Philip J. Jewsbury

A review of protein-small molecule docking methods

The binding of small molecule ligands to large protein targets is central to numerous biological processes. The accurate prediction of the binding modes between the ligand and protein, (the docking problem) is of fundamental importance in modern structure-based drug design. An overview of current docking techniques is presented with a description of applications including single docking experiments and the virtual screening of databases.

Cyclin dependent kinase inhibitors

Cyclin-dependent kinases are involved in diverse cellular processes that include cell cycle control, apoptosis, neuronal physiology, differentiation, and transcription. Intensive screening and drug design based on CDK/inhibitor co-crystal structures and on SAR studies have led to the identification and characterization of a large variety of chemical inhibitors of CDKs. Although they all act by competing with ATP for binding at the catalytic site of the kinase, their kinase selectivity varies greatly and remains to be studied in most cases. The requirement for CDKs in many physiological processes justifies their evaluation as potential therapeutic targets against a much larger scope of diseases than initially anticipated.Cell-cycle dysregulation is one of the cardinal characteristics of neoplastic cells. For this reason, small molecule inhibitors targeting cyclin-dependent kinases (CDKs), of which flavopiridol is a prototype, have been the focus of extensive interest in cancer therapy. In addition to inhibiting cell-cycle progression, these agents exhibit a variety of other activities, including the induction of cell death. Recently, several novel mechanisms of action have been ascribed to the CDK inhibitor flavopiridol, including interference with transcription, most likely through disruption of P-TEFb (i.e. the CDK9/cyclin T complex), and induction of apoptosis, possibly a consequence of downregulation of various anti-apoptotic proteins. It has also been observed that combining CDK inhibitors with either conventional cytotoxic drugs or novel signal transduction modulators dramatically promotes neoplastic cell death in a variety of preclinical models. Efforts are underway to uncover inhibitors that selectively target specific CDKs and to develop these as a new generation of antitumour drugs. For all of these reasons, it is likely that interest in CDK inhibitors as antineoplastic agents will continue for the foreseeable future.

Structure-Based Design of a Potent Purine-Based Cyclin-Dependent Kinase Inhibitor

Aberrant control of cyclin-dependent kinases (CDKs) is a central feature of the molecular pathology of cancer. Iterative structure-based design was used to optimize the ATP- competitive inhibition of CDK1 and CDK2 by O6-cyclohexylmethylguanines, resulting in O6-cyclohexylmethyl-2-(4′- sulfamoylanilino)purine. The new inhibitor is 1,000-fold more potent than the parent compound (Ki values for CDK1 = 9 nM and CDK2 = 6 nM versus 5,000 nM and 12,000 nM, respectively, for O6-cyclohexylmethylguanine). The increased potency arises primarily from the formation of two additional hydrogen bonds between the inhibitor and Asp 86 of CDK2, which facilitate optimum hydrophobic packing of the anilino group with the specificity surface of CDK2. Cellular studies with O6-cyclohexylmethyl-2-(4′- sulfamoylanilino) purine demonstrated inhibition of MCF-7 cell growth and target protein phosphorylation, consistent with CDK1 and CDK2 inhibition. The work represents the first successful iterative synthesis of a potent CDK inhibitor based on the structure of fully activated CDK2–cyclin A. Furthermore, the potency of O6-cyclohexylmethyl-2-(4′- sulfamoylanilino)purine was both predicted and fully rationalized on the basis of protein–ligand interactions.

Imidazo[1,2-A]Pyridines: A Potent and Selective Class of Cyclin-Dependent Kinase Inhibitors Identified Through Structure-Based Hybridisation

High-throughput screening identified the imidazo[1,2-a]pyridine and bisanilinopyrimidine series as inhibitors of the cyclin-dependent kinase CDK4. Comparison of their experimentally-determined binding modes and emerging structure-activity trends led to the development of potent and selective imidazo[1,2-a]pyridine inhibitors for CDK4 and in particular CDK2.

Discovery of 4-{4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity.

ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways. We describe the discovery and synthesis of a series of sulfonylmorpholinopyrimidines that show potent and selective ATR inhibition. Optimization from a high quality screening hit within tight SAR space led to compound 6 (AZ20) which inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC50 of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC50 of 50 nM. Compound 6 potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses 6 leads to significant growth inhibition of LoVo xenografts grown in nude mice. Compound 6 is a useful compound to explore ATR pharmacology in vivo.

Cyclin-Dependent Kinase 4 Inhibitors as a Treatment for Cancer. Part 1: Identification and Optimisation of Substituted 4,6-Bis Anilino Pyrimidines

Using a high-throughput screening campaign, we identified the 4,6-bis anilino pyrimidines as inhibitors of the cyclin-dependent kinase, CDK4. Herein we describe the further chemical modification and use of X-ray crystallography to develop potent and selective in vitro inhibitors of CDK4.

FDS: Flexible ligand and receptor docking with a continuum solvent model and soft‐core energy function

The docking of flexible small molecule ligands to large flexible protein targets is addressed in this article using a two‐stage simulation‐based method. The methodology presented is a hybrid approach where the first component is a dock of the ligand to the protein binding site, based on deriving sets of simultaneously satisfied intermolecular hydrogen bonds using graph theory and a recursive distance geometry algorithm. The output structures are reduced in number by cluster analysis based on distance similarities. These structures are submitted to a modified Monte Carlo algorithm using the AMBER‐AA molecular mechanics force field with the Generalized Born/Surface Area (GB/SA) continuum model. This solvent model is not only less expensive than an explicit representation, but also yields increased sampling. Sampling is also increased using a rotamer library to direct some of the protein side‐chain movements along with large dihedral moves. Finally, a softening function for the nonbonded force field terms is used, enabling the potential energy function to be slowly turned on throughout the course of the simulation. The docking procedure is optimized, and the results are presented for a single complex of the arabinose binding protein. It was found that for a rigid receptor model, the X‐ray binding geometry was reproduced and uniquely identified based on the associated potential energy. However, when side‐chain flexibility was included, although the X‐ray structure was identified, it was one of three possible binding geometries that were energetically indistinguishable. These results suggest that on relaxing the constraint on receptor flexibility, the docking energy hypersurface changes from being funnel‐like to rugged. A further 14 complexes were then examined using the optimized protocol. For each complex the docking methodology was tested for a fully flexible ligand, both with and without protein side‐chain flexibility. For the rigid protein docking, 13 out of the 15 test cases were able to find the experimental binding mode; this number was reduced to 11 for the flexible protein docking. However, of these 11, in the majority of cases the experimental binding mode was not uniquely identified, but was present in a cluster of low energy structures that were energetically indistinguishable. These results not only support the presence of a rugged docking energy hypersurface, but also suggest that it may be necessary to consider the possibility of more than one binding conformation during ligand optimization.

Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo

A nanoparticle formulation of an Aurora B kinase inhibitor uses ion pairing to achieve controlled release and efficacious, nontoxic target inhibition in tumors. Accurin nanoparticles dutifully deliver drug A class of drugs, called kinase inhibitors, could stop cancer in its tracks…if only these drugs could reach the tumors, stay for a while, and not be toxic. Hypothesizing that a nanoparticle formulation would solve the inhibitors’ woes, Ashton and colleagues investigated several different compositions of so-called Accurins—polymeric particles that encapsulate charged drugs through ion pairing. An Aurora B kinase, once formulated in Accurins, demonstrated a much-improved therapeutic index and preclinical efficacy compared with its parent molecule, when administered to rats and mice bearing human tumors. The Accurins allowed for sustained release of the drug over days, and did not have the same blood toxicity seen with the parent drug. A phase 1 trial is the next step for this nanomedicine, and additional preclinical studies will reveal whether such nanoformulations can improve the tolerability and efficacy of the broader class of molecularly targeted cancer therapeutics, including cell cycle inhibitors. Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans.

4-Alkoxy-2,6-diaminopyrimidine derivatives: Inhibitors of cyclin dependent kinases 1 and 2

The cyclin dependent kinase (cdk) inhibitor NU6027, 4-cyclohexylmethoxy-5-nitroso-pyrimidine-2,6-diamine (IC(50) vs cdk1/cyclinB1=2.9+/-0.1 microM and IC(50) vs cdk2/cyclinA3=2.2+/-0.6 microM), was used as the basis for the design of a series of 4-alkoxy-2,6-diamino-5-nitrosopyrimidine derivatives. The synthesis and evaluation of 21 compounds as potential inhibitors of cyclin-dependent kinases 1 and 2 is described and the structure-activity relationships relating to NU6027 have been probed. Simple alkoxy- or cycloalkoxy-groups at the O(4)-position were tolerated, with the 4-(2-methylbutoxy)-derivative (IC(50) vs cdk1/cyclinB1=12+/-2 microM and cdk2/cyclinA3=13+/-4 microM) retaining significant activity. Substitutions at the N(6) position were not tolerated. Replacement of the 5-nitroso substituent with ketone, oxime and semicarbazone groups essentially abolished activity. However, the derivative bearing an isosteric 5-formyl group, 2,6-diamino-4-cyclohexylmethoxy-pyrimidine-5-carbaldehyde, showed modest activity (IC(50) vs cdk1/cyclinB1=35+/-3 microM and cdk2/cyclinA3=43+/-3 microM). The X-ray crystal structure of the 5-formyl compound bound to cdk2 has been determined to 2.3A resolution. The intramolecular H-bond deduced from the structure with NU6027 bound to cdk2 is not evident in the structure with the corresponding formyl compound. Thus the parent compound, 4-cyclohexylmethoxy-5-nitrosopyrimidine-2,6-diamine (NU6027), remains the optimal basis for future structure-activity studies for cyclin-dependent kinase inhibitors in this series.

REPLACE: a strategy for iterative design of cyclin-binding groove inhibitors.

We describe a drug‐design strategy termed REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) in which nonpeptidic surrogates for specific determinants of known peptide ligands are identified in silico by using a core peptide‐bound protein structure as a design anchor. In the REPLACE application example, we present the effective replacement of two critical binding motifs in a lead protein–protein interaction inhibitor pentapeptide with more druglike phenyltriazole and diphenyl ether groups. These were identified through docking of fragment libraries into the volume of the cyclin‐binding groove of CDK2/cyclin A vacated through truncation of the inhibitor peptide‐binding determinants. Proof of concept for this strategy was obtained through the generation of potent peptide–small‐molecule hybrids and by the confirmation of inhibitor‐binding modes in X‐ray crystal structures. This method therefore allows nonpeptide fragments to be identified without the requirement for a high‐sensitivity binding assay and should be generally applicable in replacing amino acids as individual residues or groups in peptide inhibitors to generate pharmaceutically acceptable lead molecules.