Mario Varasi

PHA-680632, a novel Aurora kinase inhibitor with potent antitumoral activity.

Purpose: Aurora kinases play critical roles during mitosis in chromosome segregation and cell division. The aim of this study was to determine the preclinical profile of a novel, highly selective Aurora kinase inhibitor, PHA-680632, as a candidate for anticancer therapy. Experimental Design: The activity of PHA-680632 was assayed in a biochemical ATP competitive kinase assay. A wide panel of cell lines was evaluated for antiproliferative activity. Cell cycle analysis. Immunohistochemistry, Western blotting, and Array Scan were used to follow mechanism of action and biomarker modulation. Specific knockdown of the targets by small interfering RNA was followed to validate the observed phenotypes. Efficacy was determined in different xenograft models and in a transgenic animal model of breast cancer. Results: PHA-680632 is active on a wide range of cancer cell lines and shows significant tumor growth inhibition in different animal tumor models at well-tolerated doses. The mechanism of action of PHA-680632 is in agreement with inhibition of Aurora kinases. Histone H3 phosphorylation in Ser10 is mediated by Aurora B kinase, and our kinetic studies on its inhibition by PHA-680632 in vitro and in vivo show that phosphorylation of histone H3 is a good biomarker to follow activity of PHA-680632. Conclusions: PHA-680632 is the first representative of a new class of Aurora inhibitors with a high potential for further development as an anticancer therapeutic. On treatment, different cell lines respond differentially, suggesting the absence of critical cell cycle checkpoints that could be the basis for a favorable therapeutic window.

(R,S)-3,4-dichlorobenzoylalanine (FCE 28833A) causes a large and persistent increase in brain kynurenic acid levels in rats

Kynurenic acid is an endogenous excitatory amino-acid receptor antagonist with neuroprotective and anticonvulsant properties. We demonstrate here that systemic administration of the new and potent kynurenine 3-hydroxylase inhibitor (R,S)-3,4-dichlorobenzoylalanine (FCE 28833A) causes a dose-dependent elevation in endogenous kynurenine and kynurenic acid levels in rat brain tissue. In hippocampal microdialysates, peak increases of 10- and 80-fold above basal kynurenic acid concentrations, respectively, were obtained after a single oral or intraperitoneal administration of 400 mg/kg FCE 28833A. After intraperitoneal treatment with FCE 28833A, extracellular brain kynurenic acid levels remained significantly elevated for at least 22 h, rendering this compound a far more effective enhancer of kynurenic acid levels than the previously described kynurenine 3-hydroxylase blocker m-nitrobenzoylalanine. FCE 28833A and similar molecules may have therapeutic value in diseases which are linked to a hyperfunction of excitatory amino-acid receptors.

PNU-151774E protects against kainate-induced status epilepticus and hippocampal lesions in the rat.

Kainic acid-induced multifocal status epilepticus in the rat is a model of medically intractable complex partial seizures and neurotoxicity. The exact mechanisms of kainic acid epileptogenic and neurotoxic effects are unknown, but enhanced glutamate release seems to be an important factor. PNU-151774E ((S)-(+)-2-(4-(3-fluorobenzyloxy) benzylamino) propanamide, methanesulfonate) is a broad-spectrum new anticonvulsant with Na+ channel-blocking and glutamate release inhibiting properties. We have examined the effect of pretreatment with this compound on both seizure activity and hippocampal neuronal damage induced by systemic injection of kainic acid in rats. Lamotrigine, a recently developed anticonvulsant with similar glutamate release inhibitory properties, was tested for comparison, together with diazepam as reference standard, on the basis of its anticonvulsant and neuroprotectant properties in this animal model. PNU-151774E, lamotrigine (10, 30 mg/kg; i.p.) and diazepam (20 mg/kg; i.p.) were administered 15 min before kainic acid (10 mg/kg; i.p.). In the vehicle-treated group, kainic acid injection caused status epilepticus in 86% of animals. Hippocampal neuronal cell loss was 66% in the CA4 hippocampal area at 7 days after kainic acid administration. Diazepam inhibited both seizures and neurotoxicity. Lamotrigine reduced hippocampal neuronal cell loss at both doses, even when it did not protect from seizures, although it showed a trend toward protection. On the other hand PNU-151774E protected from both hippocampal neurodegeneration and status epilepticus. Thus, these data support the concept that seizure prevention and neuroprotection might not be tightly coupled. Glutamate release inhibition may play a major role in neuroprotection, but an additional mechanism(s) of action might be relevant for the anticonvulsant activity of PNU-151774E in this model.

Fragment-based identification of Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) plays a key role in stress response and protection of the cell against the effects of mutation. Herein we report the identification of an Hsp90 inhibitor identified by fragment screening using a high‐concentration biochemical assay, as well as its optimisation by in silico searching coupled with a structure‐based drug design (SBDD) approach.

Discovery of a Novel Hsp90 Inhibitor by Fragment Linking

Over the past decade, fragment screening has become an increasingly popular method for hit identification in drug discovery. Many methods can be successfully employed to identify fragment hits, however confirmation of a fragment hit and detailed analysis of the binding pose to its target is usually achieved by X-ray crystallographic analysis of the fragment protein complex. Subsequent optimisation involves the evolution of fragments to fully exploit binding pockets of a target or, where the chemistry of combining fragments is tractable, by the linking of fragments. Previously, we reported results from a fragment evolution process based on heat shock protein 90 (Hsp90) fragment hits; in the present report, we describe a fragment-linking approach that resulted in the rapid improvement in the level of Hsp90 inhibition. Hsp90 is a molecular chaperone with ATPase activity involved in the stabilisation of numerous client proteins including those involved in oncogenic transformations, such as BRaf. As such there continues to be considerable interest in the discovery of Hsp90 inhibitors. Previously, we reported the analysis of multiple crystal structures of diverse fragment complexes of Hsp90 derived from the primary fragment screen that demonstrated the flexibility of Hsp90 in the region of the adenosine binding site (see Figure 1). In particular, Hsp90 was found to adopt a helical conformation in the region of Asn 105 to Ile 110 in the presence of a subset of the fragments, including compound 2. The Hsp90–2 complex structure is essentially the same as the “closed” conformation previously reported for Hsp90 in the presence of geldanamycin. The helical conformation of Hsp90 creates a compact and well-defined pocket adjacent to the adenosine binding site. Other fragments, such as fragment 1, exclusively bind to the adenosine pocket and do not trigger the opening of the helical pocket. Both fragments 1 and 2 displayed relatively low inhibitory potencies against Hsp90 (IC50 = 1500 mm

Synthesis and Biological Evaluation of N-Hydroxyphenylacrylamides and N-Hydroxypyridin-2-ylacrylamides as Novel Histone Deacetylase Inhibitors

The histone deacetylases (HDACs) are able to regulate gene expression, and histone deacetylase inhibitors (HDACi) emerged as a new class of agents in the treatment of cancer as well as other human disorders such as neurodegenerative diseases. In the present investigation, we report on the synthesis and biological evaluation of compounds derived from the expansion of a HDAC inhibitor scaffold having N-hydroxy-3-phenyl-2-propenamide and N-hydroxy-3-(pyridin-2-yl)-2-propenamide as core structures and containing a phenyloxopropenyl moiety, either unsubstituted or substituted by a 4-methylpiperazin-1-yl or 4-methylpiperazin-1-ylmethyl group. The compounds were evaluated for their ability to inhibit nuclear HDACs, as well as for their in vitro antiproliferative activity. Moreover, their metabolic stability in microsomes and aqueous solubility were studied and selected compounds were further characterized by in vivo pharmacokinetic experiments. These compounds showed a remarkable stability in vivo, compared to hydroxamic acid HDAC inhibitors that have already entered clinical trials. The representative compound 30b showed in vivo antitumor activity in a human colon carcinoma xenograft model.

Optimization of 6,6-Dimethyl Pyrrolo[3,4-C]Pyrazoles: Identification of Pha-793887, a Potent Cdk Inhibitor Suitable for Intravenous Dosing.

We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.

Derivatives of kynurenine as inhibitors of rat brain kynurenine aminotransferase

Abstract The structural requirements of the catalytic site of kynurenine aminotransferase (KAT), the enzyme responsible for the conversion of l -kynurenine (KYN) to kynurenic acid (KYNA), were examined using analogs and derivatives of KYN. KYNA production from KYN was monitored in rat brain homogenates and brain tissue slices. Modification of KYNs acylalanine side chain or its ring amino group resulted in compounds which did not substantially affect KYNA synthesis. Ring chlorination in positions 3, 4, 5 and 6 yielded KYN analogs which interfered with KYNA production. l -5-Cl-KYN was the most active of the chlorinated kynurenines, and one of the most potent of several other 5-substituted kynurenines. l -5-Cl-KYN was an excellent substrate of KAT, yielding 6-Cl-KYNA. Finally, in kinetic studies, l -5-Cl-KYN ( K i = 5.4 μM) was found to have an approximately five times higher affinity to the enzyme than the natural substrate KYN ( K m = 28 μM).

Kynurenic Acid-Enhancing And Anti-Ischemic Effects of the Potent Kynurenine 3-Hydroxylase Inhibitor Fce 28833 in Rodents

When injected into the brain of experimental animals, quinolinic acid (QUIN) and kynurenic acid (KYNA) act as a neurotoxin and neuroprotectant, respectively, and these effects are mediated by excitatory amino acid (EAA) receptors (Stone, 1993). Since their identification in the mammalian brain, QUIN and KYNA have therefore been proposed to play roles as endogenous modulators of EAA receptors. Both compounds are metabolites of the kynurenine pathway in the periphery and in the brain, and a large number of studies have been performed to examine their source, metabolism and disposition in the central nervous system in physiological and pathological conditions (see Stone, 1993, for review). In particular, an increased formation of QUIN was observed in several models of nerve cell damage, such as excitotoxin lesions (Speciale et al., 1987), spinal cord trauma (Popovich et al., 1994) and global ischemia (Heyes and Nowak, 1990; Saito et al., 1993), and in humans infected with the HIV virus (Heyes et al., 1991). This enhancement of QUIN production was suggested to contribute to the loss of neurological function. On the other hand, KYNA was found to be likely involved in cerebral self-defence (Schwarcz et al., 1992). This is supported both by occurrence of cell death following the pharmacologically induced decrease in brain KYNA (McMaster et al., 1991) and by the increase in cerebral KYNA production following acute excitotoxic (Schwarcz et al., this volume) or convulsive (Wu and Schwarcz, 1994; Baran et al., 1995) insults.

6-Substituted Pyrrolo[3,4-c]pyrazoles: An Improved Class of CDK2 Inhibitors

We have recently reported a new class of CDK2/cyclin A inhibitors based on a bicyclic tetrahydropyrrolo[3,4‐c]pyrazole scaffold. The introduction of small alkyl or cycloalkyl groups in position 6 of this scaffold allowed variation at the other two diversity points. Conventional and polymer‐assisted solution phase chemistry provided a way of generating compounds with improved biochemical and cellular activity. Optimization of the physical properties and pharmacokinetic profile led to a compound which exhibited good efficacy in vivo on A2780 human ovarian carcinoma.