Combinatorial ligand design targeted at protein families

We describe a method to create ligands specific for a given protein family. The method is applied to generate ligand candidates for the cyclin-dependent kinase (CDK) family. The CDK family of proteins is involved in regulating the cell cycle by alternately activating and deactivating the cell's progression through the cycle. CDKs are activated by association with cyclin and are inhibited by complexation with small molecules. X-ray crystal structures are available for three of the thirteen known CDK family members: CDK2, CDK5 and CDK 6. In this work, we use novel computational approaches to design ligand candidates that are potentially inhibitory across the three CDK family members as well as more specific molecules which can potentially inhibit one or any combination of two of the three CDK family members. We define a new scoring term, SpecScore, to quantify the potential inhibitory power of the generated structures. According to a search of the World Drug Alerts, the highest scoring SpecScore molecule that is specific for the three CDK family members shows very similar chemical characteristics and functional groups to numerous molecules known to deactivate several members of the CDK family.     

Cyclin-Dependent Kinase 4 and 6 Inhibitors in Cell Cycle Dysregulation for Breast Cancer Treatment

In cell development, the cell cycle is crucial, and the cycle progression’s main controllers are endogenous CDK inhibitors, cyclin-dependent kinases (CDKs), and cyclins. In response to the mitogenic signal, cyclin D is produced and retinoblastoma protein (Rb) is phosphorylated due to activated CDK4/CDK6. This causes various proteins required in the cell cycle progression to be generated. In addition, complexes of CDK1-cyclin A/B, CDK2-cyclin E/A, and CDK4/CDK6-cyclin D are required in each phase of this progression. Cell cycle dysregulation has the ability to lead to cancer. Based on its role in the cell cycle, CDK has become a natural target of anticancer therapy. Therefore, understanding the CDK structures and the complex formed with the drug, helps to foster the development of CDK inhibitors. This development starts from non-selective CDK inhibitors to selective CDK4/CDK6 inhibitors, and these have been applied in clinical cancer treatment. However, these inhibitors currently require further development for various hematologic malignancies and solid tumors, based on the results demonstrated. In drug development, the main strategy is primarily to prevent and asphyxiate drug resistance, thus a determination of specific biomarkers is required to increase the therapy’s effectiveness as well as patient selection suitability in order to avoid therapy failure. This review is expected to serve as a reference for early and advanced-stage researchers in designing new molecules or repurposing existing molecules as CDK4/CDK6 inhibitors to treat breast cancer.     

In silico study of porphyrin-anthraquinone hybrids as CDK2 inhibitor

Cyclin-Dependent Kinases (CDKs) are known to play crucial roles in controlling cell cycle progression of eukaryotic cell and inhibition of their activity has long been considered as potential strategy in anti-cancer drug research. In the present work, a series of porphyrin-anthraquinone hybrids bearing meso-substituents, i.e. either pyridine or pyrazole rings were designed and computationally evaluated for their Cyclin Dependent Kinase-2 (CDK2) inhibitory activity using molecular docking, molecular dynamics simulation, and binding free energy calculation. The molecular docking simulation revealed that all six porphyrin hybrids were able to bind to ATP-binding site of CDK2 and interacted with key residues constituted the active cavity of CDK2, while molecular dynamics simulation indicated that all porphyrins bound to CDK2 were stable for 6 ns. The binding free energies predicted by MM-PBSA method showed that most compounds exhibited higher affinity than that of native ligand (4-anilinoquinazoline, DTQ) and the affinity of mono-H₂PyP-AQ was about three times better than that of DTQ, indicating its potential to be advanced as a new CDK2 inhibitor.     

Bisarylmaleimides & the Corresponding Indolocarbazoles as Kinase Inhibitors

Cyclin dependent kinases (CDKs) have recently raised considerable attention because of their central role in the regulation of cell cycle progression. A high incidence of genetic mutation of CDK substrates and deregulation of CDK modulators were found in a number of disease states, particularly in cancer. A novel series of unsymmetrical substituted indolocarbazoles were synthesized and their kinase inhibitory capability was evaluated in vitro. 6-Substituted indolocarbazoles were found to b…     

Small molecules as inhibitors of cyclin-dependent kinases

Cell division (mitosis) is one of the basic requirements for multicellular oranisms. The capability of a cell to replicate enables a complex assembly to be created. Faulty regulation of the control mechanism in the cell cycle leads to an excessive cell proliferation and is the cause of cancer. The key position of the cyclin-dependent kinases (CDKs) and their direct partners, as well as the fact that the majority of malign illnesses show defects in at least one of these key players of the cell cycle, is of great interest for the development of low-molecular-weight CDK inhibitors. In this Review an overview of the different structural classes of ATP-competitive inhibitors of CDKs are given, whose devlopment was aimed at battling cancer. The Review shows how far the development of selective CDK inhibitors has progressed and to what extent the expectations for such drugs have so far been fulfilled.     

Understanding and modulating cyclin-dependent kinase inhibitor specificity: molecular modeling and biochemical evaluation of pyrazolopyrimidinones as CDK2/cyclin A and CDK4/cyclin D1 inhibitors

Summary Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.     

Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors

BACKGROUND: Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6, 9-trisubstituted purines remain unverified. RESULTS: To address this issue, purvalanol B (95. ) and an N6-methylated, CDK-inactive derivative (95M. ) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95. matrix. Casein kinase 1 (CK1) was identified as a principal 95. matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries. CONCLUSIONS: That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds.     

Rational inhibitor design and iterative screening in the identification of selective plasmodial cyclin dependent kinase inhibitors

New chemical classes of compounds must be introduced into the malaria drug development pipeline in an effort to develop new chemotherapy options for the fight against malaria. In this review we describe an iterative approach designed to identify potent inhibitors of a kinase family that collectively functions as key regulators of the cell cycle. Cyclin-dependent protein kinases (CDKs) are attractive drug targets in numerous diseases and, most recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. Our approach uses experimental and virtual screening methodologies to identify and refine chemical inhibitors and increase the success rate of discovering potent and selective inhibitors. The active pockets of the plasmodial CDKs are unique in terms of size, shape and amino acid composition compared with those of the mammalian orthologues. These differences exemplified through the use of screening assays, molecular modeling, and crystallography can be exploited for inhibitor design. To date, several classes of compounds including quinolines and oxindoles have been identified as selective inhibitors of the plasmodial CDK7 homologue, Pfmrk. From these initial studies and through the iterative rational drug design process, more potent, selective, and most importantly, chemically unique compound classes have been identified as effective inhibitors of the plasmodial CDKs and the malarial parasite.     

Effects of the cyclin-dependent kinase 10 (CDK10) on the tamoxifen sensitivity of keloid samples

Cyclin-dependent kinase 10 (CDK10) is a cell cycle regulating protein kinase, which has just been discriminated in recent years. In this paper, mRNA and protein expression of CDK10 were first investigated by a comparative study between 23 human keloid tissue samples and their adjacent normal skin. To further address its potential as a therapeutic target in the treatment of keloid, a plasmid expressing the CDK10 gene was transfected into keloid fibroblast. The effects on tamoxifen-induced apoptosis were then investigated using Western blot assay and flow cytometry. Results showed that there is a generally down-regulated expression of CDK10 in keloid compared to normal skin samples. Transfection with the recombinant CDK10 plasmid significantly decreased the viability of cells and increased the apoptosis rates. Tamoxifen sensitivity in keloid fibroblasts was observed after treatment with the recombinant CDK10 plasmid. The results suggested that CDK10 may play an important role in enhancement of tamoxifen efficiency, and its expression may have a synergistic effect on keloid treatments.     

Detection of 210 kDa receptor protein for a leaf-movement factor by using novel photoaffinity probes

Circadian rhythmic plant leaf-movement, called nyctinasty, is controlled by a time-course change in the internal concentration of the leaf-movement factor in the plant body. We revealed that specific binding proteins (210 and 180 kDa) for the leaf-movement factor, potassium lespedezate (1), are contained in the plasma membrane of the plant motor cell by using novel synthetic photoaffinity probes. These proteins are localized on the motor cell in the plant body, and would be potential receptors for the leaf-movement factor to control the leaf-movement. Our study is a rare successful result of the detection of membrane receptors by using a synthetic photoaffinity probe designed on a biologically active natural product. And these results also advance a guideline for probe design towards successful photoaffinity labeling.     

Acid-activatible micelleplex delivering siRNA-PD-L1 for improved cancer immunotherapy of CDK4/6 inhibition

Cyclin-dependent kinases 4 and 6 inhibitors(CDK4/6i) have been demonstrated to trigger antitumor immunity for tumor regression. However, the therapeutic performance of CDK4/6i-meadiated cancer immunotherapy was impaired by the immunosuppressive tumor microenvironment(ITM) due to overexpression of programmed death ligand 1(PD-L1) on the surface of cancer cell membrane. To improve the immunotherapeutic performance of CDK4/6i, we herein developed endosomal acidactivatable micelleplex for si RNA delivery and PD-L1 knockdown in the tumor cells in vitro and in vivo. We further demonstrated that the combination of PD-L1 knockdown and CDK4/6 inhibition facilitated intratumoral infiltration of cytotoxic T lymphocytes(CTLs), and elicited protective immune response and efficiently suppressed tumor growth in vivo. This study revealed the importance of molecular design of the micelleplex for highly efficient si RNA delivery, which might provide a novel insight for RNAi-based cancer immunotherapy.     

Differential binding of inhibitors to active and inactive CDK2 provides insights for drug design

The cyclin-dependent kinases (CDKs) have been characterized in complex with a variety of inhibitors, but the majority of structures solved are in the inactive form. We have solved the structures of six inhibitors in both the monomeric CDK2 and binary CDK2/cyclinA complexes and demonstrate that significant differences in ligand binding occur depending on the activation state. The binding mode of two ligands in particular varies substantially in active and inactive CDK2. Furthermore, energetic analysis of CDK2/cyclin/inhibitors demonstrates that a good correlation exists between the in vitro potency and the calculated energies of interaction, but no such relationship exists for CDK2/inhibitor structures. These results confirm that monomeric CDK2 ligand complexes do not fully reflect active conformations, revealing significant implications for inhibitor design while also suggesting that the monomeric CDK2 conformation can be selectively inhibited.     

One-Step Synthesis of Photoaffinity Probes for Live-Cell MS-Based Proteomics

We present a one-step Ugi reaction protocol for the expedient synthesis of photoaffinity probes for live-cell MS-based proteomics. The reaction couples an amine affinity function with commonly used photoreactive groups, and a variety of handle functionalities. Using this technology, a series of pan-BET (BET: bromodomain and extra-terminal domain) selective bromodomain photoaffinity probes were obtained by parallel synthesis. Studies on the effects of photoreactive group, linker length and irradiation wavelength on photocrosslinking efficiency provide valuable insights into photoaffinity probe design. Optimal probes were progressed to MS-based proteomics to capture the BET family of proteins from live cells and reveal their potential on- and off-target profiles.     

Development of Ubiquitin‐Based Probe for Metalloprotease Deubiquitinases

Deubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn²⁺ in their active sites and are an integral part of distinct cellular protein complexes. Little is known about these enzymes because of the lack of specific probes. Described here is a Ub‐based probe that contains a ubiquitin moiety modified at its C‐terminus with a Zn²⁺ chelating group based on 8‐mercaptoquinoline, and a modification at the N‐terminus with either a fluorescent tag or a pull‐down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe binds to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from a HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail and allow better understanding of their biochemical processes.     

Structure-based design of oxygen-linked macrocyclic kinase inhibitors: discovery of SB1518 and SB1578, potent inhibitors of Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3)

Macrocycles from our Aurora project were screened in a kinase panel and were found to be active on other kinase targets, mainly JAKs, FLT3 and CDKs. Subsequently these compounds became leads in our JAK2 project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. This residue is conserved in most CDKs resulting in potent pan CDK inhibition. One of the main project objectives was to achieve JAK2 potency with 100-fold selectivity against CDKs. Macrocycles with an ether linker have potent JAK2 activity with the ether oxygen forming a hydrogen bond to Ser936. A hydrogen bond to the equivalent residues of JAK3 and most CDKs cannot be formed resulting in good selectivity for JAK2 over JAK3 and CDKs. Further optimization of the macrocyclic linker and side chain increased JAK2 and FLT3 activity as well as improving DMPK properties. The selective JAK2/FLT3 inhibitor 11 (Pacritinib, SB1518) has successfully finished phase 2 clinical trials for myelofibrosis and lymphoma. Another selective JAK2/FLT3 inhibitor, 33 (SB1578), has entered phase 1 clinical development for the non-oncology indication rheumatoid arthritis.     

Molecular dynamics simulations on the inhibition of Cyclin-Dependent Kinases 2 and 5 in the presence of activators

Interests in CDK2 and CDK5 have stemmed mainly from their association with cancer and neuronal migration or differentiation related diseases and the need to design selective inhibitors for these kinases. Molecular dynamics (MD) simulations have not only become a viable approach to drug design because of advances in computer technology but are increasingly an integral part of drug discovery processes. It is common in MD simulations of inhibitor/CDK complexes to exclude the activator of the CDKs in the structural models to keep computational time tractable. In this paper, we present simulation results of CDK2 and CDK5 with roscovitine using models with and without their activators (cyclinA and p25). While p25 was found to induce slight changes in CDK5, the calculations support that cyclinA leads to significant conformational changes near the active site of CDK2. This suggests that detailed and structure-based inhibitor design targeted at these CDKs should employ activator-included models of the kinases. Comparisons between P/CDK2/cyclinA/roscovitine and CDK5/p25/roscovitine complexes reveal differences in the conformations of the glutamine around the active sites, which may be exploited to find highly selective inhibitors with respect to CDK2 and CDK5.