Cyclin-dependent kinases (CDKs) play an important role in the cell-division cycle. Synthetic inhibitors of CDKs are based on 2,6,9-trisubstituted purines and are developed as potential anticancer drugs; however, they have low solubility in water. In this study, we proved that the pharmaco-chemical properties of purine-based inhibitors can be improved by appropriate substitution with the adamantane moiety. We prepared ten new purine derivatives with adamantane skeletons that were linked at position 6 using phenylene spacers of variable geometry and polarity. We demonstrated that the adamantane skeleton does not compromise the biological activity, and some of the new purines displayed even higher inhibition activity towards CDK2/cyclin E than the parental compounds. These findings were supported by a docking study, which showed an adamantane scaffold inside the binding pocket participating in the complex stabilisation with non-polar interactions. In addition, we demonstrated that β-cyclodextrin (CD) increases the drug's solubility in water, although this is at the cost of reducing the biochemical and cellular effect. Most likely, the drug concentration, which is necessary for target engagement, was decreased by competitive drug binding within the complex with β-CD.

• Keywords
• 2,6,9-trisubstituted purine, adamantane, cyclin-dependent kinase, cytotoxicity, molecular docking, β-cyclodextrin,
• MeSH
• Adamantane chemistry   MeSH
• beta-Cyclodextrins chemistry   MeSH
• K562 Cells MeSH
• Cyclin-Dependent Kinase 2 antagonists & inhibitors   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Purines chemistry   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adamantane MeSH
• beta-Cyclodextrins MeSH
• CDK2 protein, human MeSH Browser
• Cyclin-Dependent Kinase 2 MeSH
• Protein Kinase Inhibitors MeSH
• Antineoplastic Agents MeSH
• Purines MeSH

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional processing of mRNA expressed from extrachromosomal DNA.

• MeSH
• Cell Line MeSH
• Cell Cycle drug effects   MeSH
• Chlorocebus aethiops MeSH
• Cyclin-Dependent Kinases antagonists & inhibitors   MeSH
• DNA, Viral MeSH
• Phosphorylation drug effects   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Kidney drug effects   metabolism   MeSH
• Humans MeSH
• RNA Processing, Post-Transcriptional drug effects   MeSH
• Promoter Regions, Genetic drug effects   MeSH
• Purines pharmacology   MeSH
• RNA Polymerase II genetics   metabolism   MeSH
• Roscovitine MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cyclin-Dependent Kinases MeSH
• DNA, Viral MeSH
• Protein Kinase Inhibitors MeSH
• olomoucine II MeSH Browser
• Purines MeSH
• RNA Polymerase II MeSH
• Roscovitine MeSH

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.

• MeSH
• ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
• ATP-Binding Cassette Transporters metabolism   MeSH
• Biological Transport MeSH
• Cell Line MeSH
• Drug Resistance, Neoplasm drug effects   MeSH
• Neoplasm Proteins metabolism   MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism   MeSH
• ATP Binding Cassette Transporter, Subfamily B MeSH
• Dogs MeSH
• Purines pharmacokinetics   MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 6-((3-chloro)anilino)-2-(isopropyl-2-hydroxyethylamino)-9-isopropylpurine MeSH Browser
• ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
• ATP-Binding Cassette Transporters MeSH
• ABCB1 protein, human MeSH Browser
• ABCG2 protein, human MeSH Browser
• Neoplasm Proteins MeSH
• olomoucine II MeSH Browser
• ATP Binding Cassette Transporter, Subfamily B, Member 1 MeSH
• ATP Binding Cassette Transporter, Subfamily B MeSH
• Purines MeSH

BACKGROUND: Olomoucine II, the most recent derivative of roscovitine, is an exceptionally potent pharmacological inhibitor of cyclin-dependent kinase activities. Here, we report that olomoucine II is also an effective antiviral agent. METHODS: Antiviral activities of olomoucine II were tested on a range of human viruses in in vitro assays that evaluated viral growth and replication. RESULTS: Olomoucine II inhibited replication of a broad range of wild-type human viruses, including herpes simplex virus, human adenovirus type-4 and human cytomegalovirus. Olomoucine II also inhibited replication of vaccinia virus and herpes simplex virus mutants resistant to conventional acyclovir treatment. This report is the first demonstration of a poxvirus being sensitive to a cyclin-dependent kinase inhibitor. The antiviral effects of olomoucine II could be observed at lower concentrations than with roscovitine, although both were short-term. A remarkable observation was that olomoucine II, when used in combination with the DNA polymerase inhibitor cidofovir, was able to almost completely eliminate the spread of infectious adenovirus type-4 progeny from infected cells. CONCLUSIONS: Our results show that when targeting two complementary antiviral mechanisms, strongly additive effects could be observed.

• MeSH
• Antiviral Agents pharmacology   MeSH
• Cyclin-Dependent Kinases antagonists & inhibitors   MeSH
• Virus Physiological Phenomena drug effects   MeSH
• Inhibitory Concentration 50 MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• Purines pharmacology   MeSH
• Virus Replication drug effects   MeSH
• Viruses enzymology   growth & development   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antiviral Agents MeSH
• Cyclin-Dependent Kinases MeSH
• Protein Kinase Inhibitors MeSH
• olomoucine II MeSH Browser
• Purines MeSH