Ferdinando Maria Milazzo

Heparanase: a rainbow pharmacological target associated to multiple pathologies including rare diseases

In recent years, heparanase has attracted considerable attention as a promising target for innovative pharmacological applications. Heparanase is a multifaceted protein endowed with enzymatic activity, as an endo-β-D-glucuronidase, and nonenzymatic functions. It is responsible for the cleavage of heparan sulfate side chains of proteoglycans, resulting in structural alterations of the extracellular matrix. Heparanase appears to be involved in major human diseases, from the most studied tumors to chronic inflammation, diabetic nephropathy, bone osteolysis, thrombosis and atherosclerosis, in addition to more recent investigation in various rare diseases. The present review provides an overview on heparanase, its biological role, inhibitors and possible clinical applications, covering the latest findings in these areas.

Synthesis and Evaluation of New Hsp90 Inhibitors Based on a 1,4,5-Trisubstituted 1,2,3-Triazole Scaffold

Ruthenium catalyzed 1,3-cycloaddition (click chemistry) of an azido moiety installed on dihydroxycumene scaffold with differently substituted aryl propiolates gave a new family of 1,4,5-trisubstituted triazole carboxylic acid derivatives that showed high affinity toward Hsp90 associated with cell proliferation inhibition, both in nanomolar range. The 1,5 arrangement of the resorcinol, the aryl moieties, and the presence of an alkyl (secondary) amide in position 4 of the triazole ring were essential to get high activity. Docking simulations suggested that the triazoles penetrate the Hsp90 ATP binding site. Some 1,4,5-trisubstituted triazole carboxamides induced dramatic depletion of the examined client proteins and a very strong increase in the expression levels of the chaperone Hsp70. In vitro metabolic stability and in vivo preliminary studies on selected compounds have shown promising results comparable to the potent Hsp90 inhibitor NVP-AUY922. One of them, (compound 18, SST0287CL1) was selected for further investigation as the most promising drug candidate.

In Vivo Imaging Reveals Selective Peroxisome Proliferator Activated Receptor Modulator Activity of the Synthetic Ligand 3-(1-(4-Chlorobenzyl)-3-t-butylthio-5-isopropylindol-2-yl)-2,2-dimethylpropanoic acid (MK-886)

We report here the finding of a new pharmacological activity of a well known antagonist of peroxisome proliferator-activated receptors (PPARs). PPARs belong to the family of nuclear receptors playing a relevant role in mammalian physiology and are currently believed to represent a major target for the development of innovative drugs for metabolic and inflammatory diseases. In the present study, the application of reporter animal technology was instrumental to obtain the global pharmacological profiling indispensable to unraveling 3-(1-(4-chlorobenzyl)-3-t-butylthio-5-isopropylindol-2-yl)-2,2-dimethylpropanoic acid (MK-886)-selective PPAR modulator (SPPARM) activity not underlined by previous traditional, cell-based studies. The results of this study, demonstrating the usefulness of reporter mice, may open new avenues for the development of innovative drugs for cardiovascular, endocrine, neural, and skeletal systems characterized by limited side effects.

Novel 3,4-isoxazolediamides as potent inhibitors of chaperone heat shock protein 90.

A structural investigation on the isoxazole scaffold led to the discovery of 3,4-isoxazolediamide compounds endowed with potent Hsp90 inhibitory properties. We have found that compounds possessing a nitrogen atom directly attached to the C-4 heterocycle ring possess in vitro Hsp90 inhibitory properties at least comparable to those of the structurally related 4,5-diarylisoxazole derivatives. A group of compounds from this series of diamides combine potent binding affinity and cell growth inhibitory activity in both series of alkyl- and aryl- or heteroarylamides, with IC50 in the low nanomolar range. The 3,4-isoxazolediamides were also very effective in causing dramatic depletion of the examined client proteins and, as expected for the Hsp90 inhibitors, always induced a very strong increase in the expression levels of the chaperone Hsp70. In vivo studies against human epidermoid carcinoma A431 showed an antitumor effect of morpholine derivative 73 comparable to that induced by the reference compound 10.

Metabolic Approach to the Enhancement of Antitumor Effect of Chemotherapy: A Key Role of Acetyl-L-Carnitine

Purpose: Acetyl-l-carnitine (ALC) plays a relevant role in energy metabolism and stress response because of its function in the complex metabolic system regulating the acetyl-CoA levels that provide a source of acetyl groups for metabolic and acetylation-regulated processes. Because acetylation may influence p53 activity/stability and, therefore, the response to platinum compounds, this study was designed to investigate the effect of ALC in combination with platinum compounds. Experimental Design: The antiproliferative and antitumor activity studies were done in a panel of human tumor cell lines with functional or defective p53. The antimetastatic drug efficacy was investigated in the s.c. growing H460/M tumor subline, which is able to generate lung metastases. Results: ALC enhanced the sensitivity to cisplatin of tumor cells with functional p53. The sensitization by ALC was reflected in an improved in vivo antitumor efficacy of the combination over cisplatin alone in wild-type p53 lung tumors. ALC did not increase the cisplatin efficacy in the p53-mutant SW620 tumor. ALC exhibited a significant antimetastatic activity, and this effect was better exploited in combination with the histone deacetylase inhibitor, ST3595. The in vivo ALC/cisplatin combination caused the activation of p53, associated with protein acetylation and induction of target genes. Conclusions: ALC was effective in enhancing the antitumor potential of platinum compounds in wild-type p53 tumors. ALC, alone and in combination with a histone deacetylase inhibitor, exhibited an outstanding antimetastatic activity. Both effects, likely mediated by protein acetylation, may have implications for platinum-based therapy and combinations with histone deacetylase inhibitors. Clin Cancer Res; 16(15); 3944–53. ©2010 AACR.

Kinetic analysis and molecular modeling of the inhibition mechanism of roneparstat (SST0001) on human heparanase

Heparanase is a β-d-glucuronidase which cleaves heparan sulfate chains in the extracellular matrix and on cellular membranes. A dysregulated heparanase activity is intimately associated with cell invasion, tumor metastasis and angiogenesis, making heparanase an attractive target for the development of anticancer therapies. SST0001 (roneparstat; Sigma-Tau Research Switzerland S.A.) is a non-anticoagulant 100% N-acetylated and glycol-split heparin acting as a potent heparanase inhibitor, currently in phase I in advanced multiple myeloma. Herein, the kinetics of heparanase inhibition by roneparstat is reported. The analysis of dose-inhibition curves confirmed the high potency of roneparstat (IC50 ≈ 3 nM) and showed, at higher concentrations, a Hill coefficient consistent with the engagement of two molecules of inhibitor. A homology model of human heparanase GS3 construct was built and used for docking experiments with inhibitor fragments. The model has high structural similarity with the recently reported crystal structure of human heparanase. Different interaction schemes are proposed, which support the hypothesis of a complex binding mechanism involving the recruitment of one or multiple roneparstat chains, depending on its concentration. In particular, docking solutions were obtained in which (i) a single roneparstat molecule interacts with both heparin-binding domains (HBDs) of heparanase or (ii) two fragments of roneparstat interact with either HBD-1 or HBD-2, consistent with the possibility of different inhibitor:enzyme binding stoichiometries. This study provides unique insights into the mode of action of roneparstat as well as clues of its interaction with heparanase at a molecular level, which could be exploited to design novel potential inhibitor molecules.

7-Azaindole-1-carboxamides as a new class of PARP-1 inhibitors

7-Azaindole-1-carboxamides were designed as a new class of PARP-1 inhibitors. The compounds displayed a variable pattern of target inhibition profile that, in part, paralleled the antiproliferative activity in cell lines characterized by homologous recombination defects. A selected compound (1l; ST7710AA1) showed significant in vitro target inhibition and capability to substantially bypass the multidrug resistance mediated by Pgp. In antitumor activity studies against the MX1 human breast carcinoma growth in nude mice, the compound exhibited an effect similar to that of Olaparib in terms of tumor volume inhibition when used at a lower dose than the reference compound. Treatment was well tolerated, as no deaths or significant weight losses were observed among the treated animals.

ST7612AA1, a thioacetate-ω(γ-lactam carboxamide) derivative selected from a novel generation of oral HDAC inhibitors.

A systematic study of medicinal chemistry aimed at identifying a new generation of HDAC inhibitors, through the introduction of a thiol zinc-binding group (ZBG) and of an amide-lactam in the ω-position of the polyethylene chain of the vorinostat scaffold, allowed the selection of a new class of potent pan-HDAC inhibitors (pan-HDACis). Simple, highly versatile, and efficient synthetic approaches were used to synthesize a library of these new derivatives, which were then submitted to a screening for HDAC inhibition as well as to a preliminary in vitro assessment of their antiproliferative activity. Molecular docking into HDAC crystal structures suggested a binding mode for these thiol derivatives consistent with the stereoselectivity observed upon insertion of amide-lactam substituents in the ω-position. ST7612AA1 (117), selected as a drug candidate for further development, showed an in vitro activity in the nanomolar range associated with a remarkable in vivo antitumor activity, highly competitive with the most potent HDAC inhibitors, currently under clinical trials. A preliminary study of PK and metabolism is also illustrated.

Novel PARP-1 inhibitors based on a 2-propanoyl-3H-quinazolin-4-one scaffold.

Poly(ADP-ribose)polymerase-I (PARP-1) enzyme is involved in maintaining DNA integrity and programmed cell death. A virtual screening of commercial libraries led to the identification of five novel scaffolds with inhibitory profile in the low nanomolar range. A hit-to-lead optimization led to the identification of a group of new potent PARP-1 inhibitors, acyl-piperazinylamides of 3-(4-oxo-3,4-dihydro-quinazolin-2-yl)-propionic acid. Molecular modeling studies highlighted the preponderant role of the propanoyl side chain.

Preclinical antitumor activity of SST0116CL1: a novel heat shock protein 90 inhibitor.

4-Amino substituted resorcino-isoxazole (SST0116CL1) (property of Sigma-Tau Research Switzerland S.A.) is a potent, second generation, small-molecule heat shock protein 90 inhibitor (Hsp90i). SST0116CL1 binds to the ATP binding pocket of Hsp90, and interferes with Hsp90 chaperone function thus resulting in client protein degradation and tumor growth inhibition. The aim of the study was to assess SST0116CL1 in various solid and haematological tumors. The antitumor properties of SST0116CL1 were assessed using in vitro cell proliferation and client protein degradation assays and in vivo different tumor xenograft models. Pharmacokinetic (PK) data were also generated in tumor-bearing mice to gain an understanding of optimal dosing schedules and regimens. SST0116CL1 was shown to inhibit recombinant Hsp90α and to induce the destabilization of different client proteins, often overexpressed and constitutively activated in different types of hematological or solid human tumors. In preclinical in vivo studies, it was revealed to induce antitumor effects in murine models of leukemia and of gastric and ovarian carcinoma. A modulation of PD biomarkers in terms of downregulation of Hsp90 client proteins in tumor-bearing mice was found. SST0116CL1 is a new clinical candidate for cancer therapy. The antitumor property of SST0116CL1, likely due to direct inhibition of the Hsp90 enzymatic activity, may prove to be a critical attribute as the compound enters phase I clinical trials.