Cellular and Molecular Mechanisms of R/S-Roscovitine and CDKs Related Inhibition under Both Focal and Global Cerebral Ischemia: A Focus on Neurovascular Unit and Immune Cells.

Ischemic stroke is the second leading cause of death worldwide. Following ischemic stroke, Neurovascular Unit (NVU) inflammation and peripheral leucocytes infiltration are major contributors to the extension of brain lesions. For a long time restricted to neurons, the 10 past years have shown the emergence of an increasing number of studies focusing on the role of Cyclin-Dependent Kinases (CDKs) on the other cells of NVU, as well as on the leucocytes. The most widely used CDKs inhibitor, (R)-roscovitine, and its (S) isomer both decreased brain lesions in models of global and focal cerebral ischemia. We previously showed that (S)-roscovitine acted, at least, by modulating NVU response to ischemia. Interestingly, roscovitine was shown to decrease leucocytes-mediated inflammation in several inflammatory models. Specific inhibition of roscovitine majors target CDK 1, 2, 5, 7, and 9 showed that these CDKs played key roles in inflammatory processes of NVU cells and leucocytes after brain lesions, including ischemic stroke. The data summarized here support the investigation of roscovitine as a potential therapeutic agent for the treatment of ischemic stroke, and provide an overview of CDK 1, 2, 5, 7, and 9 functions in brain cells and leucocytes during cerebral ischemia.

Structure of cyclin-dependent kinase 2 (CDK2) in complex with the specific and potent inhibitor CVT-313.

CVT-313 is a potent CDK2 inhibitor that was identified by screening a purine-analogue library and is currently in preclinical studies. Since this molecule has the potential to be developed as a CDK2 inhibitor for cancer therapy, the potency of CVT-313 to bind and stabilize CDK2 was evaluated, together with its ability to inhibit aberrant cell proliferation. CVT-313 increased the melting temperature of CDK2 by 7°C in thermal stabilization studies, thus indicating its protein-stabilizing effect. CVT-313 inhibited the growth of human lung carcinoma cell line A549 in a dose-dependent manner, with an IC50 of 1.2 µM, which is in line with the reported biochemical potency of 0.5 µM. To support the further chemical modification of CVT-313 and to improve its biochemical and cellular potency, a crystal structure was elucidated in order to understand the molecular interaction of CVT-313 and CDK2. The crystal structure of CDK2 bound to CVT-313 was determined to a resolution of 1.74 Šand clearly demonstrated that CVT-313 binds in the ATP-binding pocket, interacting with Leu83, Asp86 and Asp145 directly, and the binding was further stabilized by a water-mediated interaction with Asn132. Based on the crystal structure, further modifications of CVT-313 are proposed to provide additional interactions with CDK2 in the active site, which may significantly increase the biochemical and cellular potency of CVT-313.

Cooperativity Between Orthosteric Inhibitors and Allosteric Inhibitor 8-Anilino-1-Naphthalene Sulfonic Acid (ANS) in Cyclin-Dependent Kinase 2.

While kinases have been attractive targets to combat many diseases, including cancer, selective kinase inhibition has been challenging, because of the high degree of structural homology in the active site, where many kinase inhibitors bind. We have previously discovered that 8-anilino-1-naphthalene sulfonic acid (ANS) binds an allosteric pocket in cyclin-dependent kinase 2 (Cdk2). Here, we detail the positive cooperativity between ANS and orthosteric Cdk2 inhibitors dinaciclib and roscovitine, which increase the affinity of ANS toward Cdk2 5-fold to 10-fold, and the relatively noncooperative effects of ATP. We observe these effects using a fluorescent binding assay and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR), where we noticed a shift from fast exchange to slow exchange upon ANS titration in the presence of roscovitine but not with an ATP mimic. The discovery of cooperative relationships between orthosteric and allosteric kinase inhibitors could further the development of selective kinase inhibitors in general.

Kinase inhibitor roscovitine as a PB2 cap-binding inhibitor against influenza a virus replication.

In this study, we examined the impact of roscovitine, a cyclin-dependent kinase inhibitor (CDKI) that has entered phase I and II clinical trials, on influenza A viruses (IAVs) and its antiviral mechanism. The results illustrated that roscovitine inhibited multiple subtypes of influenza strains dose-dependently, including A/WSN/1933(H1N1), A/Aichi/2/68 (H3N2) and A/FM1/47 (H1N1) with IC50 value of 3.35 ± 0.39, 7.01 ± 1.84 and 5.99 ± 1.89 µM, respectively. Moreover, roscovitine suppressed the gene transcription and genome replication steps in the viral life cycle. Further mechanistic studies indicated that roscovitine reduced viral polymerase activity and bound specifically to the viral PB2cap protein by fluorescence polarization assay (FP) and surface plasmon resonance (SPR). Therefore, we believed roscovitine, as a PB2cap inhibitor, was a prospective antiviral agent to be developed as therapeutic treatment against influenza A virus infection.

Platinum(II)-oxalato complexes of seliciclib (CYC202) derivatives show different cellular effects and lesser adverse effects in mouse lymphoma model than cisplatin.

This work presents a deeper pharmacological evaluation of two formerly prepared and characterized, and highly in vitro cytotoxic platinum(II) oxalato complexes [Pt(ox)(L1)2] (1) and [Pt(ox)(L2)2] (2), containing the derivatives of cyclin-dependent kinase inhibitor (CDKi) seliciclib ((R)-roscovitine, CYC202) coordinating as N-donor carrier ligands, i.e., 2-(1-ethyl-2-hydroxyethylamino)-N6-(4-methoxybenzyl)-9-isopropyladenine (L1) and 2-chloro-N6-(2,4-dimethoxybenzyl)-9-isopropyladenine (L2). The positive results of in vitro cytotoxicity screening on human cancer cell lines (HeLa, HOS, A2780, A2780R, G361 and MCF7 with IC50 at low micromolar levels) published previously, motivated us to perform extended preclinical in vitro experiments to reveal the mechanisms associated with the induction of cancer cell death. In addition, the in vivo antitumor activity was evaluated using the mouse lymphocytic leukaemia L1210 model. The obtained results revealed that complex 1 exceeds the antitumor effect of cisplatin (as for the extension of life-span of mice) and shows far less adverse effects as compared to reference drug cisplatin. The in vitro and ex vivo studies of cellular effects and molecular mechanisms of cell death induction showed that the mechanism of action of complex 1 is essentially different from that of cisplatin. The obtained results showed a possible way how to obtain antitumor active platinum(II) oxalato complexes with better therapeutic profile than contemporary used platinum-based therapeutics.

Structural analogues of roscovitine rescue the intracellular traffic and the function of ER-retained ABCB4 variants in cell models.

Adenosine triphosphate binding cassette transporter, subfamily B member 4 (ABCB4) is the transporter of phosphatidylcholine at the canalicular membrane of hepatocytes. ABCB4 deficiency, due to genetic variations, is responsible for progressive familial intrahepatic cholestasis type 3 (PFIC3) and other rare biliary diseases. Roscovitine is a molecule in clinical trial that was shown to correct the F508del variant of cystic fibrosis transmembrane conductance regulator (CFTR), another ABC transporter. In the present study, we hypothesized that roscovitine could act as a corrector of ABCB4 traffic-defective variants. Using HEK and HepG2 cells, we showed that roscovitine corrected the traffic and localisation at the plasma membrane of ABCB4-I541F, a prototypical intracellularly retained variant. However, roscovitine caused cytotoxicity, which urged us to synthesize non-toxic structural analogues. Roscovitine analogues were able to correct the intracellular traffic of ABCB4-I541F in HepG2 cells. Importantly, the phospholipid secretion activity of this variant was substantially rescued by three analogues (MRT2-235, MRT2-237 and MRT2-243) in HEK cells. We showed that these analogues also triggered the rescue of intracellular traffic and function of two other intracellularly retained ABCB4 variants, i.e. I490T and L556R. Our results indicate that structural analogues of roscovitine can rescue genetic variations altering the intracellular traffic of ABCB4 and should be considered as therapeutic means for severe biliary diseases caused by this class of variations.

Roscovitine and purvalanol A effectively reverse anthracycline resistance mediated by the activity of aldo-keto reductase 1C3 (AKR1C3): A promising therapeutic target for cancer treatment.

Members of the short-chain dehydrogenase/reductase (SDR) and aldo-keto reductase (AKR) superfamilies mediate the reduction of anthracyclines to their less potent C-13 alcohol metabolites. This reductive metabolism has been recognized as one of the most important factors that trigger anthracycline resistance in cancer cells. In our study, two purine analogues, purvalanol A and roscovitine, were identified as effective inhibitors of aldo-keto reductase 1C3 (AKR1C3), an enzyme that is overexpressed in many cancer types and is also a key player in tumour cell resistance to anthracyclines. Purvalanol A and roscovitine potently inhibited human recombinant AKR1C3 (Ki = 5.5 µM and 1.4 µM, respectively) and displayed similar activity in experiments with intact cells. Ligand-protein docking calculations suggested that both inhibitors occupied a part of the cofactor-binding site. Furthermore, we demonstrated that the combination of daunorubicin with purvalanol A or roscovitine exhibited a synergistic effect in AKR1C3 overexpressing cells. Based on these findings, it is possible to presume that purvalanol A and roscovitine may have the potential to enhance the therapeutic effectiveness and safety of anthracyclines via inhibition of AKR1C3.