The nuclear protein 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, as testified by the number of ...candidates in clinical testing that unselectively target both PARP-1 and its closest isoform PARP-2. The goal of our program was to find a PARP-1 selective inhibitor that would potentially mitigate toxicities arising from cross-inhibition of PARP-2. Thus, an HTS campaign on the proprietary Nerviano Medical Sciences (NMS) chemical collection, followed by SAR optimization, allowed us to discover 2-1-(4,4-difluorocyclohexyl)piperidin-4-yl-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118, 20by). NMS-P118 proved to be a potent, orally available, and highly selective PARP-1 inhibitor endowed with excellent ADME and pharmacokinetic profiles and high efficacy in vivo both as a single agent and in combination with Temozolomide in MDA-MB-436 and Capan-1 xenograft models, respectively. Cocrystal structures of 20by with both PARP-1 and PARP-2 catalytic domain proteins allowed rationalization of the observed selectivity.
Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are ...phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.
Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, ...and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.
Cdc7 kinase promotes and regulates DNA replication in eukaryotic organisms. Multiple mechanisms modulating kinase activity in response to DNA replication stress have been reported, supporting the ...opposing notions that Cdc7 either plays an active role under these conditions or, conversely, is a final target inactivated by a checkpoint response. We have developed new immnunological reagents to study the properties of human Cdc7 kinase in cells challenged with the ribonucleotide reductase inhibitor hydroxyurea or with the DNA topoisomerase II inhibitor etoposide. We show that Cdc7·Dbf4 and Cdc7·Drf1 complexes are stable and active in multiple cell lines upon drug treatment, with Cdc7·Dbf4 accumulating on chromatin-enriched fractions. Cdc7 depletion by small interfering RNA in hydroxyurea and etoposide impairs hyper-phosphorylation of Mcm2 at specific Cdc7-dependent phosphorylation sites and drug-induced hyper-phosphorylation of chromatin-bound Mcm4. Furthermore, sustained inhibition of Cdc7 in the presence of these drugs increases cell death supporting the notion that the Cdc7 kinase plays a role in maintaining cell viability during replication stress.
Cdc7 kinase has recently emerged as an attractive target for cancer therapy and low-molecular-weight inhibitors of Cdc7 kinase have been found to be effective in the inhibition of tumor growth in ...animal models. In this paper, we describe synthesis and structure−activity relationships of new 1H-pyrrolo2,3-bpyridine derivatives identified as inhibitors of Cdc7 kinase. Progress from (Z)-2-phenyl-5-(1H-pyrrolo2,3-bpyridin-3-ylmethylene)-3,5-dihydro-4H-imidazol-4-one (1) to (Z)-2-(benzylamino)-5-(1H-pyrrolo2,3-bpyridin-3-ylmethylene)-1,3-thiazol-4(5H)-one (42), a potent ATP mimetic inhibitor of Cdc7 kinase with IC50 value of 7 nM, is also reported.
Abstract
NMS-P293 is a novel, potent and selective small molecule inhibitor of poly (ADP-ribose) polymerase (PARP-1 Kd = 2 nM).
NMS-P293 inhibits the poly-ADP ribosylation (PAR) induced by DNA ...damage, leading to the inability of homologous repair deficient (HRD) tumor cells to repair damage and to apoptotic death, while sparing normal cells.
NMS-P293 significantly differs from other PARP inhibitors in several respects. Firstly, while possessing potent in vitro and in vivo efficacy in HRD tumors (e.g. BRCA, PTEN loss), it does not induce “DNA trapping”, i.e. the formation of PARP-chromatin complexes. Secondly, NMS-293 is not a substrate of PgP mediated drug efflux, believed to be a mechanism of resistance to current PARP inhibitors. Finally, NMS-P293 extensively crosses the blood brain barrier in rats and mice, with a brain/plasma ratio of 4-10, significantly higher than that of major comparators and opening the potential for treating primary brain tumors and brain metastasis with PARP inhibitor therapy. Extensive preclinical profiling revealed that NMS-P293 possesses favourable drug-like characteristics, which include high in vitro cross-species metabolic stability, lack of cytochrome interaction, and excellent pharmacokinetic profile, with low clearance and nearly complete oral bioavailability in both rodents and non rodents. Preclinical efficacy and PK/PD studies were conducted to assess the mechanism of action of NMS-P293 in an HRD setting, the MDA-MD-436 (BRCA1 mutant) human breast cancer xenograft model. Inhibition of PAR was observed in tumors following single oral administration of 50 mg/kg NMS-P293, with a > 95% decrease of this biomarker persisting for > 24 h. Inverse correlation was observed between intratumoral PAR levels and NMS-P293 concentrations in plasma and in tumor. Significantly, levels of NMS-P293 in tumor were consistently higher (> 3.5-fold) than in plasma, with an intratumoral concentration of > 2 μM at 24 h, in line with the prolonged PAR inhibition. In agreement with these PD effects, NMS-P293, orally administered as single agent, induced potent dose-related growth inhibition of BRCA mutated tumor model, with cured animals. Furthermore, NMS-P293 also possesses potent synergistic efficacy and tolerability in combination with temozolomide (TMZ) in glioblastoma (GBM) tumor models, including TMZ resistant MGMT hypomethylated GBMs.
The unique preclinical features of NMS-P293, which includes a highly permissive profile in GLP drug safety studies, render this compound an appealing candidate for clinical development, both as a single agent and in drug combination therapy regimes.
Citation Format: Alessia Montagnoli, Gianluca Papeo, Sonia Rainoldi, Francesco Caprera, Marina Ciomei, Eduard Felder, Daniele Donati, Antonella Isacchi, Arturo Galvani. NMS-P293, a PARP-1 selective inhibitor with no trapping activity and high CNS penetration, possesses potent in vivo efficacy and represents a novel therapeutic option for brain localized metastases and glioblastoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4843.
Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs NAD+ to modify substrate proteins via the attachment of ...poly(ADP-ribose) chains. PARP-1 is a well established target in oncology, as testified by the number of marketed drugs (e.g., Lynparza, Rubraca, Zejula, and Talzenna) used for the treatment of ovarian, breast, and prostate tumors. Efforts in investigating an uncharted region of the previously identified isoindolinone carboxamide series delivered (S)-13 (NMS-P515), a potent inhibitor of PARP-1 both in biochemical (K d: 0.016 μM) and cellular (IC50: 0.027 μM) assays. Cocrystal structure allowed explaining NMS-P515 stereospecific inhibition of the target. After having ruled out potential loss of enantiopurity in vitro and in vivo, NMS-P515 was synthesized in an asymmetric fashion. NMS-P515 ADME profile and its antitumor activity in a mouse xenograft cancer model render the compound eligible for further optimization.
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 >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
Cdc7 is an essential serine/threonine protein kinase that licenses initiation of DNA synthesis at replication origins, promotes cell cycle checkpoint activation in response to replication ...stress and is involved in additional aspects of DNA metabolism, including repair mechanisms. Cdc7 is therefore a potential target for cancer therapy, with a distinct mechanism of action from known drugs that inhibit DNA replication. Overexpression of Cdc7 is found in various types of cancer, suggesting that its deregulated activity may promote survival of cancer cells, as well as tumor progression. In particular, its overexpression in breast cancer is linked to triple negative, HER2 positive subtypes, genomic instability, arrested tumor differentiation and enhanced survival. Depletion through RNA silencing, or inhibition of the kinase activity of Cdc7 induces S-phase arrest and apoptosis in cancer cell lines, while non-transformed epithelial cells arrest in G1, remain viable, and are able to resume cell proliferation on recovery of Cdc7 activity.
We have previously reported the identification of NMS-P354, an orally bioavailable Cdc7 inhibitor which promotes tumor growth inhibition in preclinical cancer models and was very helpful in validating Cdc7 as potential target.
Here, we describe the characterization of NMS-P862, a follow -on, novel, potent (IC50: 9 nM) and more selective Cdc7 inhibitor. Profiling of anti-proliferative activity against more than one hundred human tumor cell lines indicated relatively high activity (IC50< 0.5 μM) against triple negative or HER2 positive/double negative breast cancer lines, as well as against prostate cancer cell lines. In vivo, oral administration of NMS-P862 resulted in dose-dependent antitumor activity, including for example tumor regression in a carcinogen (7,12-dimethylbenzαantracene) induced rat breast cancer model, for which we observed 9/12 objective responses, assessed as per RECIST criteria.
A favorable ADME profile with high oral bioavailability, together with permissive therapeutic safety margins in test species at efficacious exposures, indicate that NMS-P862 is suitable for further development, affording the possibility to target cancer via a novel mechanism which we believe is suited to both single agent and opportune drug combination approaches.
Citation Format: Alessia Montagnoli, Maria Menichincheri, Nadia Amboldi, Dario Ballinari, Marina Ciomei, Francesco Fiorentini, Rosita Lupi, Daniele Pezzetta, Sonia Rainoldi, Daniele Pezzetta, Eduard Felder, Antonella Isacchi, Enrico Pesenti, Arturo Galvani. NMS-P862, a novel orally available selective small molecule Cdc7 inhibitor with antitumor efficacy in breast cancer. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4539. doi:10.1158/1538-7445.AM2014-4539
Abstract
PARP-1 and PARP-2 are nuclear enzymes activated by DNA strand breaks and are involved in recruitment of DNA repair proteins to sites of damage. PARP-1 and PARP-2 share partially overlapping ...functions, as shown by the embryonic lethality of double knockout mice compared to animals with single ablation either gene, which are viable. Despite this partial functional redundancy, PARP-1, but not PARP-2, inhibition has been shown to be synthetically lethal with defects in homologous recombination such as BRCA gene mutations. All PARP inhibitors that are currently under clinical investigation inhibit both PARP-1 and PARP-2, due to the high sequence similarity between the catalytic domains of the two enzymes. A third member of the family, PARP-3, has lower homology with PARP-1 and -2 and is much less well characterized, although it is known that its activity is stimulated by double strand breaks and that it promotes DNA repair through an error prone repair pathway, non-homologous end joining (NHEJ). PARP-3 inhibition is thus presumed to be synthetically lethal with other, alternative DNA repair pathway deficiencies.
We report preclinical characterization of NMS-P648, a selective PARP-1/-3 inhibitor, with a dissociation constants (KD), of 0.9 nM on PARP-1 and with greater than four hundred-fold less affinity for PARP-2, as assessed by direct binding assay. In cells, NMS-P648 inhibits hydrogen peroxide induced poly ADP-ribose (PAR) synthesis with an IC50 of 1 nM, confirming expected mechanism of action and indicating that inhibition of PARP-2 is not required for this process.
NMS-P648 has favourable ADME properties, including stability in liver microsome incubation assays, low efflux ratio in the CACO2 assay and an excellent pharmacokinetic profile, with complete oral bioavailability and exposure levels that increase proportionally with dose.
Testing NMS-P648 on a panel of more than 100 human tumor cell lines using a 2D colony forming assay format revealed that 7 cell lines were highly sensitive to the compound (IC50 <0.5 μM). As expected for an inhibitor of DNA repair, NMS-P648 induced S-phase cell cycle arrest and the formation of nuclear γH2AX foci in sensitive cell lines, suggesting the induction of stalled or damaged replication forks, followed by cell death. Responsive lines were of diverse tissue origin and included BRCA mutated lines, as well as lines resistant to standard chemotherapeutic agents and to known PARP 1/2 inhibitors, indicating a unique activity profile. Translational and bioinformatic studies are ongoing, aimed at identifying determinants of NMS-P648 sensitivity that might drive patient population selection.The discovery that potent PARP-1/-3 dual inhibitors such as NMS-P648 possess a unique sensitivity profile provides an innovative opportunity in the field of therapeutic intervention in DNA repair processes.
Citation Format: Alessia Montagnoli, Gianluca Papeo, Sonia Rainoldi, Alessandra Cirla, Antonella Ciavolella, Clara Albanese, Michele Modugno, Roberta Bosotti, Alessio Somaschini, Giovanni Carapezza, Rosita Lupi, Dario Ballinari, Marina Ciomei, Enrico Pesenti, Daniele Donati, Antonella Isacchi, Arturo Galvani. Preclinical characterization of NMS-P648, a novel and potent PARP-1/-3 inhibitor. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2851. doi:10.1158/1538-7445.AM2015-2851
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