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New antibodies-drug conjugate (ADC) payloads overcoming chemoresistance and killing also poorly proliferating tumors at well-tolerated doses are much desired. Duocarmycins are a well-known class of highly potent cytotoxic agents, with DNA minor groove-binding and alkylation properties, active also in chemoresistant tumors. Although different duocarmycin derivatives have been used during the years as payloads for ADC production, unfavorable physicochemical properties impaired the production of ADCs with optimal features. Optimization of the toxin to balance reactivity and stability features and best linker selection allowed us to develop the novel duocarmycin-like payload-linker NMS-P945 suitable for conjugation to mAbs with reproducible drug-antibody ratio (DAR) >3.5. When conjugated to trastuzumab, it generated an ADC with good internalization properties, ability to induce bystander effect and immunogenic cell death. Moreover, it showed strong target-driven activity in cells and cytotoxic activity superior to trastuzumab deruxtecan tested, in parallel, in cell lines with HER2 expression. High in vivo efficacy with cured mice at well-tolerated doses in HER2-driven models was also observed. A developed pharmacokinetic/pharmacodynamic (PK/PD) model based on efficacy in mice and cynomolgus monkey PK data, predicted tumor regression in patients upon administration of 2 doses of trastuzumab-NMS-P945-ADC at 0.5 mg/kg. Thus, considering the superior physicochemical features for ADC production and preclinical results obtained with the model trastuzumab ADC, including bystander effect, immunogenic cell death and activity in chemoresistant tumors, NMS-P945 represents a highly effective, innovative payload for the creation of novel, next-generation ADCs., (©2023 American Association for Cancer Research.)

Background Pharmacological inhibition of polo-like kinase 1 (PLK1) represents a new approach for the treatment of solid tumors. This study was aimed at determining the first cycle dose-limiting toxicities (DLTs) and related maximum tolerated dose (MTD) of NMS-1286937, a selective ATP-competitive PLK1-specific inhibitor. Secondary objectives included evaluation of its safety and pharmacokinetic (PK) profile in plasma, its antitumor activity, and its ability to modulate intracellular targets in biopsied tissue. Methods This was a Phase I, open-label, dose-escalation trial in patients with advanced/metastatic solid tumors. A treatment cycle comprised 5 days of oral administration followed by 16 days of rest, for a total of 21 days (3-week cycle). Results Nineteen of 21 enrolled patients with confirmed metastatic disease received study medication. No DLTs occurred at the first 3 dose levels (6, 12, and 24 mg/m 2 /day). At the subsequent dose level (48 mg/m 2 /day), 2 of 3 patients developed DLTs. An intermediate level of 36 mg/m 2 /day was therefore investigated. Four patients were treated and two DLTs were observed. After further cohort expansion, the MTD and recommended phase II dose (RP2D) were determined to be 24 mg/m 2 /day. Disease stabilization, observed in several patients, was the best treatment response observed. Hematological toxicity (mostly thrombocytopenia and neutropenia) was the major DLT. Systemic exposure to NMS-1286937 increased with dose and was comparable between two cycles of treatment following oral administration of the drug. Conclusions This study successfully identified the MTD and DLTs for NMS-1286937 and characterized its safety profile.

The prolyl isomerase PIN1, a critical modifier of multiple signalling pathways, is overexpressed in the majority of cancers and its activity strongly contributes to tumour initiation and progression. Inactivation of PIN1 function conversely curbs tumour growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening, we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinone-mimicking drug that generates reactive oxygen species and DNA damage, inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer phenotypes in vitro and growth of lung metastasis in vivo.

Two small-molecule-drug conjugates (SMDCs, 6 and 7) featuring lysosomally cleavable linkers (namely the Val-Ala and Phe-Lys peptide sequences) were synthesized by conjugation of the αvβ3-integrin ligand cyclo[DKP-RGD]-CH2NH2 (2) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP-RGD]-PTX conjugate with a nonpeptide "uncleavable" linker (8) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified αVβ3-integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8, which possesses a nonpeptide "uncleavable" linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF-CEM (αVβ3-) and its subclone CCRF-CEM αVβ3 (αVβ3+). Fairly effective integrin targeting was displayed by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate (6), which was found to differentially inhibit proliferation in antigen-positive CCRF-CEM αVβ3 versus antigen-negative isogenic CCRF-CEM cells. The total lack of activity displayed by the "uncleavable" cyclo[DKP-RGD]-PTX conjugate (8) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Polo-like kinase 1 (PLK1) has emerged as a prognostic factor in various neoplasms, but only scarce data have been reported for high-grade osteosarcoma (OS). In this study, we assessed PLK1 expression and the efficacy of PLK1 inhibitor NMS-P937 in OS., Methods: PLK1 expression was assessed on 21 OS clinical samples and on a panel of human OS cell lines. In vitro efficacy of NMS-P937 was evaluated on nine drug-sensitive and six drug-resistant human OS cell lines, either as single agent or in combination with the drugs used in chemotherapy for OS., Results: PLK1 expression was higher in OS clinical samples and cell lines compared to normal human tissue. A higher PLK1 expression at diagnosis appeared to be associated with an unfavourable clinical outcome. PLK1 silencing produced growth inhibition, cell cycle retardation and apoptosis induction in human OS cell lines. NMS-P937 proved to be highly active in both drug-sensitive and drug-resistant cell lines, with the only exception of ABCB1-overexpressing, Doxorubicin (DX)-resistant variants. However, in these cells, the association of NMS-P937 with DX was able to revert DX-resistance by negatively interfering with ABCB1 transport activity. NMS-P937 was also able to decrease clonogenic and migration ability of human OS cell lines., Conclusion: PLK1 can be proposed as a new candidate target for OS. Targeting PLK1 in OS with NMS-P937 in association with conventional chemotherapeutic drugs may be a new interesting therapeutic option, since this approach has proved to be active against drug resistant cells.

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.

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., (©2013 AACR.)

As part of our drug discovery effort, we identified and developed 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as PLK1 inhibitors. We now report the optimization of this class that led to the identification of NMS-P937, a potent, selective and orally available PLK1 inhibitor. Also, in order to understand the source of PLK1 selectivity, we determined the crystal structure of PLK1 with NMS-P937. The compound was active in vivo in HCT116 xenograft model after oral administration and is presently in Phase I clinical trials evaluation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cdc7 serine/threonine kinase is a key regulator of DNA synthesis in eukaryotic organisms. Cdc7 inhibition through siRNA or prototype small molecules causes p53 independent apoptosis in tumor cells while reversibly arresting cell cycle progression in primary fibroblasts. This implies that Cdc7 kinase could be considered a potential target for anticancer therapy. We previously reported that pyrrolopyridinones (e.g., 1) are potent and selective inhibitors of Cdc7 kinase, with good cellular potency and in vitro ADME properties but with suboptimal pharmacokinetic profiles. Here we report on a new chemical class of 5-heteroaryl-3-carboxamido-2-substituted pyrroles (1A) that offers advantages of chemistry diversification and synthetic simplification. This work led to the identification of compound 18, with biochemical data and ADME profile similar to those of compound 1 but characterized by superior efficacy in an in vivo model. Derivative 18 represents a new lead compound worthy of further investigation toward the ultimate goal of identifying a clinical candidate.

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.

Cdc7 kinase is a key regulator of the S-phase of the cell cycle, known to promote the activation of DNA replication origins in eukaryotic organisms. Cdc7 inhibition can cause tumor-cell death in a p53-independent manner, supporting the rationale for developing Cdc7 inhibitors for the treatment of cancer. In this paper, we conclude the structure-activity relationships study of the 2-heteroaryl-pyrrolopyridinone class of compounds that display potent inhibitory activity against Cdc7 kinase. Furthermore, we also describe the discovery of 89S, [(S)-2-(2-aminopyrimidin-4-yl)-7-(2-fluoro-ethyl)-1,5,6,7-tetrahydropyrrolo[3,2-c]pyridin-4-one], as a potent ATP mimetic inhibitor of Cdc7. Compound 89S has a Ki value of 0.5 nM, inhibits cell proliferation of different tumor cell lines with an IC50 in the submicromolar range, and exhibits in vivo tumor growth inhibition of 68% in the A2780 xenograft model.

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 is an essential protein that promotes DNA replication in eukaryotic organisms. Genetic evidence indicates that Cdc7 inhibition can cause selective tumor-cell death in a p53-independent manner, supporting the rationale for developing Cdc7 small-molecule inhibitors for the treatment of cancers. In this paper, the synthesis and structure-activity relationships of 2-heteroaryl-pyrrolopyridinones, the first potent Cdc7 kinase inhibitors, are described. Starting from 2-pyridin-4-yl-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one, progress toward a simple scaffold, tailored for Cdc7 inhibition, is reported.