Nilla Avanzi

Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death

VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.

Targeting the Mitotic Checkpoint for Cancer Therapy with Nms-P715, an Inhibitor of Mps1 Kinase.

MPS1 kinase is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. It has been found aberrantly overexpressed in a wide range of human tumors and is necessary for tumoral cell proliferation. Here we report the identification and characterization of NMS-P715, a selective and orally bioavailable MPS1 small-molecule inhibitor, which selectively reduces cancer cell proliferation, leaving normal cells almost unaffected. NMS-P715 accelerates mitosis and affects kinetochore components localization causing massive aneuploidy and cell death in a variety of tumoral cell lines and inhibits tumor growth in preclinical cancer models. Inhibiting the SAC could represent a promising new approach to selectively target cancer cells.

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity.

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.

The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition.

The NTRK1 gene encodes Tropomyosin‐related kinase A (TRKA), the high‐affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3‐NTRK1) resulting in expression of the oncogenic chimeric protein TPM3‐TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3‐TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3‐NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS‐P626, a novel highly potent and selective TRKA inhibitor. NMS‐P626 suppressed TPM3‐TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors.

Alkylsulfanyl-1,2,4-triazoles, a new class of allosteric valosine containing protein inhibitors. Synthesis and structure-activity relationships.

Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase family that is involved in several biological processes and plays a central role in the ubiquitin-mediated degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this respect, VCP has recently received a great deal of attention as a potential new target for cancer therapy. In this paper, the discovery and structure-activity relationships of alkylsulfanyl-1,2,4-triazoles, a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class of potential anticancer agents.

NMS-P937, an orally available, specific small-molecule polo-like kinase 1 inhibitor with antitumor activity in solid and hematologic malignancies.

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.

Entrectinib, a Pan-TRK, ROS1 and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications

Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non–small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drugs pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628–39. ©2016 AACR.

Discovery of Entrectinib: A New 3-Aminoindazole as a Potent Anaplastic Lymphoma Kinase (Alk), C-Ros Oncogene 1 Kinase (Ros1), and Pan-Tropomyosin Receptor Kinases (Pan-Trks) Inhibitor.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.

Detection of traces of a trisulphide derivative in the preparation of a recombinant truncated interleukin-6 mutein.

A new mutein of interleukin-6, called delta 22-IL-6 Cys 3,4, characterized by the deletion of the first 22 amino acids at the N-terminal end and by the substitution of the first two cysteines (Cys23 and Cys29) with serine residues, was produced in Escherichia coli and was found to maintain the structural and functional properties of the human native form. A partially purified preparation still showed in isoelectric focusing a minor acidic component (pI 6.10) and a more basic component (pI 6.70), the native form having a pI of 6.56. This preparation was further fractionated in a multi-compartment electrolyser with isoelectric membranes, which allowed the collection of the more alkaline species for characterization. Mass spectra of the pI 6.70 form gave an additional mass of 32 atomic mass units (amu), suggesting the addition of two oxygen atoms (a potential oxidation of two methionine residues to sulphoxide). However, the five methionine residues in this higher pI form were identified after enzymatic hydrolysis and peptide mapping and were found to be in a reduced state. In addition, the pI 6.70 form was quickly converted into the native form by mild reductive treatment. On digestion and fingerprinting, the peptide from residues 50 to 65 of the pI 6.70 species (containing the only two cysteine residues of the molecule) exhibited a more hydrophobic behaviour in reversed-phase high-performance liquid chromatography and retained a mass increase of 32 amu. These experimental findings more likely suggest the addition of an extra sulphur atom to the only disulphide bridge to give an unusual protein trisulphide molecule.

Dual Targeting of CDK and Tropomyosin Receptor Kinase Families by the Oral Inhibitor PHA-848125, an Agent with Broad-Spectrum Antitumor Efficacy

Altered expression and activity of cyclin-dependent kinase (CDK) and tropomyosin receptor kinase (TRK) families are observed in a wide variety of tumors. In those malignancies with aberrant CDK activation, the retinoblastoma protein (pRb) pathway is deregulated, leading to uncontrolled cell proliferation. Constitutive activation of TRKs is instead linked to cancer cell survival and dissemination. Here, we show that the novel small-molecule PHA-848125, a potent dual inhibitor of CDKs and TRKs, possesses significant antitumor activity. The compound inhibits cell proliferation of a wide panel of tumoral cell lines with submicromolar IC50. PHA-848125–treated cells show cell cycle arrest in G1 and reduced DNA synthesis, accompanied by inhibition of pRb phosphorylation and modulation of other CDK-dependent markers. The compound additionally inhibits phosphorylation of TRKA and its substrates in cells, which functionally express this receptor. Following oral administration, PHA-848125 has significant antitumor activity in various human xenografts and carcinogen-induced tumors as well as in disseminated primary leukemia models, with plasma concentrations in rodents in the same range as those found active in inhibiting cancer cell proliferation. Mechanism of action was also confirmed in vivo as assessed in tumor biopsies from treated mice. These results show that the dual CDK-TRK inhibitor PHA-848125 has the potential for being a novel and efficacious targeted drug for cancer treatment. Mol Cancer Ther; 9(8); 2243–54. ©2010 AACR.