ATR and ATM kinases recognize this damage and activate proteins involved in cell cycle checkpoint, DNA repair, and apoptosis. Cancer cells have a high DSB burden, and therefore rely on DSB repair for survival. Therefore, targeting DSB repair can sensitize cancer cells to DNA-damaging agents. This review focuses on ATM and ATR, their roles in DNA damage and repair pathways, challenges in targeting them, and inhibitors that are in current clinical trials.

Exploring SPK98 for the Selective Sensitization of ATM- or P53-Deficient Cancer Cells.

Frequent mutation in the ATM/P53 signaling pathway has been documented in many human cancers. Reportedly, cancer cells with deficient P53/ATM pathways depend on functional Ataxia-telangiectasia and Rad3-related (ATR) protein for survival. This has prompted research in developing ATR inhibitors for the selective sensitization of cancer cells that are P53/ATM-deficient, but no clinical success has been attained thus far. This study explores the therapeutic potential of SPK98, an analogue of Torin2 in P53- and ATM-deficient cancer cells. Furthermore, the prospect of improving the therapeutic outcome of the genotoxic agent was also explored. SPK98 was shown to inhibit full-length human ATR protein purified from HEK293T cells. Cellular investigation using SPK98 demonstrated that it selectively sensitizes P53- and ATM-deficient cells at low concentrations compared to P53-/ATM-proficient cells. Furthermore, SPK98 drives the cancer cells toward cell death by promoting the formation of DNA double-strand breaks. Taken together, our findings suggest that SPK98 is a promising therapeutic molecule for P53- or ATM-deficient malignancy that merits additional preclinical investigation.

Synthesis, kinetics and cellular studies of new phenothiazine analogs as potent human-TLK inhibitors.

The alterations in the expression patterns of protein kinases often implicate human cancer initiation and progression. Human tousled-like kinases (TLKs), both TLK1/1B and TLK2, are evolutionary kinases found in cell signaling pathways and are involved in DNA repair, replication, and chromosomal integrity. Several reports have demonstrated the numerous roles of TLK1B in the development and progression of cancer via its interactions with different partners, and this direct association has made them viable molecular targets for cancer therapy. Previous studies have shown phenothiazines to be potent TLK1B inhibitors. Herein, we report the design and synthesis of a class of phenothiazine molecules and their biological inhibitory effect on hTLK1B/KD through in vitro kinase assays, cellular assays, and in silico studies. We identified a few inhibitors with better inhibition and physio-chemical properties than the reported TLK1B inhibitors using a recombinant human tousled-like kinase 1B-kinase domain (hTLK1B-KD). Very interestingly, inhibitory activity with LNCap cells was found to be on the sub-nanomolar level. Our attempts to study the newly designed phenothiazine analogs, as well as generate a stable catalytically active hTLK1B-KD in high yield, represent a fundamental step towards the structure-based design of future TLK-specific inhibitors.

Benzothiazole and Chromone Derivatives as Potential ATR Kinase Inhibitors and Anticancer Agents.

Despite extensive studies and the great variety of existing anticancer agents, cancer treatment remains an aggravating and challenging problem. Therefore, the development of novel anticancer drugs with a better therapeutic profile and fewer side effects to combat this persistent disease is still necessary. In this study, we report a novel series of benzothiazole and chromone derivatives that were synthesized and evaluated for their anticancer activity as an inhibitor of ATR kinase, a master regulator of the DDR pathway. The cell viability of a set of 25 compounds was performed using MTT assay in HCT116 and HeLa cell lines, involving 72 h incubation of the compounds at a final concentration of 10 µM. Cells incubated with compounds 2c, 7h and 7l were found to show viability ≤50%, and were taken forward for dose-response studies. Among the tested compounds, three of them (2c, 7h and 7l) showed higher potency, with compound 7l exhibiting the best IC50 values in both the cell lines. Compounds 2c and 7l were found to be equally cytotoxic towards both the cell lines, namely, HCT116 and HeLa, while compound 7h showed better cytotoxicity towards HeLa cell line. For these three compounds, an immunoblot assay was carried out in order to analyze the inhibition of phosphorylation of Chk1 at Ser 317 in HeLa and HCT116 cells. Compound 7h showed inhibition of pChk1 at Ser 317 in HeLa cells at a concentration of 3.995 µM. Further analysis for Chk1 and pChk1 expression was carried out in Hela cells by treatment against all the three compounds at a range of concentrations of 2, 5 and 10 µM, wherein compound 7h showed Chk1 inhibition at 2 and 5 µM, while pChk1 expression was observed for compound 7l at a concentration of 5 µM. To support the results, the binding interactions of the compounds with the ATR kinase domain was studied through molecular docking, wherein compounds 2c, 7h and 7l showed binding interactions similar to those of Torin2, a known mTOR/ATR inhibitor. Further studies on this set of molecules is in progress for their specificity towards the ATR pathway.

Small molecule NSAID derivatives for impairing powerhouse in cancer cells.

Mitochondrion emerged as an important therapeutic target for anti-cancer strategy due to its involvement in cancer progression and development. However, progress of novel small molecules for selective targeting of mitochondria in cancer cells remained a major challenge. To address this, herein, through a concise synthetic strategy, we have synthesized a small molecule library of indomethacin and ibuprofen (non-steroidal anti-inflammatory drugs, NSAIDs) derivatives having triarylphosphonium moiety for mitochondria localization. Two of the library members were identified to induce mitochondrial damage through outer membrane permeabilization (MOMP) followed by generation of reactive oxygen species (ROS) leading to the remarkable MCF7 breast cancer cell death through apoptosis. These novel mitochondria targeted NSAID derivatives could open a new direction in understanding mitochondrial biology towards anti-cancer therapeutics in future.

Mutants of Helicobacter pylori IMPDH: Kinetics and in silico Studies to Determine the Structural and Functional Role of Key Amino Acids.

The emergence of antibiotic-resistant strains of Helicobacter pylori necessitates the development of novel therapeutic strategies to fight against its infection. Recently, the enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) has emerged as a promising target to treat bacterial infections due to its crucial role in the de novo purine biosynthesis pathway. The differences between the prokaryotic and eukaryotic IMPDHs, in the NAD+ binding domain and flap region, allow the identification of pathogen-specific inhibitors. In the present study, seven point mutants of wild type Helicobacter pylori IMPDH are constructed by site-directed mutagenesis, and characterized using in silico and kinetic studies. Point mutations in the NAD+ binding domain and the flap region are shown to impart significant changes in the enzyme's structure and function. In addition, the product inhibition characteristics of the Arg396-Tyr397 dyad (RY dyad) show that both the residues are important for water activation in the reaction. The results obtained are beneficial for the design and development of small-molecule inhibitors, capable of species-specific inhibition.

Tetrathiomolybdate and Tetraselenotungstate as Sulfur/Selenium Transfer Reagents: Applications in the Synthesis of New Thio/Seleno Sugars.

Sulfur and selenium containing sugars have gained prominence in the last two decades because of their importance in several biological applications. These type of carbohydrate scaffolds are also challenging targets for synthesis. In this personal note, we have summarised the results of our investigation over the last 20 years on the use of two reagents, benzyltriethylammonium tetrathiomolybdate and tetraethylammonium tetraselenotungstate, in efficient transfer of sulfur and selenium respectively to the synthesis of a number of carbohydrate derivatives.

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.

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy.

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.

The Endeavours in RAS Inhibition - the Past, Present, and Future.

KRAS mutations are known to be the most recurrent gain-of-function changes instigated in patients with cancer. The RAS gene family is often mutated in most of the human cancers, and the pursuit of inhibitors that bind to mutant RAS continues as a foremost target. RAS is a small GTPase that controls numerous cellular functions, including cell proliferation, growth, survival, and gene expression. RAS is hence closely engaged in cancer pathogenesis. The recent achievements in the discovery of RAS inhibitors imply that the inhibition of RAS oncogene may soon go into clinical trials. This review article describes the role of RAS in cancer drug discovery, the diverse methodologies used to develop direct or indirect RAS inhibitors, and emphasize the current accomplishments in the progress of novel RAS inhibitors. In short, this review focuses on the different attributes of RAS that have been targeted by a range of inhibitors consisting of membrane localization, the active form of RAS, downstream regulator binding, and nucleotide exchange binding. A detailed explanation of RAS and its involvement in cancer drug discovery together with historical aspects are mentioned first followed by a brief outline of the different approaches to target RAS.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase.

BACKGROUND: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. OBJECTIVE: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. METHODS: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. RESULTS: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. CONCLUSION: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

Design, synthesis and biological evaluation of new Myo-inositol derivatives as potential RAS inhibitors.

Ras is a small family of GTPases that control numerous cellular functions like cell proliferation, growth, survival, gene expression, and is closely engaged in cancer pathogenesis. The ras-targeted methodology entails a holy grail in oncology. Nevertheless, there are no specific molecules reported targeting the same, although it is a known oncogene for more than three decades. In this study, we have designed and synthesized new phosphate derivatives of Myo-inositol to inhibit the oncogenic KRAS pathway in breast cancer cells, which has been validated by cellular and theoretical studies. The synthesized compound 1b (C2-O-phosphate derivative of Myo-inositol 1,3,5-orthobenzoate) inhibited the downstream signaling pathway of oncogenic KRAS, RAF/MEK/ERK. Furthermore, we also found that this compound induced necrosis/apoptosis and causes cell cycle arrest. This class of molecules may work as a potential inhibitor of breast cancer caused by a mutation in KRAS and its downstream proteins. Though the efficacy of the molecules is in the micromolar scale, they have not been explored previously for RAS inhibition. Impressive preliminary results are presented in this article which could be further explored for its detailed biological studies to get better candidates as RAS inhibitors.

Evolution of PIKK family kinase inhibitors: A new age cancer therapeutics.

Phosphatidylinositol-3 kinase-related kinases (PIKKs) belong to a family of atypical serine/threonine kinases in humans. They actively participate in a diverse set of cellular functions such as meiotic, V(D)J recombination, chromosome maintenance, DNA damage sensing and repair, cell cycle progression and arrest. ATR, ATM, DNA-PKcs, mTOR and hSMG are the members of the PIKK family that play an important role in in cancer cell proliferation, autophagy, and cell survival to radio and chemotherapy. Thereby targeting these PIKK kinases in cancer along with chemo/radiotherapy agents, can help in differential cytotoxicity towards cancer cell over the normal cell. In this review, we compile the various small molecule kinase inhibitors with respect to structural and strategic targeting of PIKK family members. Rapalogs, AZD8055, AZD2014, OSI-027, INK-128, MLN0128, VX970, NVP-BEZ235, Torin2, AZ20, and AZ31 are the diverse scaffolds which have successfully made into the pre-clinical trials either as mono or combinatorial therapy for the treatment of various human cancers. Their synthesis and pre-clinical trial highlight the challenges associated in the development process.

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.

Inhibitors of inosine 5'-monophosphate dehydrogenase as emerging new generation antimicrobial agents.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a vital enzyme involved in the de novo synthesis of guanine nucleotides. IMPDH catalyzes a crucial step of converting IMP into XMP that is further converted into GMP. Microbial infections rely on the rapid proliferation of bacteria, and this requires the rate-limiting enzyme IMPDH to expand the guanine nucleotide pool and hence, IMPDH has recently received lots of attention as a potential target for treating infections. Owing to the structural and kinetic differences in the host IMPDH and bacterial IMPDH, a selective targeting is possible and is a crucial feature in the development of new potent and selective inhibitors of bacterial IMPDH. Earlier screening of small molecules revealed a structural requirement for the bacterial/protozoal IMPDH. Early optimization of benzimidazole and benzoxazole scaffolds led to the discovery of new potent and selective inhibitors of pathogenic IMPDH. Further research is vastly focused on the development of highly potent and selective inhibitors of various bacterial IMPDHs. Such studies reveal the importance of this excellent target for treating infectious diseases. The current review focuses on the recent developments in the discovery and development of selective inhibitors of bacterial/protozoal IMPDH with emphasis on the inhibition mechanism and structure-activity relationship.

Design, synthesis and biological evaluation of Helicobacter pylori inosine 5'-monophosphate dehydrogenase (HpIMPDH) inhibitors. Further optimization of selectivity towards HpIMPDH over human IMPDH2.

Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes a crucial step in guanine nucleotide biosynthesis, thereby governing cell proliferation. In contrast to mammalian IMPDHs, microbial IMPDHs are relatively less explored as potential targets for antimicrobial drug discovery. In continuation with our previous work, here we report the discovery of moderately potent and highly selective Helicobacter pylori IMPDH (HpIMPDH) inhibitors. The present study is mainly focused around our previously identified, modestly potent and relatively nonselective (for HpIMPDH over human IMPDH2) hit molecule IX (16i). In an attempt to optimize the selectivity for the bacterial enzyme, we screened a set of 48 redesigned new chemical entities (NCEs) belonging to 5-aminoisobenzofuran-1(3H)-one series for their in vitro HpIMPDH and human IMPDH2 inhibition. A total of 12 compounds (hits) demonstrated ≥70% HpIMPDH inhibition at 10 µM concentration; none of the hits were active against hIMPDH2. Compound 24 was found to be the most potent and selective molecule (HpIMPDH IC50 = 2.21 µM) in the series. The study reaffirmed the utility of 5-aminoisobenzofuran-1(3H)-one as a promising scaffold with great potential for further development of potent and selective HpIMPDH inhibitors.

Synthesis and In Vitro Enzymatic Studies of New 3-Aryldiazenyl Indoles as Promising Helicobacter pylori IMPDH Inhibitors.

BACKGROUND & OBJECTIVE: Helicobacter pylori infection is one of the primary causes of peptic ulcer followed by gastric cancer in the world population. Due to increased occurrences of multi-drug resistance to the currently available antibiotics, there is an urgent need for a new class of drugs against H. pylori. Inosine 5'-monophosphate dehydrogenase (IMPDH), a metabolic enzyme plays a significant role in cell proliferation and cell growth. It catalyses guanine nucleotide synthesis. IMPDH enzyme has been exploited as a target for antiviral, anticancer and immunosuppressive drugs. Recently, bacterial IMPDH has been studied as a potential target for treating bacterial infections. Differences in the structural and kinetic parameters of the eukaryotic and prokaryotic IMPDH make it possible to target bacterial enzyme selectively. METHODS: In the current work, we have synthesised and studied the effect of substituted 3-aryldiazenyl indoles on Helicobacter pylori IMPDH (HpIMPDH) activity. The synthesised molecules were examined for their inhibitory potential against recombinant HpIMPDH. RESULTS: In this study, compounds 1 and 2 were found to be the most potent inhibitors amongst the database with IC50 of 0.8 ± 0.02µM and 1 ± 0.03 µM, respectively. CONCLUSION: When compared to the most potent known HpIMPDH inhibitor molecule C91, 1 was only four-fold less potent and can be a good lead for further development of selective and potent inhibitors of HpIMPDH.

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.