Cdc2-related Kinase Research Articles

Glide Docking, Autodock, Binding Free Energy and Drug-Likeness Studies for Prediction of Potential Inhibitors of Cyclin-Dependent Kinase 14 Protein in Wnt Signaling Pathway

Cyclin-dependent kinase 14 plays an essential role in multiple cancers. Cyclin-dependent kinase 14 is a serine/threonine kinase and is a member of the cell division cycle 2(cdc2) related protein kinase family, which plays a key role in promoting Wnt signaling pathway of the cell cycle and its overexpression causes various human cancers. The 3D structure of cyclin-dependent kinase 14 was built using the homology-based modeling technique. The generated model is optimized by NAMD-VMD software. The quality of stabilized CDK14 protein was checked using Ramachandran plot and ProSA servers. The potential binding site region was recognized using SiteMap and manual correlation techniques from literature studies. The virtual screening was performed with the TOSLab database of 27253 output molecules against CDK14 protein using Glide docking to assess novel chemical entities. Their binding energies were calculated from PrimeMMGSA and AutoDock. The novel lead molecules have been prioritized based on efficient binding energies (from AutoDock and PrimeMMGBSA), better glide scores, good bioavailability, and acceptable ADME properties. Thus, these are considered as CDK14 protein inhibitors for cancer therapeutics.

The CRK2-CYC13 complex functions as an S-phase cyclin-dependent kinase to promote DNA replication in Trypanosoma brucei

BackgroundFaithful DNA replication is essential to maintain genomic stability in all living organisms, and the regulatory pathway for DNA replication initiation is conserved from yeast to humans. The evolutionarily ancient human parasite Trypanosoma brucei, however, lacks many of the conserved DNA replication factors and may employ unusual mechanisms for DNA replication. Neither the S-phase cyclin-dependent kinase (CDK) nor the regulatory pathway governing DNA replication has been previously identified in T. brucei.ResultsHere we report that CRK2 (Cdc2-related kinase 2) complexes with CYC13 (Cyclin13) and functions as an S-phase CDK to promote DNA replication in T. brucei. We further show that CRK2 phosphorylates Mcm3, a subunit of the Mcm2–7 sub-complex of the Cdc45-Mcm2–7-GINS complex, and demonstrate that Mcm3 phosphorylation by CRK2 facilitates interaction with Sld5, a subunit of the GINS sub-complex of the Cdc45-Mcm2–7-GINS complex.ConclusionsThese results identify the CRK2-CYC13 complex as an S-phase regulator in T. brucei and reveal its role in regulating DNA replication through promoting the assembly of the Cdc45-Mcm2–7-GINS complex.

Inactivation of the Kinase Domain of CDK10 Prevents Tumor Growth in a Preclinical Model of Colorectal Cancer, and Is Accompanied by Downregulation of Bcl-2.

Cyclin-dependent kinase 10 (CDK10), a CDC2-related kinase, is highly expressed in colorectal cancer. Its role in the pathogenesis of colorectal cancer is unknown. This study examines the function of CDK10 in colorectal cancer, and demonstrates its role in suppressing apoptosis and in promoting tumor growth in vitro and in vivo Modulation of CDK10 expression in colorectal cancer cell lines demonstrates that CDK10 promotes cell growth, reduces chemosensitivity and inhibits apoptosis by upregulating the expression of Bcl-2. This effect appears to depend on its kinase activity, as kinase-defective mutant colorectal cancer cell lines have an exaggerated apoptotic response and reduced proliferative capacity. In vivo, inhibiting CDK10 in colorectal cancer following intratumoral injections of lentivirus-mediated CDK10 siRNA in a patient-derived xenograft mouse model demonstrated its efficacy in suppressing tumor growth. Furthermore, using a tissue microarray of human colorectal cancer tissues, the potential for CDK10 to be a prognostic biomarker in colorectal cancer was explored. In tumors of individuals with colorectal cancer, high expression of CDK10 correlates with earlier relapse and shorter overall survival. The findings of this study indicate that CDK10 plays a role in the pathogenesis in colorectal cancer and may be a potential therapeutic target for treatment. Mol Cancer Ther; 16(10); 2292-303. ©2017 AACR.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

MiR-455 inhibits breast cancer cell proliferation through targeting CDK14

Breast cancer is the most frequently occurring cancer in women worldwide, microRNAs (miRNAs) play critical role in the initiation and progression of breast cancer. Here, we studied the effect of miR-455 on cell proliferation of breast cancer, and found that miR-455 was downregulated in breast cancer tissues and cells. Its overexpression inhibited cell proliferation, whereas its knockdown promoted cell proliferation of breast cancer. We found a Cdc2-related protein kinase CDK14 was the target of miR-455, when the 3’UTR of CDK14 was cloned into luciferase reporter vector and transfected into cells, miR-455 mimic could inhibit the luciferase activity in a dose-dependent manner, miR-455 inhibitor increased the luciferase activity, but the mutant miR-455 mimic couldn’t change the luciferase activity, suggesting miR-455 directly bound to the 3’UTR of CDK14. Meanwhile, we also found miR-455 inhibited Cyclin D1 expression and promoted p21 expression, confirming miR-455 inhibited cell proliferation. Double knockdown of miR-455 and CDK14 inhibited the proliferation of breast cancer cell, confirming miR-455 inhibiting cell proliferation by targeting CDK14. Moreover, miR-455 levels were negatively correlated with CDK14 levels in breast cancer tissues. Our finding revealed miR-455 inhibits breast cancer cell proliferation through targeting CDK14, it might be a target for breast cancer therapy.

Cyclin-Dependent Kinase CRK9, Required for Spliced Leader trans Splicing of Pre-mRNA in Trypanosomes, Functions in a Complex with a New L-Type Cyclin and a Kinetoplastid-Specific Protein.

In eukaryotes, cyclin-dependent kinases (CDKs) control the cell cycle and critical steps in gene expression. The lethal parasite Trypanosoma brucei, member of the phylogenetic order Kinetoplastida, possesses eleven CDKs which, due to high sequence divergence, were generically termed CDC2-related kinases (CRKs). While several CRKs have been implied in the cell cycle, CRK9 was the first trypanosome CDK shown to control the unusual mode of gene expression found in kinetoplastids. In these organisms, protein-coding genes are arranged in tandem arrays which are transcribed polycistronically. Individual mRNAs are processed from precursor RNA by spliced leader (SL) trans splicing and polyadenylation. CRK9 ablation was lethal in cultured trypanosomes, causing a block of trans splicing before the first transesterification step. Additionally, CRK9 silencing led to dephosphorylation of RNA polymerase II and to hypomethylation of the SL cap structure. Here, we tandem affinity-purified CRK9 and, among potential CRK9 substrates and modifying enzymes, discovered an unusual tripartite complex comprising CRK9, a new L-type cyclin (CYC12) and a protein, termed CRK9-associated protein (CRK9AP), that is only conserved among kinetoplastids. Silencing of either CYC12 or CRK9AP reproduced the effects of depleting CRK9, identifying these proteins as functional partners of CRK9 in vivo. While mammalian cyclin L binds to CDK11, the CRK9 complex deviates substantially from that of CDK11, requiring CRK9AP for efficient CRK9 complex formation and autophosphorylation in vitro. Interference with this unusual CDK rescued mice from lethal trypanosome infections, validating CRK9 as a potential chemotherapeutic target.

2,6,9-Trisubstituted purines as CRK3 kinase inhibitors with antileishmanial activity in vitro.

Here we describe the leishmanicidal activities of a library of 2,6,9-trisubstituted purines that were screened for interaction with Cdc2-related protein kinase 3 (CRK3) and subsequently for activity against parasitic Leishmania species. The most active compound inhibited recombinant CRK3 with an IC50 value of 162 nM and was active against Leishmania major and Leishmania donovani at low micromolar concentrations in vitro. Its mode of binding to CRK3 was investigated by molecular docking using a homology model.

An inhibitor-driven study for enhancing the selectivity of indirubin derivatives towards leishmanial Glycogen Synthase Kinase-3 over leishmanial cdc2-related protein kinase 3.

BackgroundIn search of new antiparasitic agents for overcoming the limitations of current leishmaniasis chemotherapy, we have previously shown that 6-bromoindirubin-3'-oxime (6BIO) and several other 6-substituted analogues of indirubin, a naturally occurring bis-indole present in mollusks and plants, displayed reverse selectivity from the respective mammalian kinases, targeting more potently the leishmanial Cyclin-Dependent Kinase-1 (CDK1) homologue [cdc2-related protein kinase 3 (LCRK3)] over leishmanial Glycogen Synthase Kinase-3 (LGSK-3). This reversal of selectivity in Leishmania parasites compared to mammalian cells makes the design of specific indirubin-based LGSK-3 inhibitors difficult. In this context, the identification of compounds bearing specific substitutions that shift indirubin inhibition towards LGSK-3, previously found to be a potential drug target, over LCRK3 is imperative for antileishmanial targeted drug discovery.MethodsA new in-house indirubin library, composed of 35 compounds, initially designed to target mammalian kinases (CDKs, GSK-3), was tested against Leishmania donovani promastigotes and intracellular amastigotes using the Alamar blue assay. Indirubins with antileishmanial activity were tested against LGSK-3 and LCRK3 kinases, purified from homologous expression systems. Flow cytometry (FACS) was used to measure the DNA content for cell-cycle analysis and the mode of cell death. Comparative structural analysis of the involved kinases was then performed using the Szmap algorithm.ResultsWe have identified 7 new indirubin analogues that are selective inhibitors of LGSK-3 over LCRK3. These new inhibitors were also found to display potent antileishmanial activity with GI50 values of <1.5 μΜ. Surprisingly, all the compounds that displayed enhanced selectivity towards LGSK-3, were 6BIO analogues bearing an additional 3'-bulky amino substitution, namely a piperazine or pyrrolidine ring. A comparative structural analysis of the two aforementioned leishmanial kinases was subsequently undertaken to explain and rationalize the selectivity trend determined by the in vitro binding assays. Interestingly, the latter analysis showed that selectivity could be correlated with differences in kinase solvation thermo dynamics induced by minor sequence variations of the otherwise highly similar ATP binding pockets.ConclusionsIn conclusion, 3'-bulky amino substituted 6-BIO derivatives, which demonstrate enhanced specificity towards LGSK-3, represent a new scaffold for targeted drug development to treat leishmaniasis.

PFTK1 interacts with cyclin Y to activate non-canonical Wnt signaling in hepatocellular carcinoma

PFTK1 is a Cdc2-related protein kinase that is frequently upregulated in human hepatocellular carcinoma (HCC) where it correlates with metastatic features and motile phenotypes. To understand the modulated pathway underlining the PFTK1 action, here we show a physical interaction between PFTK1 and cyclin Y (CCNY) in promoting noncanonical Wnt signaling. In HCC cells, we found PFTK1 forms a direct complex with CCNY, and together readily upregulate key components of Wnt signaling (Dvl2 and Naked1). Exogenous expression of PFTK1 and CCNY activated Rho GTPases, which are known targets of the noncanonical path. In line with Rho GTPases activation, we also found marked actin polymerizations in cells with PFTK1–CCNY co-expressions. Our findings highlight a PFTK1–CCNY complex in activating noncanonical Wnt signaling in HCC cells.

The cooperative roles of PHO80‐like cyclins in regulating the G1/S transition and posterior cytoskeletal morphogenesis in Trypanosoma brucei

Cyclins and cyclin-dependent kinases (CDKs) represent the fundamental, crucial regulators of the cell division cycle in eukaryotes. Trypanosoma brucei expresses a large number of cyclins and Cdc2-related kinases (CRKs). However, how these cyclins and CRKs cooperate to regulate cell cycle progression remains poorly understood. Here, we carry out directional yeast two-hybrid assays to identify the interactions between the 10 cyclins and the 11 CRKs and detect a total of 26 cyclin-CRK pairs, among which 20 pairs are new. Our current efforts are focused on four PHO80-like cyclins, CYC2, CYC4, CYC5 and CYC7, and their physical and functional interactions with CRK1. Silencing of the four cyclins and CRK1 leads to the increase of G1 cells and defective DNA replication, suggesting their important roles in promoting the G1/S transition. Additionally, CYC2-, CYC7- and CRK1-deficient cells possess an elongated posterior that is filled with newly assembled microtubules. Further, we show that the four cyclins display distinct subcellular localizations and half-lives, suggesting that they likely undergo distinct regulation. Altogether, our results demonstrate the involvement of four CRK1-associated cyclins, CYC2, CYC4, CYC5 and CYC7, in promoting the G1/S transition and the requirement of CYC2 and CYC7 in maintaining posterior cytoskeletal morphogenesis during the G1/S transition.

Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK)-Cyclin Complex in Trypanosoma brucei.

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.

Characterization of Plasmodium falciparum cdc2-related kinase and the effects of a CDK inhibitor on the parasites in erythrocytic schizogony

The cell cycle of Plasmodium is unique among major eukaryotic cell cycle models. Cyclin-dependent kinases (CDKs) are thought to be the key molecular switches that regulate cell cycle progression in the parasite. However, little information is available about Plasmodium CDKs. The present study was performed to investigate the effects of a CDK inhibitor, olomoucine, on the erythrocytic growth of Plasmodium falciparum. This agent inhibited the growth of the parasite at the trophozoite/schizont stage. Furthermore, we characterized the Plasmodium CDK homolog, P. falciparum cdc2-related kinase-1 (Pfcrk-1), which is a potential target of olomoucine. We synthesized a functional kinase domain of Pfcrk-1 as a GST fusion protein using a wheat germ protein expression system, and examined its phosphorylation activity. The activity of this catalytic domain was higher than that of GST-GFP control, but the same as that of a kinase-negative mutant of Pfcrk-1. After the phosphatase treatment, the labeling of [γ-32P]ATP was abolished. Recombinant human cyclin proteins were added to these kinase reactions, but there were no differences in activity. This report provides important information for the future investigation of Plasmodium CDKs.

Identification of essential and non-essential protein kinases by a fusion PCR method for efficient production of transgenic Trypanosoma brucei

Manipulation of gene expression has been used to elucidate gene function, explore fundamental biological processes and to identify potential drug targets in Trypanosoma brucei. We show in bloodstream forms that CDC2-related kinase CRK12 (Tb11.01.4130) is essential since transcriptional inactivation in conditional null mutants is lethal but 19 other protein kinases are not essential since null mutants are viable. We did so using efficient methods for the generation of null and conditional null cell lines of T. brucei by approaches that generate transfection constructs with large targeting sequences and which use reliable transfection and selection conditions. These methods, which are described in detail in the supplementary material, employ multiple oligonucleotides and PCR reactions and several transfections but are cost effective and can simultaneously generate 24 transfectants thus shifting the rate limiting experimental steps from the production of cell lines to their analysis.

Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1

Conserved from yeast to mammals, phosphorylation of the heptad repeat sequence Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) in the carboxy-terminal domain (CTD) of the largest RNA polymerase II (RNA Pol II) subunit, RPB1, mediates the enzyme's promoter escape and binding of RNA-processing factors, such as the m(7)G capping enzymes. The first critical step, Ser(5) phosphorylation, is carried out by cyclin-dependent kinase 7 (CDK7), a subunit of the basal transcription factor TFIIH. Many early-diverged protists, such as the lethal human parasite Trypanosoma brucei, however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. The T. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression of T. brucei cdc2-related kinase 9 (CRK9) leads to a loss of RPB1 phosphorylation. Surprisingly, this event did not impair RNA Pol II transcription or cotranscriptional m(7)G capping. Instead, we observed that CRK9 silencing led to a block of spliced leader (SL) trans splicing, an essential step in trypanosome mRNA maturation, that was caused by hypomethylation of the SL RNA's unique cap4.

Interaction of Cyclin-Dependent Kinase 12/CrkRS with Cyclin K1 Is Required for the Phosphorylation of the C-Terminal Domain of RNA Polymerase II

CrkRS (Cdc2-related kinase, Arg/Ser), or cyclin-dependent kinase 12 (CKD12), is a serine/threonine kinase believed to coordinate transcription and RNA splicing. While CDK12/CrkRS complexes were known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II), the cyclin regulating this activity was not known. Using immunoprecipitation and mass spectrometry, we identified a 65-kDa isoform of cyclin K (cyclin K1) in endogenous CDK12/CrkRS protein complexes. We show that cyclin K1 complexes isolated from mammalian cells contain CDK12/CrkRS but do not contain CDK9, a presumed partner of cyclin K. Analysis of extensive RNA-Seq data shows that the 65-kDa cyclin K1 isoform is the predominantly expressed form across numerous tissue types. We also demonstrate that CDK12/CrkRS is dependent on cyclin K1 for its kinase activity and that small interfering RNA (siRNA) knockdown of CDK12/CrkRS or cyclin K1 has similar effects on the expression of a luciferase reporter gene. Our data suggest that cyclin K1 is the primary cyclin partner for CDK12/CrkRS and that cyclin K1 is required to activate CDK12/CrkRS to phosphorylate the CTD of RNA Pol II. These properties are consistent with a role of CDK12/CrkRS in regulating gene expression through phosphorylation of RNA Pol II.

High-throughput screening with the Eimeria tenella CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a

The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs.

Clinical and biological significance of Cdk10 in hepatocellular carcinoma

Cyclin-dependent kinase 10 (Cdk10) is a Cdc2-related kinase and plays an essential role in the progression from the G2 to M phase of the cell cycle. However, relative little is known about its expression pattern, clinical relevance, and biological function in hepatocellular carcinoma (HCC). In the present study, we investigated the mRNA and protein expression levels of Cdk10 in 127 pairs of HCC samples and adjacent nontumorous liver tissues and evaluated its clinical significance. Additionally, we assessed the effects of restoration of Cdk10 on cell proliferation and drug sensitivity in HCC cells. We showed that the Cdk10 mRNA and protein expression was markedly decreased in HCC samples compared to adjacent nontumorous liver tissues. Quantitative real-time polymerase chain reaction and immunohistochemical studies revealed that reduced Cdk10 expression was significantly associated with alpha-fetoprotein levels, tumor size, and tumor stage. Ectopic expression of Cdk10 reduced HCC cell proliferation, blocked the cell cycle at the G0-G1 phase, as well as inhibited cell migration and anchorage-independent growth. Additionally, Cdk10 overexpression enhanced the chemosensitivity of HCC cells to cisplatin and epidoxorubicin, two chemotherapeutic agents commonly used in HCC. These data collectively demonstrate that reduced Cdk10 expression is closely linked to HCC development and progression. Restoration of its expression may have therapeutic benefits in treating this malignancy.

The Cdc45·Mcm2–7·GINS Protein Complex in Trypanosomes Regulates DNA Replication and Interacts with Two Orc1-like Proteins in the Origin Recognition Complex

Accurate DNA replication requires a complex interplay of many regulatory proteins at replication origins. The CMG (Cdc45·Mcm2-7·GINS) complex, which is composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the heterohexameric origin recognition complex (ORC) and functions as a replicative helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. However, unlike archaea, trypanosomes possess components of the eukaryote-like CMG complex, but whether they form an active helicase complex, associate with the ORC, and regulate DNA replication remains unknown. Here, we demonstrated that the CMG complex is formed in vivo in trypanosomes and that Mcm2-7 helicase activity is activated by the association with Cdc45 and the GINS complex in vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, indicating that nuclear exclusion of Cdc45 constitutes one mechanism for preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 associate with Mcm2-7 via interactions with Mcm3. All together, we identified the Cdc45·Mcm2-7·GINS complex as the replicative helicase that interacts with two Orc1-like proteins in the unusual origin recognition complex in trypanosomes.

Anti-leishmanial activity of disubstituted purines and related pyrazolo[4,3-d]pyrimidines

We report here results of screening directed to finding new anti-leishmanial drugs among 2,6-disubstituted purines and corresponding 3,7-disubstituted pyrazolo[4,3-d]pyrimidines. These compounds have previously been shown to moderately inhibit human cyclin-dependent kinases. Since some compounds reduced viability of axenic amastigotes of Leishmania donovani, we screened them for interaction with recombinant leishmanial cdc-2 related protein kinase (CRK3/CYC6), an important cell cycle regulator of the parasitic protozoan. Eighteen pairs of corresponding isomers were tested for viability of amastigotes and for inhibition of CRK3/CYC6 kinase activity. Some compounds (9A, 12A and 13A) show activity against amastigotes with EC(50) in a range 1.5-12.4μM. Structure-activity relationships for the tested compounds are discussed and related to the lipophilicity of the compounds.

High Throughput Screens Yield Small Molecule Inhibitors of Leishmania CRK3:CYC6 Cyclin-Dependent Kinase

Background Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.Methodology/Principal FindingsIn order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.Conclusions/SignificanceThe pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.