Paolo Polucci

Rational design, synthesis and biological evaluation of thiadiazinoacridines: A new class of antitumor agents

A series of potential DNA-binding antitumor agents, 3-[omega-(alkylamino)alkyl]-6-nitro-thiadiazino[3,4,5-kl]acridines 12 and 1,3-di[omega-(alkylamino)alkyl]-6-nitro-thiadiazino[3,4,5-kl]acridines 13, has been prepared by cyclization with SOCl(2) of 1-[[omega-(alkylamino)alkyl]amino]-9-imino-4-nitro-9,10-dihydroacridines 16 or 1-[[omega-(alkylamino)alkyl]amino]-9-[omega-(alkylamino)alkyl]imino-4-nitro-9,10-dihydroacridines 17, respectively. The non-covalent DNA-binding properties of 12, 13 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives toward six tumor cell lines, including human colon adenocarcinoma (HT29) and human ovarian carcinoma (A2780 sensitive, A2780cisR cisplatin-resistant, CH1, CH1cisR cisplatin-resistant, and SKOV-3) cells, are described and compared to that of reference drugs. In vivo antitumor activity of some selected derivatives, endowed with relevant cytotoxic activity against murine leukemia P388 are reported. The 3-[2-(dimethylamino)ethyl]-6-nitro-2,7-dihydro-3H-2 lambda(4)-thiadiazino[3,4,5-kl]acridin-2-one (12d) has been identified as a new lead in the development of anticancer tetracyclic acridine derivatives.

NMS-E973, a Novel Synthetic Inhibitor of Hsp90 with Activity against Multiple Models of Drug Resistance to Targeted Agents, Including Intracranial Metastases

Purpose: Recent developments of second generation Hsp90 inhibitors suggested a potential for development of this class of molecules also in tumors that have become resistant to molecular targeted agents. Disease progression is often due to brain metastases, sometimes related to insufficient drug concentrations within the brain. Our objective was to identify and characterize a novel inhibitor of Hsp90 able to cross the blood–brain barrier (BBB). Experimental Design: Here is described a detailed biochemical and crystallographic characterization of NMS-E973. Mechanism-based anticancer activity was described in cell models, including models of resistance to kinase inhibitors. Pharmacokinetics properties were followed in plasma, tumor, liver, and brain. In vivo activity and pharmacodynamics, as well as the pharmacokinetic/pharmacodynamic relationships, were evaluated in xenografts, including an intracranially implanted melanoma model. Results: NMS-E973, representative of a novel isoxazole-derived class of Hsp90 inhibitors, binds Hsp90α with subnanomolar affinity and high selectivity towards kinases, as well as other ATPases. It possesses potent antiproliferative activity against tumor cell lines and a favorable pharmacokinetic profile, with selective retention in tumor tissue and ability to cross the BBB. NMS-E973 induces tumor shrinkage in different human tumor xenografts, and is highly active in models of resistance to kinase inhibitors. Moreover, consistent with its brain penetration, NMS-E973 is active also in an intracranially implanted melanoma model. Conclusions: Overall, the efficacy profile of NMS-E973 suggests a potential for development in different clinical settings, including tumors that have become resistant to molecular targeted agents, particularly in cases of tumors which reside beyond the BBB. Clin Cancer Res; 19(13); 3520–32. ©2013 AACR.

Fragment-Based Hit Discovery and Structure-Based Optimization of Aminotriazoloquinazolines as Novel Hsp90 Inhibitors.

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.

Discovery of Nms-E973 as Novel, Selective and Potent Inhibitor of Heat Shock Protein 90 (Hsp90).

Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.

Abstract 2082: NMS-E668, a potent and selective RET kinase inhibitor characterized by specificity towards VEGFR2 and high antitumor efficacy against RET-driven models

RET, a receptor tyrosine kinase (RTK) expressed mainly in neural crest-derived tissues, plays a role in cell growth and differentiation and its physiological activation depends upon binding to the GDNF family. Genetic aberrations leading to constitutive RET activation are well-established as oncogenic events. Activating point mutations of RET, for example, are present in ca. 70% of medullary thyroid carcinoma patients including all hereditary cases, while RET gene rearrangements resulting in production of activated RET fusion proteins occur in approximately 10% of sporadic papillary thyroid carcinomas. More recently, recurring RET gene rearrangements have also been found in 1-2 % of lung adenocarcinomas and subsets of other solid tumors including colorectal and salivary gland carcinomas. Thus RET kinase represents an actionable therapeutic target in multiple clinical settings with high medical need. Consequently several small-molecule inhibitors targeting this kinase have been explored in clinical settings. A common feature of most advanced agents is their lack of selectivity and in particular their potent cross-reactivity against VEGFR2, an RTK whose inhibition is associated with serious, dose-limiting cardiovascular toxicity. Indeed, the high homology between the two kinases renders identification of ATP competitive compounds that selectively inhibit RET over VEGFR2 a highly challenging task. Here we describe the preclinical activity of NMS-E668, a potent and selective ATP-competitive RET inhibitor characterized by favorable activity, selectivity and ADME profiles. Biochemically, NMS-E668 has an excellent selectivity profile against a panel of >50 kinases, notably including >10-fold selectivity over VEGFR2. Selectivity of NMS-E668 for RET vs. VEGFR2 was confirmed in NIH-3T3 cells engineered to express activated forms of these kinases. NMS-E668 potently (IC50 circa 50 nM) and selectively inhibited proliferation of RET-dependent tumor cells, including TT medullary carcinoma cells harboring a RET C634W activating point mutation and LC2/ad lung carcinoma cells bearing the oncogenic fusion protein CCDC6-RET. NMS-E668 also potently inhibited IL3-independent growth of Ba/F3 cells expressing KIF5B-RET, the RET rearrangement that is most commonly found in lung adenocarcinomas. Cellular mechanism studies confirmed that NMS-E668 inhibits RET autophosphorylation and downstream signaling at doses consistent with antiproliferative activity. Tested in vivo against KIF5B-RET-driven Ba/F3 tumors, NMS-E668 displayed >90% tumor growth inhibition accompanied by target modulation following oral administration at 10 and 20 mg/kg, with prolonged tumor regressions observed at the higher dose. Thus NMS-E668, a potent and VEGFR2-sparing RET inhibitor is an innovative and highly promising candidate for further development. Citation Format: Elena Ardini, Patrizia Banfi, Nilla Avanzi, Marina Ciomei, Paolo Polucci, Alessandra Cirla, Antonella Ermoli, Ilaria Motto, Elena Casale, Giulia Canevari, Cinzia Cristiani, Sonia Troiani, Federico Riccardi Sirtori, Nadia Amboldi, Dario Ballinari, Francesco Caprera, Eduard Felder, Arturo Galvani, Daniele Donati, Antonella Isacchi, Maria Menichincheri. NMS-E668, a potent and selective RET kinase inhibitor characterized by specificity towards VEGFR2 and high antitumor efficacy against RET-driven models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2082. doi:10.1158/1538-7445.AM2017-2082

Abstract 2091: Identification of a highly potent, selective and orally available RET inhibitor with antitumor efficacy in RET-dependent tumor models.

The RET proto-oncogene encodes a receptor tyrosine kinase that is mainly expressed in neural crest-derived tissues where it plays an important role in cell differentiation, growth and survival. Germline activating point mutations of RET are associated with multiple endocrine neoplasia type 2 (MEN2), an inherited cancer syndrome characterized by development of medullary thyroid carcinoma (MTC), pheochromocytoma and parathyroid hyperplasia and are present in circa 50% of sporadic cases of MTC. More recently, a chromosomal rearrangement of the RET gene was identified in a subset of lung adenocarcinomas (1-2%) which is reported to result in expression of a fusion protein containing constitutively active RET kinase domain fused to the N-terminal portion of the kinesin KIF5B. We recently presented data on NMS-173, a very potent RET inhibitor characterized by an excellent in vivo activity profile upon i.v. administration. Here we describe the identification and the preclinical characterization of NMS-616, a novel analogue belonging to the same chemical class, suitable for oral administration. NMS-616 is a highly potent (IC50: 2 nM), ATP competitive RET inhibitor, characterized by high selectivity when tested against a panel of more than 50 kinases. NMS-616 potently blocked proliferation of MTC cell lines, such as the TT human cell line, which endogenously harbours constitutively activated RET, concomitant with abrogation of RET autophosphorylation and signaling pathway activation. The compound also inhibited the IL-3 independent proliferation of RET-driven Ba/F3 cells, with down-modulation of RET autophosphorylation and downstream signalling pathways. NMS-616 is characterized by a good in vitro ADME profile and in vivo pharmacokinetic parameters in the mouse, including oral bioavailability. When tested in vivo in a murine subcutaneous xenograft model employing TT cells, NMS-616 displayed dose-dependent tumor growth inhibition following daily oral administration at 50 and 100 mg/Kg, with tumor regression observed at both dose levels. In addition ex vivo analysis demonstrated dose-dependent target modulation following a single administration and the maintenance of RET phosphorylation inhibition for at least 24 hours. Citation Format: Elena Ardini, Patrizia Banfi, Francesca Quartieri, Paolo Polucci, Nilla Avanzi, Dario Ballinari, Laura Mancini, Edward Felder, Daniele Donati, Arturo Galvani, Enrico Pesenti, Antonella Isacchi, Maria Menichincheri. Identification of a highly potent, selective and orally available RET inhibitor with antitumor efficacy in RET-dependent tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2091. doi:10.1158/1538-7445.AM2013-2091

Abstract 2940: Identification of potent VCP/p97/CDC48 inhibitors with distinct biochemical mechanisms including a reversible, allosteric inhibitor that activates the unfolded protein response, induce autophagy and cancer cell death

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Valosin Containing Protein (VCP), also called p97 in mammals and cdc48 in yeast, is an ubiquitously expressed and essential AAA ATPase important in specific cellular processes including Endoplasmic Reticulum Associated Degradation (ERAD), Golgi reformation, membrane fusion and autophagy. VCP is a hexameric complex formed by six identical protomers, each composed of three domains: an N-terminal domain responsible for the interaction with co-factors and adaptor proteins, and two AAA ATPase domains, D1 and D2. VCP acts as a protein-directed molecular machine that converts energy derived from ATP hydrolysis into mechanical force to cause disassembly of multiprotein complexes or extraction of molecules from the membrane to be delivered to the proteasome for degradation. VCP also affects autophagy and aggresome formation processes. Overall, VCP plays a key role in cellular homeostasis. The clinical success of proteasome inhibitors, and recent advances in the preclinical development of molecules that interfere with protein folding and degradation has highlighted that cancer cells can be extremely sensitive to perturbation of protein homeostasis. The availability of small molecules that specifically inhibit VCP function would help to clarify whether VCP is a valid target for cancer therapy. We found that VCP silencing by siRNA induces cancer cell death in a variety of tumor cell lines, and examined the main cellular pathways modulated upon VCP ablation, thus identifying biomarkers suitable for characterizing the cellular activity of VCP inhibitors. We then performed a High Throughput Screening campaign using recombinant VCP and identified multiple compound classes that inhibit VCP function with distinct biochemical mechanisms. Initial hits included a compound that covalently modifies VCP, representatives of two classes of ATP-sensitive inhibitors, and an inhibitor class characterized by a novel allosteric mechanism of action. Chemical expansion of initial hits resulted in improvement of biochemical potency, yielding highly active derivatives for each class, suggesting that VCP is a druggable target. Notably, the binding site of the allosteric class of inhibitors was identified by photo-affinity labeling in combination with available structural data. We will present data on a potent (30 nM) and specific compound emerging from this class that displays anti-proliferative activity related to the modulation of direct VCP biomarkers, activation of the Unfolded Protein Response (UPR) and perturbation of autophagy, which ultimately result in cancer cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2940. doi:1538-7445.AM2012-2940