Identification of cylin-dependent kinase 1 inhibitors of a new chemical type by structure-based design and database searching

We have selected cyclin-dependent kinase 1 (CDK1), an enzyme participating in the regulation of the cell cycle, as a target in our efforts to discover new antitumor agents. By exploiting available structural information, we designed an ATP-site directed ligand scaffold that allowed us to identify 4-(3-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylamino)-benzenesulfonamide as a new potent inhibitor of CDK1 in a subsequent database search. The synthesis and testing of some analogues confirmed the interest of this class of compounds as novel CDK1 inhibitors.     

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.     

Structural determinants of CDK4 inhibition and design of selective ATP competitive inhibitors

A number of selective inhibitors of the CDK4/cyclin D1 complex have been reported recently. Due to the absence of an experimental CDK4 structure, the ligand and protein determinants contributing to CDK4 selectivity are poorly understood at present. Here, we report the use of computational methods to elucidate the characteristics of selectivity and to derive the structural basis for specific, high-affinity binding of inhibitors to the CDK4 active site. From these data, the hypothesis emerged that appropriate incorporation of an ionizable function into a CDK2 inhibitor results in more favorable binding to CDK4. This knowledge was applied to the design of compounds in the otherwise CDK2-selective 2-anilino-4-(thiazol-5-yl)pyrimidine pharmacophore that are potent and highly selective ATP antagonists of CDK4/cyclin D1. The findings of this study also have significant implications in the design of CDK4 mimic structures based on CDK2.     

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.     

Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by hymenialdisine, a marine sponge constituent

BACKGROUND: Over 2000 protein kinases regulate cellular functions. Screening for inhibitors of some of these kinases has already yielded some potent and selective compounds with promising potential for the treatment of human diseases. RESULTS: The marine sponge constituent hymenialdisine is a potent inhibitor of cyclin-dependent kinases, glycogen synthase kinase-3beta and casein kinase 1. Hymenialdisine competes with ATP for binding to these kinases. A CDK2-hymenialdisine complex crystal structure shows that three hydrogen bonds link hymenialdisine to the Glu81 and Leu83 residues of CDK2, as observed with other inhibitors. Hymenialdisine inhibits CDK5/p35 in vivo as demonstrated by the lack of phosphorylation/down-regulation of Pak1 kinase in E18 rat cortical neurons, and also inhibits GSK-3 in vivo as shown by the inhibition of MAP-1B phosphorylation. Hymenialdisine also blocks the in vivo phosphorylation of the microtubule-binding protein tau at sites that are hyperphosphorylated by GSK-3 and CDK5/p35 in Alzheimer's disease (cross-reacting with Alzheimer's-specific AT100 antibodies). CONCLUSIONS: The natural product hymenialdisine is a new kinase inhibitor with promising potential applications for treating neurodegenerative disorders.     

Biochemical characterizations reveal different properties between CDK4/cyclin D1 and CDK2/cyclin A

CDK2 and CDK4 known promoter of cell cycling catalyze phosphorylation of RB protein. Enzyme specificity between two CDKs that work at a different cell cycle phase is not clearly understood. In order to define kinase properties of CDK2 and CDK4 in complex with cycline A or cycline D1 in relation to their respective role in cell cycling regulation, we examined enzymatic properties of both CDK4/cycline D1 and CDK2/cycline A in vitro. Association constant, Km for ATP in CDK4/cyclin D1 was found as 418 microM, a value unusually high whereas CDK2/cyclin A was 23 microM, a value close to most of other regulatory protein kinases. Turnover value for both CDK4/cyclin D1 and CDK2/cyclin A were estimated as 3.4 and 3.9 min(-1) respectively. Kinetic efficiency estimation indicates far over one order magnitude less efficiency for CDK4/cyclin D1 than the value of CDK2/cycline A (9.3 pM(-1) min(-1) and 170 pM(-1) min(-1) respectively). In addition, inhibition of cellular CDK4 caused increase of cellular levels of ATP, even though inhibition of CDK2 did not change it noticeably. These data suggest cellular CDK4/cyclin D1 activity is tightly associated with cellular ATP concentration. Also, analysis of phosphorylated serine/threonine sites on RB catalyzed by CDK4/cyclin D1 and CDK2/cyclin A showed significant differences in their preference of phosphorylation sites in RB C-terminal domain. Since RB is known to regulate various cellular proteins by binding and this binding is controlled by its phosphorylation, these data shown here clearly indicate significant difference in their biochemical properties between CDK4/cyclin D1 and CDK2/cyclin A affecting regulation of cellular RB function.     

Fragment-based de novo ligand design by multiobjective evolutionary optimization

GANDI (Genetic Algorithm-based de Novo Design of Inhibitors) is a computational tool for automatic fragment-based design of molecules within a protein binding site of known structure. A genetic algorithm and a tabu search act in concert to join predocked fragments with a user-supplied list of fragments. A novel feature of GANDI is the simultaneous optimization of force field energy and a term enforcing 2D-similarity to known inhibitor(s) or 3D-overlap to known binding mode(s). Scaffold hopping can be promoted by tuning the relative weights of these terms. The performance of GANDI is tested on cyclin-dependent kinase 2 (CDK2) using a library of about 14 000 fragments and the binding mode of a known oxindole inhibitor to bias the design. Top ranking GANDI molecules are involved in one to three hydrogen bonds with the backbone polar groups in the hinge region of CDK2, an interaction pattern observed in potent kinase inhibitors. Notably, a GANDI molecule with very favorable predicted binding affinity shares a 2-N-phenyl-1,3-thiazole-2,4-diamine moiety with a known nanomolar inhibitor of CDK2. Importantly, molecules with a favorable GANDI score are synthetic accessible. In fact, eight of the 1809 molecules designed by GANDI for CDK2 are found in the ZINC database of commercially available compounds which also contains about 600 compounds with identical scaffolds as those in the top ranking GANDI molecules.     

Charge optimization of the interface between protein kinases and their ligands

Examining the potential for electrostatic complementarity between a ligand and a receptor is a useful technique for rational drug design, and can demonstrate how a system prioritizes interactions when allowed to optimize its charge distribution. In this computational study, we implemented the previously developed, continuum solvent-based charge optimization theory with a simple, quadratic programming algorithm and the UHBD Poisson-Boltzmann solver. This method allows one to compute the best set of point charges for a ligand or ligand region based on the ligand and receptor shape, and the receptor partial charges, by optimizing the binding free energy obtained from a continuum-solvent model. We applied charge optimization to a fragment of the heat-stable protein kinase inhibitor (PKI) of protein kinase A (PKA), to three flavopiridol inhibitors of CDK2, and to cyclin A which interacts with CDK2 to regulate the cell cycle. We found that a combination of global (involving every charge) and local (involving only charges in a local region) optimization can give useful hints for designing better inhibitors. Although some parts of an inhibitor may already contribute significantly to binding, we found that they could still be the most important targets for modifications to obtain stronger binders. In studying the binding of flavopiridol inhibitors to CDK2, comparable binding affinity could be obtained regardless of whether the net charges of the inhibitors were constrained to -2, -1, 0, 1, or 2 during the optimization. This provides flexibility in inhibitor design when a certain net charge of the inhibitor is desired in addition to strong binding affinity. For the study of the PKA-PKI and CDK2-cyclin A interfaces, we identified residues whose charge distributions are already close to optimal and those whose charge distributions could be refined to further improve binding.     

Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor

Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. We have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monolayer wound healing. Using automated microscopy, we screened a library of approximately 16,000 compounds finding many that affected cell migration or cell morphology as well as compounds that blocked mitotic progression. We tested approximately 200 compounds in a series of subassays and chose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50 of 25 microM ( approximately 5-fold less potent than Y-27632) but has a similar specificity profile. We also profile the wound healing assay with a library of compounds with known bioactivities, revealing multiple pathways involved in the biology.     

High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.     

Discovery of novel Thieno[2,3-d]pyrimidin-4-yl hydrazone-based cyclin-dependent kinase 4 inhibitors: synthesis, biological evaluation and structure-activity relationships

The design, synthesis, and evaluation of novel thieno[2,3-d]pyrimidin-4-yl hydrazone analogues as cyclin-dependent kinase 4 (CDK4) inhibitors are described. In continuing our program aim to search for potent CDK4 inhibitors, the introduction of a thiazole group at the hydrazone part has led to marked enhancement of chemical stability. Furthermore, by focusing on the optimization at the C-4' position of the thiazole ring and the C-6 position of the thieno[2,3-d]pyrimidine moiety, compound 35 has been identified with efficacy in a xenograft model of HCT116 cells. In this paper, the potency, selectivity profile, and structure-activity relationships of our synthetic compounds are discussed.     

Optimized Target Residence Time: Type I Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R‐Spine

Skepinone‐L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP‐competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I binders for p38α MAP kinase. Type I inhibitors interfere with the R‐spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X‐ray crystallography.     

Synthesis and structure-activity relationship of N-arylrolipram derivatives as inhibitors of PDE4 isozymes

Structure activity studies of N-phenylrolipram derivatives have led to the identification of highly potent PDE4 inhibitors. The potential of these inhibitors for cellular activity was routinely assessed in an assay of fMLP induced oxidative burst in human eosinophils. Since first generation PDE4 inhibitors have been plagued with a number of unwanted side effects, parallel structure activity studies for competition with the [3H]-rolipram binding site in rat brain were performed. In this fashion 5-[4-(3-cyclopentyloxy-4-methoxyphenyl)-2-oxo-pyrrolidin-1-yl]-3-(3-methoxybenzyloxy)benzoic acid N',N'-dimethylhydrazide (22) was identified as a potent inhibitor of PDE4 which exhibits >1000 fold selectivity versus PDE3, and is a nanomolar inhibitor in all the cellular assays tested. Studies on the stereoselectivity of PDE4 inhibition of this class of rolipram based compounds revealed, that for example (S)-11 is a more potent inhibitor than (R)-11. This effect can also be observed in primary human cells where the (S)-enantiomer is about 10 fold more potent than the corresponding (R)-enantiomer.     

Predicting kinase selectivity profiles using Free-Wilson QSAR analysis

Kinases are involved in a variety of diseases such as cancer, diabetes, and arthritis. In recent years, many kinase small molecule inhibitors have been developed as potential disease treatments. Despite the recent advances, selectivity remains one of the most challenging aspects in kinase inhibitor design. To interrogate kinase selectivity, a panel of 45 kinase assays has been developed in-house at Pfizer. Here we present an application of in silico quantitative structure activity relationship (QSAR) models to extract rules from this experimental screening data and make reliable selectivity profile predictions for all compounds enumerated from virtual libraries. We also propose the construction of R-group selectivity profiles by deriving their activity contribution against each kinase using QSAR models. Such selectivity profiles can be used to provide better understanding of subtle structure selectivity relationships during kinase inhibitor design.     

ONIOM study of the nonbonding interaction of the 2PU inhibitor with the CDK2 and CDK4 cyclin‐dependant kinases

We discuss in this article, the applicability of hybrid techniques [especially the our‐own N‐layered integrated molecular orbital and molecular mechanics (ONIOM) method] to weak chemical interactions in large systems, such as the interaction of cyclin‐dependant kinases, CDK4, and CDK2, with a specific ligand (2PU) showing selectivity for CDK4. Our results show that the energies from the ONIOM calculations perfectly match our former molecular dynamics results, both for determining the amino acids which have strongest interactions with the ligand and for explaining the selectivity of 2PU towards CDK4, as compared with CDK2. We show that the ONIOM method is a good candidate for studying such interactions in large systems, even though there are still some technical and theoretical problems to solve. The calculation details will be presented together with the methodology we devised for using the ONIOM approach in such a context. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

Synthesis and application of functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors.

BACKGROUND: Purines constitute a structural class of protein ligands involved in mediating an astonishing array of metabolic processes and signal pathways in all living organisms. Synthesis of purine derivatives targeting specific purine-binding proteins in vivo could lead to versatile lead compounds for use as biological probes or drug candidates. RESULTS: We synthesized several libraries of 2,6, 9-trisubstituted purines using both solution- and solid-phase chemistry, and screened the compounds for inhibition of cyclin-dependent kinase (CDK) activity and human leukemic cell growth. Lead compounds were optimized by iterative synthesis based on structure-activity relationships (SARs), as well as analysis of several CDK-inhibitor cocrystal structures, to afford several interesting compounds including one of the most potent CDK inhibitors known to date. Unexpectedly, some compounds with similar CDK inhibitory activity arrested cellular proliferation at distinctly different phases of the cell cycle and another inhibitor directly induced apoptosis, bypassing cell-cycle arrest. Some of these compounds selectively inhibited growth of cells derived from specific tumors. CONCLUSIONS: 2,6,9-Trisubstituted purines have various and potent biological activities, despite high concentrations of competing endogenous purine ligands in living cells. Purine libraries constitute a versatile source of small molecules that affect distinct biochemical pathways mediating different cellular functions.