Rita Perego

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-Å resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix αC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II β-turn. The intermediate portion of the supersite (1353NATY) assumes a type I β-turn conformation as in an Shc–phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

Identification of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as a new class of orally and selective Polo-like kinase 1 inhibitors

Polo-like kinase 1 (Plk1) is a fundamental regulator of mitotic progression whose overexpression is often associated with oncogenesis and therefore is recognized as an attractive therapeutic target in the treatment of proliferative diseases. Here we discuss the structure-activity relationship of the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline class of compounds that emerged from a high throughput screening (HTS) campaign as potent inhibitors of Plk1 kinase. Furthermore, we describe the discovery of 49, 8-{[2-methoxy-5-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide, as a highly potent and specific ATP mimetic inhibitor of Plk1 (IC(50) = 0.007 microM) as well as its crystal structure in complex with the methylated Plk1(36-345) construct. Compound 49 was active in cell proliferation against different tumor cell lines with IC(50) values in the submicromolar range and active in vivo in the HCT116 xenograft model where it showed 82% tumor growth inhibition after repeated oral administration.

Discovery of 2-[1-(4,4-Difluorocyclohexyl)Piperidin-4-Yl]-6-Fluoro-3-Oxo-2,3-Dihydro-1H-Isoindole-4-Carboxamide (Nms-P118): A Potent, Orally Available and Highly Selective Parp- 1 Inhibitor for Cancer Therapy.

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.

Identification of candidate substrates for poly(ADP-ribose) polymerase-2 (PARP2) in the absence of DNA damage using high-density protein microarrays.

Poly(ADP‐ribose) polymerase‐2 (PARP2) belongs to the ADP‐ribosyltransferase family of enzymes that catalyze the addition of ADP‐ribose units to acceptor proteins, thus affecting many diverse cellular processes. In particular, PARP2 shares with PARP1 and, as recently highlighted, PARP3 the sole property of being catalytically activated by DNA‐strand breaks, implying key downstream functions in the cellular response to DNA damage for both enzymes. However, evidence from several studies suggests unique functions for PARP2 in additional processes, possibly mediated through its basal, DNA‐damage unstimulated ADP‐ribosylating activity. Here, we describe the development and application of a protein microarray‐based approach tailored to identify proteins that are ADP‐ribosylated by PARP2 in the absence of DNA damage mimetics and might thus represent useful entry points to the exploration of novel PARP2 functions. Several candidate substrates for PARP2 were identified and global hit enrichment analysis showed a clear enrichment in translation initiation and RNA helicase molecular functions. In addition, the top scoring candidates FK506‐binding protein 3 and SH3 and cysteine‐rich domain‐containing protein 1 were selected and confirmed in a complementary assay format as substrates for unstimulated PARP2.

Insights into PARP Inhibitors’ Selectivity Using Fluorescence Polarization and Surface Plasmon Resonance Binding Assays

PARP inhibitors are an exciting new class of antineoplastic drugs that have been proven to be efficacious as single agents in cancer settings with inherent DNA repair defects, as well as in combination with DNA-damaging chemotherapeutics. Currently, they are designed to target the catalytic domain of PARP-1, the most studied member of the family, with a key role in the DNA-damage repair process. Because PARP inhibitors are substrate (NAD+) competitors, there is a need for a deeper understanding of their cross-reactivity. This is particularly relevant for PARP-2, the PARP-1 closest homologue, for which an embryonic lethal phenotype has been observed in double knockout mice. In this study, we describe the development and validation of binding assays based on fluorescence polarization (FP) and surface plasmon resonance (SPR) techniques. PARP-1, PARP-2, PARP-3, and TNKS-1 FP displacement assays are set up by employing ad hoc synthesized probes. These assays are suitable for high-throughput screening (HTS) and selectivity profiling, thus allowing the identification of NAD+ binding site selective inhibitors. The PARP-1 and PARP-2 complementary SPR binding assays confirm displacement data and the in-depth inhibitor characterization. Moreover, these formats have the potential to be broadly applicable to other members of the PARP family.

Abstract A149: NMS-P945, a highly active payload for antibody drug conjugates generation

Antibody-drug conjugates (ADCs) are increasingly employed in different oncology settings with more than forty products in clinical evaluation at present, and two approved drugs, ado-trastuzumab emtansine and brentuximab vedotin, respectively targeting Her2 and CD30 positive tumors. Although many different antibody targets have been so far considered for this approach, only a handful of toxins have been exploited, and more than 50% of ADCs result to be conjugated to two well known tubulin binding agents, auristatin and maitansine. New toxins with a different cellular mechanism of action, possibly acting also on quiescent or slowly proliferating cells, are thus strongly needed. Duocarmycins, classical DNA minor groove alkylating agents, were studied in clinical trials as chemotherapy drugs in the nineties, but were abandoned few years later due to the poor therapeutic index of the free drugs. In this context we approached a new proprietary class of thienoindole duocarmycins derivatives as novel promising toxins for ADC generation. Here we describe the identification and related proof of concept studies for this novel chemical series, characterized by both potent antitumor activity and physicochemical properties highly compatible with deployment as antibody payloads. Extensive in vitro profiling within the thienoindole series led to the selection of a potent cytotoxic compound suitable for conjugation to humanized monoclonal antibodies upon introduction of a linker moiety (NMS-P945). Efficacy and mechanism of action studies were carried out for NMS-P945-ADCs prepared with trastuzumab showing target-directed and target-enhanced (“bystander”) cytotoxicity in Her2 positive vs. negative cancer cell lines. In vitro and in vivo activity of NMS-P945-ADC is strictly correlated to the target presence, the internalization and the lysosomal digestion of the ADC inside the tumor cells. In addition we generated and validated selective antidrug antibodies and using this tool we followed the fate of the toxin upon release from the ADC clearly showing chromatin localization, as expected for duocarmycin based molecules. In animal efficacy studies against a Her2-positive human breast cancer model, trastuzumab-NMS-P945 ADC administration yielded complete tumor regression in treated mice, with no effects on body weight gain, while unarmed trastuzumab and armed control antibody showed little and no effect, respectively. These results support further development of NMS-P945 as a new interesting payload for conjugation with targeted antibodies. Citation Format: Fabio Gasparri, Michele Caruso, Italo Beria, Nicoletta Colombo, Paolo Orsini, Rita Perego, Simona Rizzi, Ulisse Cucchi, Sonia Troiani, Federico Riccardi Sirtori, Clara Albanese, Aurelio Marsiglio, Ivan Fraietta, Francesco Sola, Marina Ciomei, Sabrina Cribioli, Carlo Visco, Eduard Rudolf Felder, Antonella Isacchi, Enrico Pesenti, Arturo Galvani, Daniele Donati, Barbara Valsasina. NMS-P945, a highly active payload for antibody drug conjugates generation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A149.

Abstract 822: Thienoindoles, a novel class of DNA minor groove alkylating agents highly suited for the generation of novel antibody drug conjugates (ADCs)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The rapidly growing field of Antibody-Drug Conjugates (ADCs) has recently spurred the study of novel drug payloads. In particular, much interest has been paid to the identification of novel cytotoxic agents, which differ in mechanism from anti-tubulin agents, currently the most commonly employed class of drug for ADC coupling. Novel drug payloads for ADC-based therapy should thus ideally possess highly potent cytotoxic activity with a diverse mechanism from tubulin agents, as well as physicochemical properties, which facilitate coupling to antibodies. Duocarmycins are a widely-described class of DNA minor groove binding alkylating agents, examples of which can exhibit picomolar activity against human tumor cell lines, as well as high activity in preclinical tumor models. The clinical development of duocarmycins as drugs in their own right has however been hampered by an extremely limited therapeutic window. Duocarmycins also typically exhibit poor physicochemical profiles, most importantly generally possessing limited aqueous solubility and a propensity to induce protein aggregation, which renders these molecules poorly tractable as optimal ADC payloads. Based on the premise that opportune modification of the duocarmycin scaffold could lead to the discovery of compounds better suited to antibody conjugation, we describe the identification and related proof of concept studies for a novel chemical series based on a proprietary thienoindole scaffold, characterized both by potent antitumor activity and by physicochemical properties that are highly compatible with deployment as antibody payloads. Extensive in vitro profiling within the thienoindole series led to selection of potent cytotoxic compounds suitable for test conjugation to humanized monoclonal antibodies, which were then submitted to in vitro/in vivo profiling. Typically, average drug/antibody ratios (DARs) of 4 could readily be achieved without induction of antibody aggregation/precipitation and without affecting natural antibody-ligand affinity. Efficacy and mechanism of action studies were carried out for thienoindole-ADCs prepared with trastuzumab and with rituximab showing target driven effects in Her2 positive vs. negative breast cancer cell lines and against CD20 positive vs. negative hematological cell lines. PK data indicated long, essentially unaffected plasma half life of our novel ADCs in the mouse. In vivo efficacy studies in the Her2-positive HCC1954 human breast cancer model showed complete tumor regression in mice treated with trastuzumab-ADC with no effects on body weight gain, while unarmed trastuzumab and armed control antibody had little and no effect, respectively. Citation Format: Barbara Valsasina, Fabio Gasparri, Italo Beria, Nicoletta Colombo, Paolo Orsini, Rita Perego, Simona Rizzi, Ulisse Cucchi, Clara Albanese, Aurelio Marsiglio, Ivan Fraietta, Marina Ciomei, Sabrina Cribioli, Carlo Visco, Eduard R. Felder, Antonella Isacchi, Enrico A. Pesenti, Arturo Galvani, Daniele Donati, Michele Caruso. Thienoindoles, a novel class of DNA minor groove alkylating agents highly suited for the generation of novel antibody drug conjugates (ADCs). [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 822. doi:10.1158/1538-7445.AM2014-822

Abstract 691: In vitro and in vivo characterization of selective orally available Parp-1 inhibitors with demonstrated antitumor efficacy in BRCA negative cancer models

Poly (ADP-ribose) polymerase 1 and 2 (Parp-1 and Parp-2) are nuclear enzymes responsible for signaling the presence of DNA damages by catalyzing the addition of ADP-ribose units to DNA, histones and various DNA repair enzymes, thus facilitating DNA repair. Parp-1 has been gaining increasing interest as a therapeutic target for cancer in combination with DNA damaging agents but also as single agent in particular tumor settings, such as BRCA mutated tumors. While Parp-1 knock out mice are viable and display only defects in DNA repair, double Parp-1 and Parp-2 knock-out are embryonic lethal indicating that the absence of Parp-1 and Parp-2 is less tolerated in normal cells. We confirmed this concept in vitro by RNAi of Parp-1 and Parp-2, showing that the inhibition of Parp-1 alone is enough to kill tumor cells but spares normal cells. Hence a selective Parp-1 inhibitor might have a better safety profile, particularly in view of a long treatment. Here we report the discovery and the in vitro and in vivo characterization of a novel class of potent, orally available Parp-1 selective small molecule inhibitors with no activity on other Parp isoforms such as Parp-2, −3 and −5a (tankyrase), which differentiates it from current clinical Parp inhibitors. The most potent compounds were further evaluated in vitro and in vivo. These compounds are highly potent in inhibiting DNA damage-induced Parp-1 activity in cells and exhibit selective anti-proliferative and pro-apoptotic activity in tumor cell lines harbouring defects in DNA repair. In vivo, the compounds show an excellent pharmacokinetic profile with almost complete oral bioavailability and demonstrate efficacy as a single agent in xenograft tumor models with DNA repair deficiencies superior to reference Parp inhibitors. These compounds are very well tolerated with no overt toxicity even after prolonged exposure and their mechanism of action is confirmed both in tumors and peripheral blood cells of treated mice. 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 691.