Frontiers in Medicinal Chemistry

The viral enzyme, HIV integrase (MW 32 kDa), is one of the three key enzymes of the pol gene of HIV. HIV integrase is involved in the integration of HIV DNA into host chromosomal DNA. There is apparently no functional equivalent of this enzyme in human cells. Integration of HIV DNA into the host cell genome apparently occurs by a carefully defined sequence of DNA tailoring (3'-processing) and coupling (joining or integration) reactions. In spite of some effort in this area targeted at the discovery of therapeutically useful inhibitors of this viral enzyme, there are no drugs for HIV/AIDS in clinical use where the mechanism of action is inhibition of HIV integrase. It is clear that new knowledge on inhibitors of this enzyme is of critical importance in the anti-HIV drug discovery area. This review focuses on the major classes of compounds that have been discovered as inhibitors of HIV integrase. Some of these compounds are nonspecific inhibitors of the enzyme while evidence suggests that others may possess some specificity. The various classes include nucleotides, oligonucleotides, dinucleotides, and miscellaneous small molecules including heterocyclic systems, natural products, diketo acids and sulfones. A major focus of the review is on discoveries from my laboratory in the area of non-natural, nuclease-resistant dinucleotide inhibitors of HIV integrase.

Macrolides are among the most clinically important antibiotics. However, many aspects of macrolide action and resistance remain obscure. In this review we summarize the current knowledge, as well as unsolved questions, regarding the principles of macrolide binding to the large ribosomal subunit and the mechanism of drug action. Two mechanisms of macrolide resistance, inducible expression of Erm methyltransferase and peptide-mediated resistance, appear to depend on specific interactions between the ribosome-bound macrolide molecule and the nascent peptide. The similarity between these mechanisms and their relation to the general mode of macrolide action is discussed and the discrepancies between currently available data are highlighted.

Thrombosis is the collective term for diseases caused by the localized accumulation of circulating blood elements within the vasculature that result in vessel occlusion. Conventional antithrombotic drugs target the coagulation pathways (e.g., heparins, warfarin, ximelagatran), platelet-dependent mechanisms (e.g., clopidogrel), or thrombi in situ (e.g., streptokinase). While marketed anticoagulants are very efficacious, they can paralyze hemostasis, which is a potentially fatal condition when left untreated. Accordingly, anticoagulants are only rarely used at fully or markedly efficacious doses, e.g., high dose heparin, for short periods of time in closely watched clinical situations. Ideally, new targets for therapy would lead to the development of agents that are specific for thrombusforming mechanisms without compromising hemostasis. However, our understanding of the molecular, cellular, and physical interactions that differentiate thrombosis vs. hemostasis is limited. Even in the absence of thrombosis-specific, targeting, new drugs should preferentially inhibit the thrombotic process at doses that are relatively safe. The symptomatology of hemostatic pathway alterations can serve as basis for rational target selection. Hemostatic disorders that are compatible with human life and potentially protective against thrombosis provide useful guidance for new pharmacologic strategies. Additionally, theoretical considerations and experimental data suggest that new strategies for antithrombotic therapy might include: 1) inhibition of intrinsic coagulation pathway activity, 2) reduction of circulating platelet count, 3) inhibition of platelet protease activated receptor-4, or 4) enhancement of endogenous protein C or thrombolytic pathways might safely improve antithrombotic therapy.

Due to the well known receptor-receptor interaction between adenosine A2A and dopamine D2 receptors in the basal ganglia, the discovery and development of potent and selective A2A adenosine receptor antagonists, became, in the last ten years, an attractive field of research to discovery new drugs for the treatment of neurodegenerative disorders, such as Parkinsons disease.

Different compounds have been investigated as A2A adenosine receptor antagonists, which could be classified in two great families: xanthine derivatives and nitrogen poliheterocyclic systems. These studies led to the discovery of some highly potent and selective A2A adenosine receptor antagonists such as ZM241385, SCH58261 and some xanthine derivatives (KW6002) which have been used as pharmacological tools for studying this receptor subtype.

Anyway most of the reported compounds showed some problems which do not permit their use in clinical studies, such as poor water solubility (SCH58261, and xanthine derivatives) or good affinity for other adenosine receptor subtypes (e.g. ZM241385 possess good affinity for A2B adenosine receptors).

Recently, a three-dimensional model of the human A2A adenosine receptor (AR) and its docked ligands was built by homology to rhodopsin and validated with sitedirected mutagenesis and the synthesis of chemically complementary agonists and antagonists. Interestingly, the molecular modeling results clearly delineated the interactions involved in the binding of agonists and antagonists, which correlated well with known experimental results.

The aim of this report is to briefly summarize the recent progress made in this attractive field of research.

Nitric oxide (NO) has been established as an important messenger molecule in various steps of brain physiology, from development to synaptic plasticity, learning and memory. However, NO has also been viewed as a major agent of neuropathology when, escaping controlled production it may directly or indirectly promote oxidative and nitrosative stress. The exact borderline between physiological, and therefore neuroprotective, and pathological, and therefore neurodegenerative, actions of NO is a matter of controversy among researchers in the field. This is reflected in the present status of drug research, that is focused on finding ways to block NO production, and therefore limit neuropathology, as well as on finding ways to increase NO availability and therefore elicit neuroprotection. As an unavoidable consequence, both classes of drugs are reported to have neurodegenerative or neuroprotective effects, depending on the models in which they are tested. Aim of the present paper is to provide the reader with a survey, as much complete as possible, on the main aspects of NO biology, from biochemistry and chemical reactivity to the molecular signals elicited in neural cells target of its neurodegenerative or neuroprotective action. In doing that, many controversial aspects related to basic biology and to neuropathology of NO are taken into account. In the final sections, main classes of drugs able to interfere with NO physiopathology are examined, in order to try to devise possible directions for future NO-based therapeutical strategies.

Glutamate receptor signaling is essential to normal synaptic function in the central nervous system. The major ionotropic glutamate receptors (AMPA, Kainic, and NMDA) have different synaptic functions depending upon cellular and subcellular localization, subunit composition, and second messenger systems linked to the receptors. In this review, we examine major advances in glutamate receptor biology whose physiology plays a central role in neurologic disease such as epilepsy and stroke. A key feature of glutamate receptor activation in neurologic disease is the downstream effects on cell survival, genetic expression of axon guidance cues, synaptic connectivity/formation of networks, and neuronal excitability. Identification of therapeutic pharmacologic targets and development of antagonists specific to the disease process remain central themes in epilepsy and stroke research.

Cannabinoids comprise three classes of compounds, the active components of Marijuana (Cannabis sativa), as well as endogenous and synthetic derivatives. To date, two distinct cannabinoid receptors (CB1 and CB2) have been discovered, but evidence for further receptor types has been brought forward. The potential use of cannabinoids for medicinal purposes has long been known, but the mechanisms of action of both exogenously applied and endogenous cannabinoids are only partly established. For nervous system disorders, cannabinoids may be useful by modulating neurotransmission and calcium homeostasis as well as by anti-inflammatory and anti-oxidant actions. Some cannabinoids can also trigger cell death, which may be of therapeutic benefit in the treatment of malignant tumours.

A number of both in vitro and in vivo models have provided promising but diverse evidence for cannabinoid protection in glutamate-mediated excitotoxicity, hypoxia and glucose deprivation, brain trauma, epilepsy and MS. Subsequent to many preclinical investigations, clinical trials are now underway in a variety of the above applications. Overall, the understanding of the therapeutic relevance of cannabinoids will rely on further investigations into the neuroprotective and neurotoxic potency of cannabinoids in animal models and humans, as much as on a further advancement of our general understanding of the endocannabinoid system and the development of specific compounds devoid of unwanted psychoactive side effects.

New treatments are currently required for the common metabolic diseases obesity and type 2 diabetes. The identification of physiological and biochemical factors that underlie the metabolic disturbances observed in obesity and type 2 diabetes is a key step in developing better therapeutic outcomes. The discovery of new genes and pathways involved in the pathogenesis of these diseases is critical to this process, however identification of genes that contribute to the risk of developing these diseases represents a significant challenge as obesity and type 2 diabetes are complex diseases with many genetic and environmental causes. A number of diverse approaches have been used to discover and validate potential new targets for obesity and diabetes. To date, DNA-based approaches using candidate gene and genome-wide linkage analysis have had limited success in identifying genomic regions or genes involved in the development of these diseases. Recent advances in the ability to evaluate linkage analysis data from large family pedigrees using variance components based linkage analysis show great promise in robustly identifying genomic regions associated with the development of obesity and diabetes. RNA-based technologies such as cDNA microarrays have identified many genes differentially expressed in tissues of healthy and diseased subjects. Using a combined approach, we are endeavouring to focus attention on differentially expressed genes located in chromosomal regions previously linked with obesity and/or diabetes. Using this strategy, we have identified Beacon as a potential new target for obesity and diabetes.

Estrogen receptor modulators are recognised as playing a central role in the regulation of many endocrine functions. In recent years, the discovery of many new members of this class of ligand together with the acquisition of detailed knowledge concerning the structure and molecular mechanism of action of this protein continues to attract widespread interest.

This work details recent advances in the science of estrogen receptor (ER) modulation, with emphasis on the discovery of novel ligands for the ER ligand binding domain (LBD) and summarises the major developments in this area of selective estrogen receptor modulators (SERMs), pure antiestrogens, and related ER modulating ligands in the period 1998-2004. A detailed examination of structural studies of the ERs is presented with analysis of the impact of such works on contemporary ligand design and the molecular pharmacology of the ER. The various classes of ER modulators are discussed on the basis of stuctural similarities including selective estrogen receptor modulators (SERMs) and 'pure' nonsteroidal antiestrogens. Additionally we review the emergence of a novel selective class of modulator - which we have termed the selective estrogen receptor subtype modulators (SERSMs) and, in a departure from LBD strategies we examine the discovery of novel peptide inhibitors of the ER which inhibit transcriptional activiation of agonist liganded receptor through interaction with coactivator recruitment proteins, and offer unique insight to the mechanism of action of all classes of ER modulators. Through examination of patent and classical literature we present a thorough and informative cross-section of the contemporary state of the art in this exciting field of pharmaceutical research.

The peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors, a set of three receptor sub-types encoded by distinct genes, function as lipid sensors to regulate a broad range of genes in many metabolically active tissues. Synthetic PPAR agonists have exhibited therapeutic benefits in treating diabetes and cardiovascular diseases. The discovery of PPAR-specific ligands has led to significant advancement in our understanding of the structure of these receptor proteins and the molecular mechanism of their ligand-dependent activation. Herein, we present both recent progress in the functional analysis of these orphan receptors and the confirmation of the PPARs as molecular targets for the development of new medicines to treat human metabolic disease.

Diabetes is among the largest contributors to global mortality through its long term complications. The worldwide epidemic of type 2 diabetes has been stimulating the quest for new concepts and targets for the treatment of this incurable disease. A new target is glycogen phosphorylase (GP), the main regulatory enzyme in the liver responsible for the control of blood glucose levels. One of several approaches to influence the action of GP is the use of glucose derivatives as active site inhibitors. This field of research commenced 10-15 years ago and, due to joint efforts in computer aided molecular design, organic synthesis, protein crystallography, and biological assays, resulted in glucopyranosylidenespiro- hydantoin 16 (Ki = 3-4 μM) as the most efficient glucose analog inhibitor of GP of that time. The present paper surveys the recent developments of this field achieved mainly in the last five years: the synthesis and evaluation of glucopyranosylidene-spiro-thiohydantoin 18 (Ki = 5 μM) which has proven equipotent with 16, and is available in gram amounts; furanosylidene- and xylopyranosylidene-spiro-(thio)hydantoins whose ineffectiveness (Ki > 10 μM) confirmed the high specificity of the catalytic site of GP towards the Dglucopyranosyl unit; “open” hydantoins like methyl N-(1-carboxamido-Dglucopyranosyl) carbamate 37 (Ki = 16 μM) and N-acyl-N'-(b-D-glucopyranosyl) ureas among them the to date best glucose analog inhibitor N-(2-naphthoyl)-N'-(b- D-glucopyranosyl)urea (35, Ki = 0.4 μM) which can also bind to the so-called new allosteric site of GP; C-(b-D-glucopyranosyl)heterocycles (tetrazole, 2-methyl- 1,3,4-oxadiazole, benzimidazole (Ki = 11 μM), and benzothiazole). Iminosugars like isofagomine (52, IC50 = 0.7 μM), noeuromycin (60, IC50 = 4 μM), and azafagomine (61, IC50 = 13.5 μM) also bind strongly to the active site of GP, however, substitution on the nitrogens makes the binding weaker. The natural product five-membered iminosugar DAB (63) exhibited IC50 ~ 0.4-0.5 μM. Azoloperhydropyridines which can be regarded iminosugar-annelated heterocycles show moderate inhibition of GP: nojiritetrazole 12 (Ki = 53 μM) is the best inhibitor and fewer nitrogens in the five-membered ring weakens the binding. Physiological investigations have been carried out with N-acetyl-b-Dglucopyranosylamine 6, spiro-thiohydantoin 18, isofagomine 52, and DAB 63 to underline the potential use of these compounds in the treatment of type 2 diabetes. Computational methods suggest to synthesize further anomerically bifunctional glucose derivatives which may be good inhibitors of GP.

Obesity is an epidemic problem in the U. S. and many other industrialized nations. Historically, the drugs used for its treatment generally targeted small-molecule neurotransmitters. As research grows to decipher the underlying molecular mechanisms of energy homeostasis, it is becoming evident that the modulating effects of neuropeptides also are critical in the regulation of appetite and metabolism. The search for drugs to modify these monoaminergic and peptidergic pathways may eventually prove successful in the treatment of obesity. While tobacco smoking has long been used as one strategy to maintain a lower body weight, especially by female smokers, its adverse associations with addiction and disease overshadow its potential use as an antiobesity agent. Potential pharmacological effects of nicotine could be better understood as the intricacies of the nicotinic acetylcholine receptor are revealed. The objectives of this review are threefold: (1) to provide an overview of the physiological effects of nicotine on body weight while focusing on the drugs that are available as antiobesity and smoking cessation agents; (2) to describe the status of knowledge of the nicotinic acetylcholine receptor as it relates to energy homeostasis and its potential as an effective treatment modality for obesity; and (3) to present the current knowledge with respect to nicotine's effects on energy homeostatic and reward-related pathways at the molecular level. A better understanding of the regulatory mechanisms underlying the pharmacological effects of nicotine on body weight will provide insights into potential targets for the development of appropriate medicines for the treatment of obesity.

Histone deacetylases (HDACs) are key enzymes in the regulation of gene expression. By maintaining the dynamic equilibrium of the acetylation status of highly conserved lysine residues on histones, they regulate chromatin remodelling and gene expression. A link between aberrant HDAC activity and cancer has been widely reported and HDAC inhibitors have been shown to inhibit the proliferation of human tumor cell lines in vitro. Furthermore, several HDAC inhibitors have exhibited potent anti-tumor activity in human xenograft models, suggesting this class of compounds to be promising novel cancer therapeutic agents. This review provides an update on the current knowledge on HDAC inhibition with a focus on the most recent progress of HDAC inhibitors in clinical development.

The aim of cancer gene therapy is to selectively kill malignant cells at the tumour site, by exploiting traits specific to cancer cells and/or solid tumours. Strategies that take advantage of biological features common to different tumour types are particularly promising, since they have wide clinical applicability. Much attention has focused on genetic methods that complement radiotherapy, the principal treatment modality, or that exploit hypoxia the most ubiquitous characteristic of most solid cancers. The goal of this review is to highlight two promising gene therapy methods developed specifically to target the tumour volume that can be readily used in combination with radiotherapy. The first approach uses radiation-responsive gene promoters to control the selective expression of a suicide gene to irradiated tissue only, leading to targeted cell killing in the presence of a prodrug. The second method utilizes oxygen-dependent promoters to produce selective therapeutic gene expression and prodrug activation in hypoxic cells, which are refractive to conventional radiotherapy. Further refining of tumour targeting can be achieved by combining radiation and hypoxia responsive elements in chimeric promoters activated by either and dual stimuli. The in vitro and in vivo studies described in this review suggest that the combination of gene therapy and radiotherapy protocols has potential for use in cancer care, particularly in cases currently refractory to treatment as a result of inherent or hypoxia-mediated radioresistance.

The development of farnesyltransferase inhibitors, a novel approach to non-cytotoxic anticancer therapy, has been an active area of research over the past decade. Compounds that have advanced to clinical trials were evolved both from substrate-based design efforts and from compound library screening hits. This review focuses on the effort at Merck to evolve inhibitors from the protein substrate of farnesyltransferase, which resulted in the identification of a non-peptide inhibitor for clinical evaluation. X-ray crystal structures of farnesyltransferase complexed with early peptidomimetic as well as later non-peptide inhibitors have validated this design approach. NMR spectroscopic methods for studying enzyme-bound inhibitor structure, in conjunction with the use of conformational constraints, were critical components of subsequent efforts to provide potent inhibitors with varying levels of farnesyltransferase and geranylgeranyltransferase-I inhibitory specificity. Several of these compounds were important tools for investigating the use of prenyltransferase inhibitors to target Ki-Rasmediated tumor growth.

Natural Products: Biosynthesis and Prospect Towards Engineering Novel Antitumor Agents

This review gives a brief account on the current status of enediyne biosynthesis and the prospective of applying combinatorial biosynthesis methods to the enediyne system for novel analog production. Methods for cloning enedlyne biosynthetic gene clusters are first reviewed. A unified paradigm for enediyne biosynthesis, characterized with (a) the enediyne PKS, (b) the enediyne PKS accessory enzymes, and (c) tailoring enzymes, is then presented. Strategies and tools for novel enediyne analog production by combinatorial biosynthesis are finally discussed. The results set the stage to decipher the molecular mechanism for enediyne biosynthesis and lay the foundation to engineer novel enediynes by combinatorial biosynthesis for future endeavor.

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases. Tumors cannot grow as a mass above few mm3 unless a new blood supply is induced. It derives that the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy.

Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors. The angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance towards inhibition.

Heparan sulfate proteoglycans are involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions. This modulation occurs through the interaction with angiogenic growth factors or with negative regulators of angiogenesis. Thus, the study of the biochemical bases of this interaction may help to design glycosaminoglycan analogs endowed with angiostatic properties.

The purpose of this review is to provide an overview of the structure/function of heparan sulfate proteoglycans in endothelial cells and to summarize the angiostatic properties of synthetic heparin-like compounds, chemically modified heparins, and biotechnological heparins.

The following review outlines the physiological outcome of VIP and VIP gene manipulations. Previously, we reviewed the various VIP receptors associated with biological functions ranging from growth regulation, sexual function, bronchodilation, vasodilation and immune interactions to neurotrophism. Recent Progress in VIP-based drug design is discussed below.

Biologically active small molecules that interact specifically with protein have tremendous values not only for the functional analysis of genes but also for the drug development. Chemical genetics/genomics-based approach has recently been developed and recognized as one of key solutions for this purpose. This review focuses on the utilization of this new research engine for the target mining and validation of angiogenesis inhibitors that are capable of regulating the growth and spreading of cancer cells. Discovery of novel targets for angiogenesis inhibitors and validation of their biological relevancy based on chemical genetics/genomics provide new insight for the biological role of targets as well as for the development of new angiogenesis inhibitors.

Results of various projects on Mexican Indian ethnobotany and some of the subsequent pharmacological and phytochemical studies are summarised focusing both on chemical-pharmacological as well as anthropological (ethnopharmacological) aspects of our research. We have identified taste and smell properties of medicinal (vs. non-medicinal) plants as important indigenous selection criteria. There exist well-defined criteria specific for each culture, which lead to the selection of a plant as a medicine. This field research has also formed a basis for studies on bioactive natural products from selected species. The bark of Guazuma ulmifolia showed antisecretory activity (cholera toxin-induced chloride secretion in rabbit distal colon in an USSING chamber). Active constituents are procyanidins with a polymerisation degree of eight or higher. Byrsonima crassifolia yielded proanthocyanidins with (+) epicatechin units and Baccharis conferta showed a dose-dependant antispasmodic effect with the effect being particularly strong in flavonoid-rich fractions. Our ethnopharmacological research led to the identification of sesquiterpene lactones (SLs) like parthenolide as potent and relatively specific inhibitors of the transcription factor NF-kB, an important mediator of the inflammatory process. The inhibitory effect of SLs is very strongly enhanced by the presence of such groups as the isoprenoid ring system, a lactone ring containing a conjugated exomethylene group (a-methylene-g-lactone) and an a,b-unsaturated cyclopentenone or a conjugated ester moieties. Our work also elucidated the NF-kB inhibiting activity of the photosensitiser phaeophorbide A from Solanum diflorum (Solanaceae) in PMA induced HeLa cells. Hyptis verticillata yielded a series of lignans as well as sideritoflavone, rosmarinic acid and (R)- 5-hydroxypyrrolidin-2-one and is rich in essential oil (rich in a-pinene, b- pinene and thymol). Other species investigated include Begonia heracleifolia, Crossopetalum gaumerii, Epaltes mexicana, Pluchea symphytifolia and Xanthosoma robustum.

Natural killer T (NKT) cells are a subset of lymphocytes that express receptors characteristic of conventional T cells together with receptors typically found on natural killer cells. A key feature of NKT cells is the expression of a semi-invariant T cell receptor that is specific for glycolipid antigens presented by the unusual major histocompatibility complex class I-like molecule CD1d. While their precise immunological functions remain unknown, NKT cells have been implicated in the regulation of adaptive immune responses, including those directed against autoantigens. These findings raise the possibility that specific stimulation of NKT cells may be exploited for therapeutic purposes. A number of laboratories have tested this hypothesis, utilizing the sea sponge-derived agent a- galactosylceramide (a-GalCer), a specific agonist of NKT cells. Administration of a-GalCer to mice results in potent activation of NKT cells, rapid and robust cytokine production, and activation of a variety of cells of the innate and adaptive immune systems. Most notably, repeated administration of a-GalCer to mice favors the generation of conventional T lymphocytes producing T helper (Th) type 2 cytokines such as IL-4 and IL-10. These findings suggest that a-GalCer can modulate inflammatory conditions that are mediated by pathogenic Th1 cells. Indeed, recent studies have demonstrated that a-GalCer modulates the development of a variety of autoimmune and inflammatory diseases. Collectively, these studies provide a solid foundation for the development of NKT cell ligands as pharmacological agents for treatment of autoimmune diseases.

Lead identification and optimization is always a challenge to the medicinal chemists in drug discovery. Numbers of simple to complex and smaller to bigger organic compounds are prepared to meet the screening purpose of biological targets. Conventional solution phase synthetic methodologies are lacking the speed to run along with the need of medicinally interesting compounds due to their long reaction time, tedious work-up and purification problems. Alternatively opted polymer-supported synthesis of combinatorial libraries has emerged as a promising tool in generating large numbers of structurally diverse molecules parallelly and rapidly. Microwave-assisted solid/liquid phase combinatorial synthetic techniques have proved their efficiencies to reduce the reaction time from days & hours to minutes & seconds and more promisingly to produce improved yields with high purities. This review briefs about the theory behind microwave chemical technology and glimpses of recent advancements in its application on polymer supported combinatorial synthesis.

Pharmacophore discovery is one of the major elements of molecular modeling in the absence of X-ray structural data. While pharmacophores initially made their debut as a means for lead discovery, more recent refinements have brought them into the domain of lead optimization, e.g. as a means to define the molecular alignment in 3D-QSAR. In this review, the experiences of over a decade of confronting and solving the challenges of pharmacophore discovery applied to actual drug discovery are summarized. Also, practical tips are described for using the author's methodology for pharmacophore discovery, DANTE.

In recent years, tools for the development of new drugs have been dramatically improved. These include genomic and proteomic research, numerous biophysical methods, combinatorial chemistry and screening technologies. In addition, early ADMET studies are employed in order to significantly reduce the failure rate in the development of drug candidates. As a consequence, the lead finding, lead optimization and development process has gained marked enhancement in speed and efficiency. In parallel to this development, major pharmaceutical companies are increasingly outsourcing many components of drug discovery research to biotech companies. All these measures are designed to address the need for a faster time to market.

New screening methodologies have significantly enhanced the drug discovery process. High throughput screening accelerates lead discovery through rapid evaluation of compound collections, which may already be biased toward a target of interest through in silico screening. This article discusses new NMR screening techniques, which have opened the door to a wide range of new lead finding and lead optimization opportunities.

Methods for the prediction of octanol/water partition coefficient, aqueous solubility and acid/base dissociation constants are described and discussed. The advantages and limitations of the different approaches are described and an indication of problem areas discussed. Available prediction software is described and listed and attempts are made to assess the likely reliability of the predictions. The concept of “drug-likeness” is introduced and put into context and models for the prediction of ADME properties and toxicity are briefly described and assessed. Software for ADME/toxicity prediction is listed and the impact of these techniques on current drug design efforts is described. Web references are given for both commercial and public domain software which is available for property prediction from chemical structure.

Cytochromes P450 (Cyt P450s) constitute the most important biotransformation enzymes involved in the biotransformation of drugs and other xenobiotics. Because drug metabolism by Cyt P450s plays such an important role in the disposition and in the pharmacological and toxicological effects of drugs, early consideration of ADME-properties is increasingly seen as essential for the discovery and the development of new drugs and drug candidates.

The primary aim of this paper is to present various computational approaches used to rationalize and predict the activity and substrate selectivity of Cyt P450s, as well as the possibilities and limitations of these approaches, now and in the future. Attention is also paid to the experimental validation of these approaches by using high-throughput screening (HTS) of affinities to drug-drug interactions at the level of Cyt P450-isoenzymes. Since human Cyt P450 2D6 is one of the most important drug metabolizing enzymes and since in this regard much pioneering work has been done with this Cyt P450, Cyt P450 2D6 is chosen as a model for this discussion.

Apart from early mechanism-based ab initio calculations on substrates of Cyt P450 2D6, pharmacophore modeling of ligands (i.e. both substrates and inhibitors) of Cyt P450 2D6 and protein homology modeling have been used succesfully for the rationalisation and prediction of metabolite formation by this Cyt P450 isoenzyme. Significant protein structure-related species differences have been reported recently.

It is concluded that not one computational approach is capable of rationalizing and reliably predicting metabolite formation by Cyt P450 2D6, but that it is rather the combination of the various complimentary approaches. It is moreover concluded, that experimental validation of the computational models and predictions is often still lacking. With the advent of novel, easily and well applicable in vitro based high throughput assays for ligand binding and turnover this limitation could be overcome soon, however. When effective links with other new and recent developments, such as bioinformatics, neural network computing, genomics and proteomics can be created, in silico rationalisation and prediction of drug metabolism by Cyt P450s is likely to become one of the key technologies in early drug discovery and development processes.