Cyclin-dependent protein kinases as therapeutic drug targets for antimalarial drug development

Cyclin-dependent protein kinases (CDKs) have been attractive drug targets for the development of anticancer therapies due to their direct and crucial role in the regulation of cellular proliferation. Following this trend, CDKs have been pursued as potential drug targets for several other diseases. Structure-based drug design programmes have focused on the plasmodial CDKs to develop new candidate antimalarial compounds. This review discusses the most recent advances relating to three Plasmodium falciparum CDKs (PfPK5, PfPK6 and Pfmrk) as they are developed as antimalarial drug targets. CDKs are highly conserved, and focus must be placed upon the amino acid differences between human and plasmodial CDKs in order to develop specific inhibitors. Comparisons of the active sites of human and parasite CDKs reveal sequence and potential structural variations. Using sequence analysis, molecular modelling and in vitro drug screening, it is possible to identify and develop inhibitors that specifically target the plasmodial CDKs. These efforts are aimed at identifying new classes of CDK inhibitors that may be exploited for antimalarial drug development.     

A three-dimensional in silico pharmacophore model for inhibition of Plasmodium falciparum cyclin-dependent kinases and discovery of different classes of novel Pfmrk specific inhibitors

The cell division cycle is regulated by a family of cyclin-dependent protein kinases (CDKs) that are functionally conserved among many eukaryotic species. The characterization of plasmodial CDKs has identified them as a leading antimalarial drug target in our laboratory. We have developed a three-dimensional QSAR pharmacophore model for inhibition of a Plasmodium falciparum CDK, known as Pfmrk, from a set of fifteen structurally diverse kinase inhibitors with a wide range of activity. The model was found to contain two hydrogen bond acceptor functions and two hydrophobic sites including one aromatic-ring hydrophobic site. Although the model was not developed from X-ray structural analysis of the known CDK2 structure, it is consistent with the structure-functional requirements for binding of the CDK inhibitors in the ATP binding pocket. Using the model as a template, a search of the in-house three-dimensional multiconformer database resulted in the discovery of sixteen potent Pfmrk inhibitors. The predicted inhibitory activities of some of these Pfmrk inhibitors from the molecular model agree exceptionally well with the experimental inhibitory values from the in vitro CDK assay.     

Oxindole-based compounds are selective inhibitors of Plasmodium falciparum cyclin dependent protein kinases

Cyclin dependent protein kinases (CDKs) have become attractive drug targets in an effort to identify effective inhibitors of the parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria. We tested known CDK inhibitors for their ability to inhibit two malarial CDKs: Pfmrk and PfPK5. Many broad spectrum CDK inhibitors failed to inhibit Pfmrk suggesting that the active site differs from other CDKs in important ways. By screening compounds in the Walter Reed chemical database, we identified oxindole-based compounds as effective inhibitors of Pfmrk (IC(50) = 1.5 microM). These compounds have low cross-reactivity against PfPK5 and human CDK1 demonstrating selectivity for Pfmrk. Amino acid comparison of the active sites of Pfmrk and PfPK5 identified unique amino acid differences that may explain this selectivity and be exploited for further drug development efforts.     

Cell cycle molecular targets in novel anticancer drug discovery

A number of potential molecular targets for novel anticancer drug discovery have been identified in cell cycle control mechanisms. Prominent among these are the regulatory proteins, cyclins and their effector counterparts the cyclin dependent kinases (CDKs). Aberrant expression of these proteins, particularly cyclins involved in the G1 phase of the cell cycle, namely the D and E cyclins, has been associated with a variety of human cancers, including breast and colorectal cancer, B-lymphoma, prostate and non-small cell lung cancer. Inhibition of CDK kinase activity has turned out to be the most productive strategy for the discovery and design novel anticancer agents specifically targeting the cell cycle. Other potentially useful cell cycle areas for exploration include cyclin-CDK interactions, Cdc25 activation of cyclin-CDK complexes, ubiquitin-mediated proteolysis of cyclins, cell cycle check point kinases like Chk1, and recently identified oncogenic cell cycle-related aurora and polo-like kinases. Potent specific inhibitors have been identified that bind to the ATP site of CDKs, mainly cyclin B-CDK1, cyclin A-CDK2, and cyclin D-CDK4 complexes, and inhibit kinase activity. X-ray crystallographic data of CDKs, and their complexes with inhibitors have played a major role in the success of drug discovery efforts. Combinatorial chemistry, highthroughput screening, functional genomics and informatics have also contributed. CDK inhibitors currently under investigation include flavopiridol, olomoucine, roscovitine, puvalanol B, the dihydroindolo[3,2-d][1]benzazepinone kenpaullone, indirubin-3 -monoxime and novel diaminothiazoles such as AG12275. The anticancer therapeutic potential of CDK inhibitors has been demonstrated in preclinical studies, and Phases I and II clinical trials in cancer patients are currently underway.     

Advances in the identification of β-secretase inhibitors

Introduction: Alzheimer's disease (AD), which is characterized by progressive intellectual deterioration, is the most common cause of dementia. β-Secretase (or BACE1) expression is a trigger for amyloid β peptide formation, a cause of AD, and thus is a molecular target for the development of drugs against AD. Many BACE1 inhibitors have been identified by academic and pharmaceutical research groups and a number of advanced technologies in drug discovery have been applied to the drug discovery.

Areas covered: The purpose of this review is to present and discuss the methodologies used for BACE1 inhibitor drug discovery via substrate- and structure-based design, high-throughput screening and fragment-based drug design. The authors also review the advantages and disadvantages of these methodologies.

Expert opinion: Many BACE1 inhibitors have been designed using X-ray crystal structure-based drug design as well as through in silico screening. Nevertheless, there are serious problems with regards to deciding the best X-ray crystal structure for designing BACE1 inhibitors through computational approaches. There are two prominent configurations of BACE1 but there is still room for improvement. Future developments may make it possible to identify BACE1 inhibitors as potential drug candidates.     

Inhibitors of cyclin dependent kinases: useful targets for cancer treatment

Cancer drug discovery is one of the most rapidly changing areas of pharmaceutical research. Uncontrolled proliferation is a hallmark of cancer cells. Over the past two decades, it has become increasingly clear that in many human cancers, hyperactivity of Cyclin Dependent Kinases (CDKs) is one of the mechanisms underlying the physiological hyper-proliferation. CDKs are serine/threonine protein kinases, which play an important role in cell-cycle regulation. Their sequential activation ensures, the correct timing and ordering of events required for cell cycle progression. Therefore, inhibition of CDKs, through the insertion of small molecules into its ATP binding pocket has emerged as a potential therapy method for cancers. Consequently, a number of small molecules with CDK inhibitory properties have been developed. Many of these have been evaluated as potent inhibitors and some are currently in clinical-trials for various types of cancer. This review reports various CDK inhibitors, natural as well as small molecules, along with their reported activities for various CDKs. It will highlight the potential for the development of novel anti-cancer molecules.     

Novel approaches in antimalarial drug discovery

Introduction: The development of new antimalarial drugs remains of the utmost importance, since Plasmodium falciparum has developed resistance against nearly all chemotherapeutics in clinical use. In an effort to contain the resistance of P. falciparum against artemisinins and to further eradication efforts, studies are ongoing to identify novel and more efficacious approaches to develop antimalarials.

Areas covered: The authors review the classical and new approaches to antimalarial drug discovery, with a special emphasis on the various stages of the parasite's life cycle and the different Plasmodium species. The authors discuss the methodologies and strategies for early efficacy testing that aim to narrow down the portfolio of promising compounds.

Expert opinion: The increased efforts in the discovery and development of new antimalarial compounds have led to the recognition of new promising hits. However, there is still major roadblock of selecting the most promising compounds and then further testing them in early clinical trials, especially in the current restricted economy. Controlled human malaria infection has much potential for speeding-up the early development process of many drug candidates including those which target the pre-erythrocytic stages.     

ATP-noncompetitive CDK inhibitors for cancer therapy: an overview

Introduction: Cyclin-dependent kinases (CDKs) are the key drivers of cell cycle progression and are often deregulated in cancer, therefore, targeting CDKs has long been pursued as a therapeutic strategy to tackle cancer. Unfortunately, however, none of the first-generation CDK inhibitors has yielded the expected efficacy to be successfully translated to the clinic mostly because, by targeting the very conserved kinase ATP-binding site resulted to be poorly specific and quite toxic.

Areas covered: Here, the authors review recent approaches aimed at developing more specific CDK inhibitors mostly through the aid of computational drug design studies and report various small molecules and peptides, which resulted in promising CDK ATP-noncompetitive inhibitors.

Expert opinion: Despite few successes, these new approaches still need additional considerations to generate effective antitumoral agents. The authors discuss some of the hurdles to overcome for a successful clinical translation.     

The hunt for antimitotic agents: an overview of structure-based design strategies

ABSTRACT

Introduction: Structure-based drug discovery offers a rational approach for the design and development of novel anti-mitotic agents which target specific proteins involved in mitosis. This strategy has paved the way for development of a new generation of chemotypes which selectively interfere with the target proteins. The interference of these anti-mitotic targets implicated in diverse stages of mitotic cell cycle progression culminates in cancer cell apoptosis.

Areas covered: This review covers the various mitotic inhibitors developed against validated mitotic checkpoint protein targets using structure-based design and optimization strategies. The protein-ligand interactions and the insights gained from these studies, culminating in the development of more potent and selective inhibitors, have been presented.

Expert opinion: The advent of structure-based drug design coupled with advances in X-ray crystallography has revolutionized the discovery of candidate lead molecules. The structural insights gleaned from the co-complex protein-drug interactions have provided a new dimension in the design of anti-mitotic molecules to develop drugs with a higher selectivity and specificity profile. Targeting non-catalytic domains has provided an alternate approach to address cross-reactivity and broad selectivity among kinase inhibitors. The elucidation of structures of emerging mitotic drug targets has opened avenues for the design of inhibitors that target cancer.     

Structure-activity relationships and inhibitory effects of various purine derivatives on the in vitro growth of Plasmodium falciparum.

The development of novel chemotherapeutic agents has become an urgent task due to the development and rapid spread of drug resistance in Plasmodium falciparum, the protozoan parasite responsible for cerebral malaria. Cyclin-dependent kinases (CDKs) are essential for the regulation of the eukaryotic cell cycle, and several enzymes of this family have been identified in P. falciparum. In recent years, a number of purine-derived kinase inhibitors have been synthesised, some of which display selective activity against CDKs. This report describes a study in which various purine derivatives were screened for in vitro antimalarial activity. The erythrocytic asexual stages of the chloroquine-resistant P. falciparum strain (FCR-3) were cultivated in vitro in the presence of the various purines, and their effect on parasite proliferation was determined by the [3H]hypoxanthine incorporation assay. Our results show considerable variation in the sensitivity of P. falciparum to the different purines, as well as a general independence from their effect on purified starfish CDK1/cyclin B activity, which has been the standard assay used to identify CDK-specific inhibitors. Two subfamilies of purines with moderate to poor activity against CDK1/cyclin B activity showed submicromolar activity against P. falciparum. Structure-activity analysis indicates that certain structural features are associated with increased activity against P. falciparum. These features can be exploited to synthesise compounds with higher activity and specificity towards P. falciparum.     

Discovery methodology for the development of direct factor VIIa inhibitors

Introduction: Heparin and warfarin have historically been the only antithrombotics available. Recently, however, newer anticoagulants have been developed. Factor VIIa (fVIIa) inhibitors represent one of the new and potentially exciting classes of anticoagulants currently under development. Indeed, several methodologies have been used to develop fVIIa inhibitors.

Areas covered: The authors highlight some of the methologies applied for the discovery of fVIIa inhibitors including phage display, isolation of endogenous peptides from hematophagous animals and the use of the 1,5-benzothiazepine molecular scaffolds and screens of large chemical libraries previously used to identify other serine protease inhibitors. Although these screens were intended to identify thrombin and factor Xa inhibitors, the compounds often had concomitant fVIIa activity. The authors also discuss the utilization of medical chemistry techniques for the discovery of these compounds.

Expert opinion: FVIIa inhibitors represent a viable option for the development of new anticoagulants. There are theoretical advantages that fVIIa inhibitors may possess over existing anticoagulants and highly specific inhibitors that possess oral bioavailability and low bleeding risk may succeed.     

Toward understanding the structural basis of cyclin-dependent kinase 6 specific inhibition

Cyclin-dependent kinases (CDKs) are key players in cell cycle control, and genetic alterations of CDKs and their regulators have been linked to a variety of cancers. Hence, CDKs are obvious targets for therapeutic intervention in various proliferative diseases, including cancer. To date, drug design efforts have mostly focused on CDK2 because methods for crystallization of its inhibitor complexes have been well established. CDK4 and CDK6, however, may be at least as important as enzymes for cell cycle regulation and could provide alternative treatment options. We describe here two complex structures of human CDK6 with a very specific kinase inhibitor, PD0332991, which is based on a pyrido[2,3-d]pyrimidin-7-one scaffold, and with the less specific aminopurvalanol inhibitor. Analysis of the structures suggests that relatively small conformational differences between CDK2 and CDK6 in the hinge region are contributing to the inhibitor specificity by inducing changes in the inhibitor orientation that lead to sterical clashes in CDK2 but not CDK6. These complex structures provide valuable insights for the future development of CDK-specific inhibitors.     

Development of novel benzotriazines for drug discovery

Background: The synthesis of novel benzotriazine heterocycles was developed independently around the same time by Bischler, Bamberger and Arndt. Over the years, different groups have reported the synthesis of benzotriazine based compounds. Objective: This literature review gives an update on recent benzotriazine compounds and their applications. Conclusion: The benzotriazine core has been used in various drug discovery projects including anticancer, anti-inflammatory and antimalarial programs. Recently, the benzotriazine core was used to develop selective kinase inhibitors targeting SRC, VEGFr2, BCR-ABL and BCR-ABL-T315I. Two benzotriazine based compounds, tirapazamine for the treatment of cancer and TG100801 for the treatment of age-related macular degeneration, have entered clinical trials.     

Cell cycle inhibitors for the treatment of NSCLC

Introduction: Lung cancer remains to be the leading cause of cancer-related death worldwide. Treatment of lung cancer still poses a significant challenge. Cell cycle is a tightly integrated process and is frequently aberrant in lung cancer. Cell cycle inhibitors have emerged as novel therapeutics, in anticipation of overcoming the unrestricted cell division and growth in lung cancer.

Areas covered: In this article, we first address the potential roles of cell cycle proteins and cell cycle deregulation in the development of lung cancer. The review then provides an overview for several major categories of cell cycle inhibitors with particular attention to their tolerability and disease control in early phases of lung cancer trials.

Expert opinion: Targeted agents against different components of cell cycle regulation, such as cyclin-dependent kinase, polo-like kinase, checkpoint kinase and aurora kinase, are currently in clinical development for lung cancer management. Their clinical benefits remain to be defined. When evaluated as single agents in lung cancer, cell cycle inhibitors are often associated with limited clinical activity and tolerable toxicities. The key challenges in the drug development are to understand resistance mechanisms and to identify predictive biomarkers that can potentially guide patient selection and optimize the utility of these targeted inhibitors.     

Advances in predicting CYP-mediated drug interactions in the drug discovery setting

Advances in high-throughput screening methodologies, biological reagents and in silico techniques relating to cytochrome p450 (CYP)-mediated drug–drug interactions have led to reduced clinical attrition rates and to the development of safer therapeutics. Greater understanding of the impact of genetic variability and CYP induction on drug interactions, particularly for low therapeutic index drugs, has facilitated improved clinical study design. This review outlines recent developments using in vitro and in silico technologies to study CYP-mediated drug interactions and describes how those tools have been combined to drive improved candidate selection and in vivo predictions early in the drug discovery process.     

Inhibitors of cyclin-dependent kinases as anti-cancer therapeutics

Initiation, progression, and completion of the cell cycle are regulated by various cyclin-dependent kinases (CDKs), which are thus critical for cell growth. Tumour development is closely associated with genetic alteration and deregulation of CDKs and their regulators, suggesting that inhibitors of CDKs may be useful anti-cancer therapeutics. Indeed, early results suggest that transformed and normal cells differ in their requirement for e.g. cyclin/CDK2 and that it may be possible to develop novel antineoplastic agents devoid of the general host toxicity observed with conventional cystostatic drugs. Numerous active-site inhibitors of CDKs have been studied; the main limitation with these ATP antagonists is kinase specificity for CDKs. However, screening of compound collections, as well as rational design based on enzyme-ligand complex crystal structures, are now yielding pre-clinical candidates, particularly certain purine and flavonoid analogues, with impressive potency and selectivity. Natural CDK inhibitors (CKIs), e.g. the tumour suppressor gene products p16(INK4), p21(WAF1), and p27(KIP1), form the starting point for the design of mechanism-based CDK inhibitors. A number of these small proteins have been dissected and inhibitory lead peptides amenable to peptidomimetic development have been identified. Conversion of these peptides into pharmaceutically useful molecules is greatly aided by the recent elucidation of CKI/CDK crystal and solution structures. Additional interaction sites on CDKs being exploited for the purposes of inhibitor design include: phosphorylation/dephosphorylation sites, macromolecular substrate binding site, CKS regulatory subunit binding sites, cyclin-binding site, cellular localisation domain, and destruction box. Finally, progress has recently been made in the application of antisense technology in order to target CDK activity.