Current Medicinal Chemistry - Volume 11, Issue 6, 2004

Recent years have seen an explosion in the number of publicly available x-ray crystal structures of protein kinases. These structures have provided a wealth of information on the regulatory mechanisms, conformational plasticity and drugability of this important family of enzymes. Drawing upon structural information, new insights into the development of protein kinase inhibitors are discussed including de-novo design, molecular templates for ATP competitive inhibitors and alternative mechanisms of inhibition. The highly conserved nature of the ATP binding site is of central concern to drug development and the concept of a selectivity profile has arisen with structure-based design emerging as a key tool for addressing the challenges of specificity. In addition, protein-ligand complexes, where the enzyme is in an inactive conformation, signify an alternate approach to protein kinase inhibition. The belief that an inactive kinase presents a less conserved target is reviewed using observations on the structural changes occurring during protein kinase regulation.

A stochastic algorithm for constructing multiple loops in homology modeling of proteins is presented. The algorithm discards variable values in iterations based on a cost function and on statistical analysis of results. Values that remain are used for constructing an ensemble of best solutions. In test cases, the stochastic algorithm retains all the best solutions, compared to an exhaustive scan of the full set of conformations. Individual loops are constructed by adding dipeptide units. Dipeptide conformations are extracted from a database of proteins and their conformations include bond lengths and all angles. Single loops are constructed from both N- and C- terminals to the center, and loop closure is evaluated by a combination of penalties for the peptide closure and Miyazawa-Jernigan (MJ) [1] residue-residue interactions with the rest of the protein. Large ensembles of each loop are clustered and re-evaluated with a refined [2] energy term. The reduced, clustered set of each loop is then employed to construct simultaneously all the loops. The algorithm was applied to construct simultaneously six loops in c-Src kinase family proteins, incorporating a total of 37-40 residues. The best RMSD for reconstructing the loops is 1.45Å for Lck (structure 1QPE in the Protein Data Bank) and 2.54Å for human c-Src (structure 1FMK). The multiple loop conformations with lowest energy have higher RMSD values, of 2.06Å and 3.09Å, respectively. The average RMSD values for the first 1000 conformations are 3.00Å and 3.46Å, respectively. Models for the “open” structures of c-Src and of Jak-2 were constructed on the basis of 1QPE. The Jak-2 model is found to be more flexible in the loops region than its c- Src counterpart.

Kinases have become a major area of drug discovery and structure-based design. Hundreds of 3D structures for more than thirty different kinases are available to the public. High structural and sequence homology within the kinase gene family makes the remaining kinases ideal targets for homology modeling and virtual screening. Somewhat surprisingly, however, the number of publications about virtual screening of kinases is very low. Therefore, rather than reviewing the field of virtual screening for kinases, we attempt here a hybrid approach of presenting what is known and common practice together with new studies on CDK2 and SRC kinase. To illustrate the challenges and pitfalls of virtual screening for kinase targets we focus on the question of how ranking is influenced by the database screened, the docking scheme, the scoring function, the activity of the compounds used for testing, and small changes in the binding pocket. In addition, a case study of finding irreversible inhibitors of ErbB2 through in silico screening is presented.

Small molecule inhibitors of KDR kinase activity have typically possessed poor intrinsic physical properties including low aqueous solubility and high lipophilicity. These features have often conferred limited cell permeability manifested in low levels of cell-based KDR inhibitory activity and oral bioavailability. Thus, the design of inhibitors with appropriate physical properties has played a critical role in the development of clinical candidates. We present a variety of structural modifications that have afforded improvements in physical properties and thereby have addressed suboptimal cellular potency and pharmacokinetics for three unique classes of KDR kinase inhibitors.

Kinases represents one of the most important family of targets in high throughput drug screening. Tyrosine kinases and serine / threonine kinases are known to play key roles in signal transduction as well as in cell growth and differentiation. Intense screening campaigns are underway in all major pharmaceuticals and large biotech companies to find kinase inhibitors for the treatment of inflammatory diseases, immunological disorders and cancer. The present contribution describes models that were developed to produce kinase assays amenable to HTS using AlphaScreen. Because of the flexibility allowed by AlphaScreen, kinase assays can be developed using direct or indirect approaches. Tyrosine kinase assays are usually performed with a direct format involving generic anti-phosphotyrosine antibodies while serine / threonine kinase assays are performed with an indirect format where specific antibodies are captured using protein A conjugated Acceptor beads. Streptavidin-coated Donor beads are used to capture either generic (ex. poly GT) or specific biotinylated substrates. Herein, are presented different methods to perform screening for inhibitors acting on the soluble β- insulin receptor tyrosine kinase (IRKD), and on p38, a member of the MAP kinase family.

Among the known angiogenic growth factors and cytokines implicated in the modulation of normal and pathological angiogenesis, the VEGF family (VEGF-A, VEGF-B, VEGF-C, VEGF-D) and their corresponding receptor tyrosine kinases [VEGFR-1 (Flt-1), VEGFR-2 (Flk-1, KDR), and VEGFR-3 (Flt-4)] play a paramount and indispensable role in regulating the multiple facets of the angiogenic and lymphangiogenic processes, as well as the induction of vascular permeability and inflammation. The receptor VEGFR-2 / KDR is the principal one through which VEGFs exert their mitogenic, chemotactic, and vascular permeabilizing effects on the host vasculature. Increased expression of VEGFs by tumor cells and VEGFR-2 / KDR and VEGFR-1 / Flt-1 by the tumor-associated vasculature are a hallmark of a variety of human and rodent tumors in vivo and correlates with tumor growth rate, micro-vessel density / proliferation, tumor metastatic potential, and poorer patient prognosis in a variety of malignancies. Approaches to disrupting the VEGF / VEGFR signaling cascade range from biological agents (soluble receptors, anti-VEGF and anti-VEGFR-2 antibodies, and VEGF transcription inhibitors) to small molecule ATP competitive VEGFR inhibitors. Examples from this latter class that are currently in clinical development include compounds from distinct chemical classes such as: indolin-2-ones, anilinoquinazolines, anilinophthalazines, isothiazoles, indolo- and indenocarbazoles. The structure activity relationships, biochemical and pharmacological profile of optimized representatives from each of these classes constitute the subject matter of this review.

We have developed a series of 4-thiophenoxy-N-(3,4,5-trialkoxyphenyl) pyrimidine-2-amines as potent and selective inhibitors of p56lck tyrosine kinase activity. In particular, the most potent inhibitor shows cellular activity in T-cell receptor (TCR) stimulated models of cytokine release, which suggests an immunomodulatory role for this class of inhibitor.

Glycogen synthase kinase 3 (GSK-3) in the 21st century emerged as one of the most attractive therapeutic target for the development of selective inhibitors as new promising drugs for unmet pathologies including Alzheimer's disease, stroke, bipolar disorders, chronic inflammatory processes, cancer and diabetes type II. The full potential of GSK-3 inhibitors is just starting to be realized but the number of candidates in development provided by both academic centres and pharmaceutical companies have increased exponentially in the last two years. This review discloses recent discoveries and developments on peptides and small molecules targeting GSK-3. Focusing attention on this exciting target could thus reap considerable clinical and economic rewards.

Two-component signal transduction systems and their expanded variants known as phosphorelays are integral elements of the virulence and antimicrobial resistance responses of a wide range of pathogenic bacteria and fungi and also regulate essential functions. As a consequence, two-component systems and phosphorelays are recognized targets for the development of novel antimicrobial agents and a number of chemically synthesized inhibitors from different chemical classes have been identified by compound library screens. However, in the majority of cases these compounds do not appear to be selective for signal transduction pathways and exert their effect by multiple mechanisms of action. The key to designing molecules to selectively disrupt signal transduction may lie with the conserved features of response regulators and the structural analysis of complexes of signaling proteins.

Antimicrobial resistance in hospital and community settings is growing at an alarming rate and has been attributed to such organisms as methicillin-resistant staphylococcus aureus, staphylococci with decreased susceptibility to vancomycin, vancomycin-resistant enterococci, multi-drug resistant pseudomonas spp., klebsiella spp., enterobacter spp, and acinetobacter spp., as well as Streptococcus pneumoniae with decreased susceptibility to penicillin and other antibacterials. To address the need for new therapies to combat resistant organisms, drug companies are refocusing their discovery efforts on developing novel agents with new mechanisms of action. The hope is that rapidly emerging technologies including combinatorial chemistry, high throughput screening, proteomics and microbial genomics will have a positive impact on antimicrobial drug discovery. These technologies should aid in the identification of novel drug targets and compounds with unique mechanisms of action other than those currently provided by the traditional antibiotics. Nucleosides are one class of compounds worthy of further investigation as antibacterials since some derivatives have shown moderate to good activity against specific bacterial strains. For example, 5'-peptidyl nucleoside derivatives can inhibit peptide deformylase, an enzyme essential for bacterial survival that is not vital to human cells. This review also includes a list of miscellaneous nucleosides that have been synthesized as potential antibacterials. More detailed investigations on structure, as it relates to the antimicrobial activity of the various classes of nucleosides, need to be conducted in order to maximize the potential of developing a potent nucleoside for the treatment of bacterial infections. This review begins with an introduction to terms followed by discussions regarding the general background and relevance for developing novel antimicrobial agents. Challenges facing the antimicrobial drug discovery process are discussed along with relevant drug targets. An overview of nucleoside chemistry as it relates to antimicrobial activity is presented, followed by a discussion of the evidence which supports the potential of this class of compounds to yield the novel antimicrobial therapies needed in the new millennium.

Obesity and type II diabetes are closely related metabolic diseases with an increasing incidence worldwide. No clear-cut pharmacological treatment for these complex metabolic disturbances is available despite current efforts. New directions and perspectives for the pharmacological or nutritional treatment of these diseases should be defined. In recent years, a growing body of evidence shows that retinoids and retinoic acid receptors are involved in the control of biological aspects (e.g. adiposity and energy expenditure mechanisms), which offers great potential for research on the treatment of obesity and type II diabetes. All-trans retinoic acid is known to inhibit adipocyte differentiation, whereas, molecules activating the retinoid Xreceptor (rexinoids) promote the differentiation of adipocytes. Treatment with rexinoids ameliorates glycemic control in rodent models of type II diabetes and obesity, although other findings indicate similar positive effects by inhibiting the receptor. Moreover, natural products of dietary origin, such as phytanic acid can activate RXR and thus, trigger adipose cell differentiation. Finally, the activation of retinoic acid receptors or retinoid X receptors has been reported to induce the gene expression of uncoupling proteins, which are mitochondrial proteins involved in the regulation of energy expenditure and fatty acid metabolism. Further research is required to exploit the capacities of the retinoid-dependent pathways of regulation of adiposity, insulin sensitivity and energy expenditure for drug development in metabolic disturbances.