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In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP ...concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.
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.
The discovery of a novel class of inhibitors of cyclin dependent kinases (CDKs) is described. Starting from compound 1, showing good potency as inhibitor of CDKs but being poorly selective against a ...panel of serine−threonine and tyrosine kinases, new analogues were synthesized. Enhancement in selectivity, antiproliferative activity against A2780 human ovarian carcinoma cells, and optimization of the physical properties and pharmacokinetic profile led to the identification of highly potent and orally available compounds. Compound 28 (PHA-848125), which in the preclinical xenograft A2780 human ovarian carcinoma model showed good efficacy and was well tolerated upon repeated daily treatments, was identified as a drug candidate for further development. Compound 28 is currently undergoing phase I and phase II clinical trials.
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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.
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.
Mutations in the kinase domain of Bcr-Abl are the most common cause of resistance to therapy with imatinib in patients with chronic myelogenous leukemia (CML). Second-generation Bcr-Abl inhibitors ...are able to overcome most imatinib-resistant mutants, with the exception of the frequent T315I substitution, which is emerging as a major cause of resistance to these drugs in CML patients. Structural studies could be used to support the drug design process for the development of inhibitors able to target the T315I substitution, but until now no crystal structure of the T315I Abl mutant has been solved. We show here the first crystal structure of the kinase domain of Abl T315I in complex with PHA-739358, an Aurora kinase inhibitor currently in clinical development for solid and hematologic malignancies. This compound inhibits in vitro the kinase activity of wild-type Abl and of several mutants, including T315I. The cocrystal structure of T315I Abl kinase domain provides the structural basis for this activity: the inhibitor associates with an active conformation of the kinase domain in the ATP-binding pocket and lacks the steric hindrance imposed by the substitution of threonine by isoleucine.
Abstract
Introduction: Choline kinase alpha (ChoKα), the first enzyme in the Kennedy pathway that catalyzes the phosphorylation of free choline to phosphocholine (PCho), is responsible for the de ...novo biosynthesis of phosphatidylcholine (PC), the major phospholipid of cellular membranes. Aberrant choline metabolic profiles and concomitant ChoKα upregulation have been described in most human malignancies (i.e. breast, lung, ovary, liver) and have been found to correlate with advanced histological tumour grade. ChoKα, depletion by siRNA or shRNA inhibits growth and migration of different tumor cell lines both in vitro and in vivo, which is not observed for the ChoKβ isoform. Choline mimetic inhibitors of ChoKα (i.e. MN58b) have been shown to have antitumor activity in preclinical models, although their efficacy is hampered by a significant toxicity, possibly due to cross-reactivity with other choline-dependent proteins (transporters, enzymes). At NMS a high throughput screening (HTS) was performed with the objective to identify ATP-mimetic ChoKα inhibitors potentially less toxic than choline-mimetic compounds.
Methods: Hits from different classes were characterized for biochemical activity on ChoKα and ChoKβ and biochemical mechanism of inhibition. The binding site of selected ATP competitive inhibitors was confirmed by co-crystallization with ChoKα. On-target mechanism of action in cells was confirmed by analysing inhibition of PCho formation.
Result: Structure-based chemical expansion of Hits from one of the prioritized classes resulted in compounds with biochemical potencies in the single digit nanomolar range displaying selectivity vs ChoKβ as well as a diverse panel of protein kinases. In several tumor cell lines, the compounds were able to inhibit the formation of PCho at a concentration in agreement with that required to achieve anti-proliferative activity. The most potent compounds were tested on a panel of 24 representative breast cancer cell lines which showed differential sensitivity towards ChoKα inhibition. Analysis of genomic (DNA and RNA) and proteomic (>50 markers) expression profiles of the breast cancer cell lines is ongoing to identify predictive biomarkers of response.
Conclusion: Medicinal chemistry expansion of a novel class of compounds identified by HTS allowed the development of potent ChoKα ATP-mimetic inhibitors able to modulate PCho levels in cells, which can be used to identify preferential sensitivity contexts. Medicinal chemistry activities are ongoing to further improve their potency and optimize their ADME/PK properties.
Citation Format: Paola Gnocchi, Francesca Quartieri, Alessandra Badari, Roberta Bosotti, Elena Casale, Emiliana Corti, Cinzia G. Cristiani, Ulisse Cucchi, Fabio Gasparri, Laura M. Gianellini, Laura M. Giorgini, Marisa Montemartini, Giuliana Mion, Marcella Nesi, Christian Orrenius, Claudia E. Re, Daniele Donati, Eduard R. Felder, Arturo Galvani, Antonella Isacchi. Identification and characterization of ATP-mimetic choline kinase inhibitors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4790.
Maternal embryonic leucine zipper kinase (MELK) is upregulated in several types of tumor, including breast, prostate, and brain tumors. Its expression is generally associated with cell survival, cell ...proliferation, and resistance to apoptosis. Therefore, the potential of MELK inhibitors as therapeutic agents is recently attracting considerable interest. Here we report the first structures of MELK in complex with AMP–PNP and with nanomolar inhibitors. Our studies shed light on the role of the MELK UBA domain, provide a characterization of the kinase active site, and identify key residues for achieving high potency, laying the groundwork for structure-based drug design efforts.
A novel series of PIM inhibitors was derived from a combined effort in natural product-inspired library generation and screening. The novel pyrrolo1,2-apyrazinones initial hits are inhibitors of PIM ...isoforms with IC50 values in the low micromolar range. The application of a rational optimization strategy, guided by the determination of the crystal structure of the complex in the kinase domain of PIM1 with compound 1, led to the discovery of compound 15a, which is a potent PIM kinases inhibitor exhibiting excellent selectivity against a large panel of kinases, representative of each family. The synthesis, structure–activity relationship studies, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Furthermore, the cellular activities including inhibition of cell growth and modulation of downstream targets are also described.
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