Abstract: Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase considered to be the master player of cell-cycle regulation during mitosis. It is indeed involved in centrosome maturation, bipolar spindle formation, chromosome separation, and cytokinesis. PLK1 is overexpressed in a variety of human tumors and its overexpression often correlates with poor prognosis. Although five different PLKs are described in humans, depletion or inhibition of kinase activity of PLK1 is sufficient to induce cell-cycle arrest and apoptosis in cancer cell lines and in xenograft tumor models. NMS-P937 is a novel, orally available PLK1-specific inhibitor. The compound shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid and hematologic tumors. NMS-P937 potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft tumor growth with clear PLK1-related mechanism of action at well-tolerated doses in mice after oral administration. In addition, NMS-P937 shows potential for combination in clinical settings with approved cytotoxic drugs, causing tumor regression in HT29 human colon adenocarcinoma xenografts upon combination with irinotecan and prolonged survival of animals in a disseminated model of acute myelogenous leukemia in combination with cytarabine. NMS-P937, with its favorable pharmacologic parameters, good oral bioavailability in rodent and nonrodent species, and proven antitumor activity in different preclinical models using a variety of dosing regimens, potentially provides a high degree of flexibility in dosing schedules and warrants investigation in clinical settings. Mol Cancer Ther; 11(4); 1006–16. ©2012 AACR.

Abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology. Inhibition of PARP-1 is synthetically lethal with loss of function of the BRCA1 and BRCA2 tumor suppressor genes, as well with additional DNA repair defects. Tumor cells harboring defects in DNA repair pathways can thus be selectively targeted with PARP-1 inhibitors, and several such compounds are at different stages of clinical investigation in diverse tumor types, either as single agents or in combination regimens. However none of these agents selectively inhibits PARP-1 within the PARP family of enzymes: all drugs currently in clinical development, as well as the vast majority of preclinical compounds described to date potently cross-inhibit PARP-2, due to the high sequence homology between the two enzymes. Although PARP-2 is reported to be involved in DNA single-strand break repair, its contribution to total cellular levels of DNA damage-induced PARP activity is minimal (5-10%). Gene ablation studies show that loss of both PARP-1 and -2 function is incompatible with normal embryonic development, while PARP-2 single knockout mice show a variety of defects, including impaired erythropoiesis, thymopoiesis adipogenesis and spermatogenesis, increased neuronal loss after ischemic damage and higher risk of pancreatitis following chemical insult. We therefore reasoned that a potent and highly selective PARP-1 inhibitor might represent a significant advancement over currently available agents targeting both PARP-1 and -2, since sparing of PARP-2 inhibition potentially limits on target side-effects and offers greater opportunity for combination with other chemotherapeutic agents. Here we describe the preclinical characterization of NMS-P293, a novel highly potent PARP-1 inhibitor possessing & gt;200-fold selectivity versus PARP-2. In cells, NMS-P293 inhibits hydrogen peroxide induced poly ADP-ribose (PAR) synthesis with an IC50 in the single digit nanomolar range, confirming expected mechanism of action in cells. NMS-P293 is selectively active on tumor cell lines defective in the HR repair pathway, such as pTEN and BRCA mutated lines, while sparing DNA repair proficient cells and normal myelocytes. NMS-P293 possesses favorable ADME properties including a low efflux ratio, high cross-species metabolic stability, low clearance and nearly complete oral bioavailability in rodents and non rodents. Oral administration to mice bearing BRCA mutated breast cancer xenografts resulted in complete tumor regressions and cures. The highly favorable preclinical characteristics of NMS-P293 make this compound a promising candidate for further development. Citation Format: Alessia Montagnoli, Sonia Rainoldi, Antonella Ciavolella, Dario Ballinari, Francesco Caprera, Lucio Ceriani, Rosita Lupi, Marina Ciomei, Eduard Felder, Antonella Isacchi, Daniele Donati, Arturo Galvani, Gianluca Papeo. NMS-P293, a novel potent and selective PARP-1 inhibitor with high antitumor efficacy and tolerability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1223.

Abstract: Most of the widely used anticancer drugs target the elongation step of DNA synthesis, either directly, for example by restricting the pool of dNTPs or by acting as chain terminators following incorporation into the nascent DNA strands, or indirectly, for example by targeting enzymes that facilitate replication fork progression, or by intercalating DNA and thereby creating a physical block to fork progression. The arrest of replication forks induced by these agents invariably results in DNA strand breakage, which, while being potentially toxic to normal proliferating tissues, is also susceptible to escape by tumor cells via activation of the ATR/ATM dependent S-phase checkpoint pathway and subsequent repair of the damaged lesion. Targeting the initiation step of DNA replication, rather than elongation, might represent a strategy to overcome activation of the S-phase checkpoint, and in this context a potentially attractive molecular target is the serine/threonine kinase Cdc-7. Cdc7 promotes DNA replication initiation via phosphorylation of one or more subunits of the MCM DNA helicase complex, thus facilitating unwinding of double-stranded DNA at replication origins. Phosphorylation of Mcm2 at Ser40 and Ser53 is uniquely dependent upon Cdc7 and is essential for initiation of DNA replication and cell growth. The altered expression of proteins involved in the initiation of DNA replication closely correlates with aggressive tumor phenotypes and is a powerful marker of clinical outcome in a variety of malignancies. In particular, Cdc7 protein levels and Cdc7 dependent activity are increased in many cancer cell lines, and in tumors compared to matched normal tissue, where correlation with negative prognosis has been observed for ovarian, breast, colon and uterine carcinomas, diffuse large B cell non-Hodgkin's and Hodgkin's lymphomas. Somatic mutations of the CDC7 gene have also been identified in colorectal and gastric carcinomas. Depletion of Cdc7 by RNA interference results in p53 independent apoptosis of tumor cells, whereas normal cells respond with reversible arrest of cell cycle progression, perhaps corresponding to a physiological checkpoint. Sustained inhibition of Cdc7 in the presence of drugs affecting the elongation step of DNA replication such as topoisomerase inhibitors or DNA intercalating agents, increases cell death. Taken together, these findings suggest Cdc7 kinase may represent a promising novel cancer target. NMS-1116354 is a potent (low nM) ATP competitive small molecule inhibitor of Cdc7 kinase activity. Tested on a panel of more than 50 kinases representative of the human kinome, NMS-1116354 was found to be highly selective, with cross-reactivity observed only for CDK9. NMS-1116354 was tested for anti-proliferative activity against a panel of 171 human cancer cell lines of both solid and hematological origin. Cancer cell proliferation was inhibited with IC50 values & lt; 1 μM in 99 cell lines. Lymphoma, multiple myeloma and breast cancer cell lines, in particular triple negative, were especially sensitive to NMS-1116354. Activity of NMS-1116354 was independent of p53 status, and was maintained in cell lines resistant to 5-FU, cisplatin, gemcitabine and doxorubicin. NMS-1116354 induced rapid (within 3 hours of treatment) and massive cell apoptosis as assessed by induction of active caspase 3 and sub-G1 accumulation. Thus, in vitro, NMS-1116354 has potent, broad and p53-independent anti-cancer activity. In mechanism of action studies in cells, NMS-1116354 inhibited pSer40-Mcm2 (a phosphorylation event which we have previously described as being a specific biomarker of Cdc7 kinase activity) at drug concentrations consistent with those required for cell proliferation inhibition and apoptosis induction. Unlike DNA elongation inhibitors, such as hydroxyurea (HU), NMS-1116354 did not induce the DNA damage checkpoint response as assessed by Chk1 and Chk2 phosphorylation. Thus, effects observed following treatment of cells with NMS-1116354 essentially recapitulated those observed after genetic ablation of the protein using anti-Cdc7 siRNA. In cells, NMS-1116354 was also found to down-regulate the pro-survival protein Mcl-1, consistent with inhibition of Cdk9, the only other tested kinase against which the compound has significant activity. Downregulation of Mcl-1 protein may thus potentially contribute to anti-tumor activity of the compound, particularly in contexts which are dependent on the anti-apoptotic activity of this protein. NMS-1116354 has excellent oral biovailalability and is well tolerated in mice after prolonged treatment. Administration of NMS-1116354 to tumor-bearing animals induced potent tumor growth inhibition, including tumor regression, in xenograft models of human breast, colon, and ovarian cancer, as well as in the transgenic TRAMP model of prostate carcinoma. Tumor regression in 7 out of 10 treated animals was also observed in the rat DMBA carcinogen induced mammary tumor model. NMS-1116354 treatment also increased survival time and induced tumor regressions in AML and multiple myeloma animal models. Ex-vivo analysis of mouse xenografts treated with NMS-1116354 showed dose dependent inhibition of Mcm2 phosphorylation and modulation of the expression of a specific set of Cdc7 regulated genes in skin and tumors after both single and repeated administration. Additionally, Mcl-1 expression was found to be down-regulated in white blood cells of treated mice, as well as in tumor cells in the case of hematological cancer models, where interestingly, down-regulation correlated with antitumor activity. Thus, NMS-1116354 has potent anti-cancer activity in vivo on several different solid and hematological cancer models, in which the compound inhibits Cdc7 kinase activity and reduces Mcl-1 protein levels. These findings confirm the dual mechanism of action of the compound in vivo and support the use of Mcm2 phosphorylation, Mcl-1 down-regulation and a specifically identified gene signature as biomarkers of target modulation in clinical trials, both in tumors and in surrogate tissues (i.e. skin and blood). In drug combination studies, NMS-1116354 exhibited synergistic effects when combined with Irinotecan, Docetaxel, anti-metabolites (Gemcitabine and 5-FU) and Bortezomib in vitro and in vivo, opening a possible path for its clinical development in combination with approved drugs. Phase I clinical trials to evaluate the safety of NMS-1116354 in cancer patients were initiated in 2009. The Phase I program currently ongoing includes clinical studies with NMS-1116354 administered orally as single agent exploring different schedules in patients with solid tumors and hematological malignancies. In conclusion, NMS-1116354 is a novel Cdc7 and Cdk9 kinase inhibitor with a unique mechanism of action which involves inhibition of initiation of DNA replication and down-regulation of the pro-survival protein Mcl-1. The compound is endowed with potent single agent antitumor activity in solid and hematological cancer models, is synergistic in combination with approved drugs, and is presently in Phase I clinical studies. Selected References Montagnoli A. et al., Drf1, a novel regulatory subunit for human Cdc7 kinase. EMBO J. 2002; 21: 3171-3181. Montagnoli A. et al., Cdc7 inhibition reveals a p53-dependent replication checkpoint that is defective in cancer cells. Cancer Res. 2004; 64: 7110-7116. Montagnoli A. et al., Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases. J Biol. Chem. 2006; 281: 10281-10290. Tenca P. et al., Cdc7 is an active kinase in human cancer cells undergoing replication stress. J Biol. Chem. 2007; 282: 208-215. Montagnoli A. et al., A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity. Nature Chem. Biol. 2008; 4: 357-365. Vanotti E. et al., Cdc7 kinase inhibitors: pyrrolopyridinones as potential antitumor agents. Synthesis and structure-activity relationships. J. Med. Chem. 2008; 51: 487-501. Menichincheri et al., First Cdc7 Kinase Inhibitors: Pyrrolopyridinones as Potent and Orally Active Antitumor Agents. 2. Lead Discovery. J. Med. Chem. 2009, 22; 293-307. Ermoli et al. Cell division cycle 7 kinase inhibitors: 1H-pyrrolopyridines, synthesis and structure-activity relationships. J. Med. Chem. 2009, 23; 4380-90. Warr MR, Shore GC., Unique biology of Mcl-1: therapeutic opportunities in cancer. Current Molecular Medicine 2008; 8: 138-147. Citation Format: Francesco Colotta, Jurgen Moll, Barbara Valsasina, Ermes Vanotti, Sonia Rainoldi, Francesco Sola, Vanessa Marchesi, Maria Menichincheri, Antonella Ciavolella, Veronica Patton, Clara Albanese, Daniele Volpi, Nilla Avanzi, Dario Ballinari, Francesco Fiorentini, Antonella Isacchi, Enrico Pesenti, Arturo Galvani, Corrado Santocanale, Alessia Montagnoli. NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr DD01-01

Abstract: We previously demonstrated that natural product‐inspired 3,4‐dihydropyrrolo[1,2‐a]pyrazin‐1(2H)‐ones derivatives delivered potent and selective PIM kinases inhibitors however with non‐optimal ADME/PK properties and modest oral bioavailability. Herein, we describe a structure‐based scaffold decoration and a stereoselective approach to this chemical class. The synthesis, structure–activity relationship studies, chiral analysis, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Compound 20c demonstrated excellent potency on PIM1 and PIM2 with exquisite kinases selectivity and PK properties that efficiently and dose‐dependently promoted c‐Myc degradation and appear to be promising lead compounds for further development.

Abstract: Aberrant activation of the MAPK-mediated pathway components, RAF-MEK-ERK, is frequently found in human cancers and clearly contributes to oncogenesis. In particular, one of the three isoforms of RAF, BRAF, presents activating somatic mutations in 70% of melanomas, 50% of thyroid cancers, 10% of colon and 20% ovarian carcinomas. The most common BRAF mutation, substitution of glutamic acid for valine at position 600 within the activation segment of the kinase domain, accounts for 90% of mutated BRAF cases, and results in elevated kinase activity with consequent enhanced promotion of cell survival and proliferation. Here we describe the in vitro and in vivo properties of a novel class of potent and selective BRAF inhibitors. Starting from a series of arylpyrazole derivatives that were found to be active against wild-type and mutated BRAF, an expansion and optimization program was undertaken. This led to the identification of both inhibitors of the active conformation and inhibitors of the inactive conformation of the enzyme, as confirmed by co-crystallography studies. Among the former category, a sub-series endowed with high potency and selectivity was identified, with compounds possessing low nM affinity for BRAFV600E and negligible cross-reactivity against a panel of 44 kinases. Members of this sub-series inhibited 72-hour proliferation of cell lines harboring the BRAFV600E mutation with IC50s below 100 nM and with greater than 100-fold selectivity compared to lines with wild-type BRAF. Consistent with this observation, inhibition of the MAPK signal transduction pathway was highly selective for cell lines with mutated BRAF. Biochemical profile, potency in cell proliferation assays, and cellular mechanism of action compared favorably among the sub-series to a reference BRAF inhibitor, PLX-4720, tested in parallel. The most potent compounds (e.g. NMS-P730 and NMS-P383) were selected for in vivo evaluation. In vivo, potent tumor growth inhibition was observed in the A375 xenograft model following oral administration, with no overt toxicity, and with corresponding modulation of the MAPK pathway, consistent with expected mechanism of action. Selection of candidates most suitable for further development is underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2522.