Carmen Di Giovanni

Inhibitors of Cdc25 phosphatases as anticancer agents: a patent review.

Importance of the field: The cell division cycle 25 (Cdc25) family of proteins are highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases, the main gatekeepers of the eukaryotic cell division cycle. The three isoforms of Cdc25, including Cdc25A, Cdc25B and Cdc25C, appear to act on different cyclin-dependent kinase/cyclin complexes at different stages of the cell cycle. Overexpression of Cdc25A and/or Cdc25B, but not Cdc25C, has been detected in numerous cancers and is often correlated with a poor clinical prognosis. Thus, inhibition of these phosphatases may represent a promising therapeutic approach in oncology. Areas covered in this review: The main focus of the present review is to describe the development of Cdc25 inhibitors over the years. We describe different compounds according to the decade of discovery and focus attention on molecules that were published in patents. What the reader will gain: Insight into the most clinically relevant therapeutic Cdc25 analogues that have been published in over 40 patents over the past 19 years. Take home message: Some Cdc25 inhibitors have suppressed in vivo the growth of human tumor xenografts in animals; this confirmed the validity of using Cdc25 phosphatase inhibition as an anticancer strategy, but side effects and toxicity remain to be investigated.

Novel Quinolinonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis, and Biological Activities

Novel quinolinonyl diketo acids were designed to obtain integrase (IN) inhibitors selectively active against the strand transfer (ST) step of the HIV integration process. Those new compounds are characterized by a single aryl diketo acid (DKA) chain in comparison to 4, a bifunctional diketo acid reported by our group as an anti-IN agent highly potent against both the 3′-processing and ST steps. Compound 6d was the most potent derivative in IN enzyme assays, while 6i showed the highest potency against HIV-1 in acutely infected cells. The selective inhibition of ST suggested the newly designed monofunctional DKAs bind the IN−DNA acceptor site without affecting the DNA donor site.

Discovery of new inhibitors of Cdc25B dual specificity phosphatases by structure-based virtual screening.

Cell division cycle 25 (Cdc25) proteins are highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases and represent attractive drug targets for anticancer therapies. To discover more potent and diverse inhibitors of Cdc25 biological activity, virtual screening was performed by docking 2.1 million compounds into the Cdc25B active site. An initial subset of top-ranked compounds was selected and assayed, and 15 were found to have enzyme inhibition activity at micromolar concentration. Among these, four structurally diverse inhibitors with a different inhibition profile were found to inhibit human MCF-7, PC-3, and K562 cancer cell proliferation and significantly affect the cell cycle progression. A subsequent hierarchical similarity search with the most active reversible Cdc25B inhibitor found led to the identification of an additional set of 19 ligands, three of which were confirmed as Cdc25B inhibitors with IC(50) values of 7.9, 4.2, and 9.9 μM, respectively.

Diarylheterocycle core ring features effect in selective COX-1 inhibition.

The COX‐1 isoenzyme plays a significant role in a variety of diseases, as it catalyzes the bioprocesses behind many health problems. Among the diarylheterocycle class of COX inhibitors, the isoxazole ring has been widely used as a central heterocycle for the preparation of potent and selective COX‐1 inhibitors such as P6 [3‐(5‐chlorofuran‐2‐yl)‐5‐methyl‐4‐phenylisoxazole]. The role of the isoxazole nucleus in COX‐1 inhibitor selectivity has been clarified by preparing a set of new diarylheterocycles with various heterocycle cores. Replacement of isoxazole with isothiazole or pyrazole gave a drastic decrease in COX‐1 inhibitory activity, whereas the introduction of an electron‐donating group (EDG) on the N‐aryl pyrazole allowed recovery of COX‐1 inhibitory activity and selectivity. The EDG‐equipped 5‐(furan‐2‐yl)‐1‐(4‐methoxyphenyl)‐3‐(trifluoromethyl)‐1H‐pyrazole (17) selectively inhibits COX‐1 activity (IC50=3.4 μM; 28 % COX‐2 inhibition at 50 μM), in contrast to its inactive analogue, 3‐(furan‐2‐yl)‐1‐phenyl‐5‐(trifluoromethyl)‐1H‐pyrazole, which does not bear the methoxy EDG. Molecular docking studies of compound 17 into the binding site of COX‐1 shed light on its binding mode.

Discovery of a novel small molecule inhibitor targeting the frataxin/ubiquitin interaction via structure-based virtual screening and bioassays.

Friedreichs ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the mitochondrial protein frataxin. Here, we report findings that frataxin is degraded via the ubiquitin-proteasomal pathway and that it is ubiquitinated at residue K(147) in Calu-6 cells. A theoretical model of the frataxin-K(147)/Ub complex, constructed by combining bioinformatics interface predictions with information-driven docking, revealed a hitherto unnoticed, potential ubiquitin-binding domain in frataxin. Through structure-based virtual screening and cell-based assays, we discovered a novel small molecule (compound (+)-11) able to prevent frataxin ubiquitination and degradation. (+)-11 was synthesized and tested for specific binding to frataxin by an UF-LC/MS based ligand-binding assay. Follow-up scaffold-based searches resulted in the identification of a lead series with micromolar activity in disrupting the frataxin/Ub interaction. This study also suggests that frataxin could be a potential target for FRDA drug development.

CDC25A and B Dual-Specificity Phosphatase Inhibitors: Potential Agents for Cancer Therapy

Members of the cell division cycle 25 (Cdc25) family of proteins are highly conserved dual specificity phosphatases, which play a fundamental role in transitions between cell cycle phases during normal cell division through the activation of CdK/cyclin complexes. Furthermore, they are important targets of checkpoints in cellular pathways in the response to DNA damage. Over the past few years, more information about the basic enzymology of the Cdc25 phosphatases has emerged, with the identification of three Cdc25 phosphatase isoforms (A, B, and C) in mammalians. In particular, the Cdc25 A and B phosphatases have oncogenic properties and are overexpressed singly in some types of cancers and together in others. Therefore, it is not surprising that the Cdc25s are interesting targets for the development of new anticancer therapeutic strategies. In this review, we examine the most important classes of reversible inhibitors that show specificity for the Cdc25 A and B phosphatases (both singly and together) and the recent advances in the design of new potent Cdc25 A and B inhibitors. Using computational methodologies, we also consider their plausible mechanisms of action.

Development of Peptidomimetic Boronates as Proteasome Inhibitors

Proteasome inhibition has emerged over the past decade as an effective therapeutic approach for the treatment of hematologic malignancies. It is a multicatalytic complex, whose proteolytic activity relies in three types of subunits: chymotrypsin-like (β5), trypsin-like (β2) and caspase-like (β1). Most important for the development of effective antitumor agents is the inhibition of the β5 subunits. In this context, the dipeptide boronate bortezomib (Velcade(®)) represents the first proteasome inhibitor approved by the FDA and the lead compound in drug discovery. This paper describes the synthesis and biological evaluation of a series of conformationally constrained pseudopeptide boronates (1-3) structurally related to bortezomib. The synthesized compounds showed a promising inhibitory profile by blocking primarily the chymotrypsin-like activity of the proteasome with Ki values in submicromolar/micromolar range. These compounds also resulted quite selective since no significant inhibition was recorded in the test against bovine pancreatic α-chymotrypsin. The obtained results were rationalized by means of docking experiments based on a model of the crystal structure of bortezomib bound to the yeast 20S proteasome providing essential insights for further optimization of this class of inhibitors.

Cdc25B Phosphatase Inhibitors in Cancer Therapy: Latest Developments, Trends and Medicinal Chemistry Perspective

The Cdc25 phosphatases (Cdc25A, Cdc25B, and Cdc25C in humans), which are responsible for dephosphorylating specific tyrosine/threonine residues on cyclin dependent kinases (CDKs), function as essential regulators of cell cycle control during normal eukaryotic cell division and as mediators of the checkpoint response in cells with DNA damage. Because overexpression of Cdc25A and Cdc25B has been linked to numerous cancers and often correlates with a poor clinical outcome, both academia and industry have devoted substantial research effort in establishing the basic underlying molecular mechanisms and in identifying novel, specific and potentially useful inhibitors of Cdc25 as potential anticancer drugs. Over the past year, dozens of research papers and patent applications describing new Cdc25 inhibitors belonging to different structural classes have been disclosed. In this review, we give an overview on the current status in the field of medicinal chemistry of Cdc25B inhibitors. In addition, molecular modeling studies aimed to clarify the molecular mechanism of inhibition as well as the pharmacophoric features critical for design of new and selective Cdc25B inhibitors are also discussed.

Identification of a new series of amides as non-covalent proteasome inhibitors

Proteasome inhibition has emerged as an important therapeutic strategy for the treatment of multiple myeloma (MM) and some forms of lymphoma, with potential application in other types of cancers. 20S proteasome consists of three different catalytic activities known as chymotrypsin-like (ChT-L), trypsin-like (T-L), and, post-glutamyl peptide hydrolyzing (PGPH) or caspase-like (C-L), which are located respectively on the β5, β2, and β1 subunits of each heptameric β rings. Currently a wide number of covalent proteasome inhibitors are reported in literature; however, the less widely investigated non-covalent inhibitors might be a promising alternative to employ in therapy, because of the lack of all drawbacks and side-effects related to irreversible inhibition. In the present work we identified a series of amides, two of which (1b and 1f) are good candidates to non-covalent inhibition of the chymotrypsin-like activity of the β5 proteasome subunit. The non-covalent binding mode was corroborated by docking simulations of the most active inhibitors 1b, 1f and 2h into the yeast 20S proteasome crystal structure.

Discovery of new small molecules targeting the vitronectin-binding site of the urokinase receptor that block cancer cell invasion.

Besides focusing urokinase (uPA) proteolytic activity on the cell membrane, the uPA receptor (uPAR) is able to bind vitronectin, via a direct binding site. Furthermore, uPAR interacts with other cell surface receptors, such as integrins, receptor tyrosine kinases, and chemotaxis receptors, triggering cell-signaling pathways that promote tumor progression. The ability of uPAR to coordinate binding and degradation of extracellular matrix (ECM) and cell signaling makes it an attractive therapeutic target in cancer. We used structure-based virtual screening (SB-VS) to search for small molecules targeting the uPAR-binding site for vitronectin. Forty-one compounds were identified and tested on uPAR-negative HEK-293 epithelial cells transfected with uPAR (uPAR-293 cells), using the parental cell line transfected with the empty vector (V-293 cells) as a control. Compounds 6 and 37 selectively inhibited uPAR-293 cell adhesion to vitronectin and the resulting changes in cell morphology and signal transduction, without exerting any effect on V-293 cells. Compounds 6 and 37 inhibited uPAR-293 cell binding to vitronectin with IC50 values of 3.6 and 1.2 μmol/L, respectively. Compounds 6 and 37 targeted S88 and R91, key residues for uPAR binding to vitronectin but also for uPAR interaction with the fMLF family of chemotaxis receptors (fMLF-Rs). As a consequence, compounds 6 and 37 impaired uPAR-293 cell migration toward fetal calf serum (FCS), uPA, and fMLF, likely by inhibiting the interaction between uPAR and FPR1, the high affinity fMLF-R. Both compounds blocked in vitro ECM invasion of several cancer cell types, thus representing new promising leads for pharmaceuticals in cancer. Mol Cancer Ther; 12(8); 1402–16. ©2013 AACR.