HIV-1 Nef is a peripheral membrane protein that affects both signal transduction and membrane trafficking in infected cells. Alterations in these cellular processes enhance the efficiency of viral replication and the pathogenesis of AIDS in vivo. The precise mechanisms by which Nef functions are not fully elucidated. Nef is not an enzyme but appears to act as a linker molecule, mediating a variety of protein-protein interactions. Structural, biochemical and mutational data have allowed tentative identification of the key interactive surfaces on Nef, their cellular partners and their roles in Nef activity. Nef contains an SH3-binding surface through which it can interact with cellular Src-family tyrosine kinases and/or activator molecules for small GTPases involved in signal transduction. This SH3-binding surface is important for the ability of Nef to facilitate the activation of host T-lymphocytes, a process which renders the cells more permissive for viral replication. Nef also contains two relatively unstructured, solvent-exposed loops, through which it interacts with the cellular proteins that coat vesicles involved in membrane trafficking. These surfaces are important for Nef-mediated alterations in the subcellular distribution of transmembrane proteins, a process which causes diverse effects, including the assembly of maximally infectious viral particles and viral evasion of the host immune system. These data provide precise molecular targets within the Nef protein. Molecules that bind these interactive surfaces are predicted to inhibit Nef activity and provide the basis for novel chemotherapeutic agents for the treatment of HIV-infection.
Peptide deformylase (PDF) catalyses the hydrolytic removal of the N-terminal formyl group from nascent ribosome-synthesised polypeptides. Its activity is essential and it is present in all eubacteria. It is also present in the organelles of some eukaryotes. PDF represents a novel class of mononuclear iron protein, utilising an Fe2+ ion to catalyse the hydrolysis of an amide bond. Due to its extreme lability, isolation and characterisation of PDF was not possible until very recently. This review will discuss the recent progress in the elucidation of the the structure and function of PDF, evaluating its suitability as a target for antibiotic design and summarising the current approaches to designing drugs that target PDF.
Saccharomyces cerevisiae has been used extensively as a model for higher eukaryotes in the study of basic cellular processes. The high degree of conservation in terms of sequence similarity and function has made this organism useful in elucidating biological pathways, both yeast and human. Among these are pathways responsible for DNA damage repair and cell cycle control. This review presents an overview of opportunities for using yeast as a model system for anticancer drug discovery. It covers screens directed against specific cancer-related targets as well as contexts created by cancer-related alterations. The methodologies covered include pharmacological and genetic screens, as well as genome-wide approaches to drug target identification.
Peptide deformylases (PDFs) constitute a growing family of hydrolytic enzymes previously believed to be unique to Eubacteria. Recent data from our laboratory have demonstrated that PDF orthologues are present in many eukaryotes, including several parasites. In this report we aim to explain why PDF could be considered to be a potent target for human and veterinary antiparasitic treatments.
Sphingosine-1-phosphate (SPP) is a polar sphingolipid metabolite that has received increasing attention as both an extracellular mediator and an intracellular second messenger. SPP is the ligand of a family of specific cell surface G-protein coupled receptors (GPCR), known as the endothelial differentiation gene-1 (EDG-1) family. These receptors, which include EDG-1, -3, -5, -6 and -8, regulate diverse processes including cell migration, angiogenesis, vascular maturation, heart development, neurite retraction and soma rounding. In addition, abundant evidence indicates that SPP also acts as an intracellular lipid messenger, regulating calcium mobilisation, cell growth and survival. The relative intracellular level of SPP and ceramide, another sphingolipid metabolite associated with cell death and cell growth arrest, is an important factor in determining cell fate. Changes in SPP and ceramide have been implicated in a number of pathological conditions in which apoptosis plays an important role, including cancer and neurodegenerative disorders, as well as in atherosclerosis and allergic responses. This review will examine the biosynthesis, metabolism and potential functions of SPP in diverse diseases in order to illuminate targets for the pharmaceutical and therapeutic manipulation of SPP levels.
Temporal lobe epilepsy (TLE), Parkinson’s disease (PD) and Huntington’s disease (HD) are neurodegenerative disorders that involve disruptions in γ-amino butyric acid (GABA) signalling. GABA is the major inhibitory neurotransmitter in the central nervous system (CNS). TLE seizures reflect excess excitation, which may result from local inhibitory circuit dysfunction. PD devastates the input to striatal GABAergic neurones and HD destroys striatal GABAergic neurones. Controlling GABA delivery to specific brain areas should benefit each of these diseases. The molecules responsible for GABA release and signalling are ideal targets for new therapies. In this paper, we discuss the role of GABA in the circuitry affected by each of these diseases and suggest potential sites for intervention. GABA is unique among neurotransmitters because it can be synthesised by either of two related enzymes. Intracellular GABA is found throughout the cytosol and in synaptic vesicles. GABA can be released either through exocytosis, or via the plasma membrane transporter. The synthesising enzyme probably determines the intracellular location and hence the mechanism for GABA release. Directing GABA synthesis, degradation, transport or receptors can control GABA signalling. We propose that new drugs and devices aimed at GABA synthesis, release and binding will offer novel and highly effective treatments for neurodegenerative diseases.
The sarcoplasmic reticulum (SR) is a major player in maintaining cardiac function, as it is intimately involved in the regulation of Ca2+-movements on a beat-to-beat basis. SR dysfunction due to abnormalities in SR protein content has been reported in different cardiac diseases such as ischaemic heart disease, myocardial infarction, congestive heart failure and various cardiomyopathies; thus the genes expressing the SR Ca2+-pump, Ca2+-channels, calsequestrin, phospholamban and other regulatory proteins are considered important targets for drug development. In our experience, ischaemic preconditioning (IP) and pharmacological therapies, such as anti-oxidants, beta-adrenergic receptor blockers, angiotensin receptor (AT-1) blockers, angiotensin converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers are effective therapies that improve cardiac performance in the failing heart by improving SR function. Accordingly, this paper is intended to shed light on the knowledge in the field of cardiac therapy targeted to improve and protect SR function.
The ETS gene family encodes unique transcription regulators that have a common ETS DNA binding domain. At least 25 distinct ETS related genes have been isolated from various species. The ETS family transcription factors are known to regulate genetic programs essential for differentiation and development processes and play diverse roles in a number of biological processes such as organogenesis and tissue remodelling during murine development, hematopoiesis, B-cell development, activation of T-cells and signal transduction, as well as osteogenesis, osteoblast differentiation and extracellular matrix mineralization. Based on the observation of overexpression of ETS related genes in various primary and metastatic tumors, their utility as potential therapeutic targets has been suggested. Antisense oligonucleotides, transdominant, and dominant-negative mutants have been exploited to target and inhibit ETS gene expression selectively. These ETS-targeted studies are being pursued to assess their antitumour effect, and hold the potential that such specific ETS-targeted inhibitors may become a viable option for cancer therapy. Collectively, these studies also demonstrate that Ets factors can regulate multiple aspects of the malignant phenotype of many tumor cells in particular neoangiogenesis and extracellular matrix-regulated (ECM-regulated) cell proliferation, motility and invasiveness.
A large number of bacterial proteins are active in extracytoplasmic locations. Targeting and membrane translocation of the vast majority of these secretory and membrane polypeptides is mediated by the Sec pathway. Protein secretion requires the co-ordinated and sequential action of targeting factors on the cis-side of the membrane, a complex membrane-embedded protein translocase and maturation enzymes on the trans-side. Recently, significant advances in the molecular genetics and biochemistry of the Sec pathway have revealed that several of the Sec pathway components are essential for bacterial viability and/or pathogenicity. Moreover, several biochemical assays and structural insights have become available. Importantly, some of the Sec components are unique to bacteria. These developments raise the possibility that the bacterial protein translocase and other Sec pathway components could become formidable targets for antibacterial drug discovery.
The transcription factor NF-kappaB has been implicated in the pathogenesis of a variety of both acute and chronic inflammatory diseases. Many agents have shown promise and potential to abrogate NF-kappaB activity in both in vitro and in vivo systems. These include antioxidants, corticosteroids, proteasome inhibitors, arachadonic acid pathway metabolites, salicylates, molecular interventions and cell-permeable peptides. Unfortunately, therapies aimed at blocking its activation have not proven clinically feasible at this time. As the complex signal transduction pathways leading to NF-kappaB activation are further elucidated, more specific inhibitors of NF-kappaB are likely to be identified.
All cells, from bacterial to human, have a common, intricate response to stress that protects them from injury. Heat shock proteins (Hsps), also known as stress proteins and molecular chaperones, play a central role in protecting cellular homeostatic processes from environmental and physiologic insult by preserving the structure of normal proteins and repairing or removing damaged ones. An understanding of the interplay between Hsps and cell stress tolerance will provide new tools for treatment and drug design that maximise preservation or restoration of health. For example, the increased vulnerability of tissues to injury in some conditions, such as ageing, diabetes mellitus and menopause, or with the use of certain drugs,, such as some antihypertensive medications, is associated with an impaired Hsp response. Additionally, diseases that are associated with tissue oxidation, free radical formation, disorders of protein folding, or inflammation, may be improved therapeutically by elevated expression of Hsps. The accumulation of Hsps, whether induced physiologically, pharmacologically, genetically, or by direct administration of the proteins, is known to protect the organism from a great variety of pathological conditions, including myocardial infarction, stroke, sepsis, viral infection, trauma, neurodegenerative diseases, retinal damage, congestive heart failure, arthritis, sunburn, colitis, gastric ulcer, diabetic complications and transplanted organ failure. Conversely, lowering Hsps in cancer tissues can amplify the effectiveness of chemo- or radiotherapy. Treatments and agents that induce Hsps include hyperthermia, heavy metals (zinc and tin), salicylates, dexamethasone, cocaine, nicotine, alcohol, alpha-adrenergic agonists, PPAR-gamma agonists, bimoclomol, geldanamycin, geranylgeranylacetone and cyclopentenone prostanoids. Compounds that suppress Hsps include quercetin (a bioflavinoid), 15-deoxyspergualin (an immunosuppressive agent) and retinoic acid. Researchers who are cognisant of the Hsp-related effects of these and other agents will be able to use them to develop new therapeutic paradigms.
The endoplasmic reticulum (ER), the first secretory compartment of eukaryotic cells, co-ordinates the biogenesis and export of all membrane-bound and soluble cargo molecules to the cell surface. ER function is now recognised to have unprecedented links with signalling pathways regulating cell growth and differentiation and host physiology. Misfolding and aggregation of newly synthesised proteins in the ER or alterations in ER processing of cargo mediated by pathogens is responsible for a broad range of diseases including cystic fibrosis, emphysema and neuropathies such as Alzheimer's disease. The central, integrative role of the ER in determining cell physiology in health and disease represents an untapped area for pharmacological intervention. This review focuses on the potential use of pharmacological agents to modulate cargo selection, folding and degradation in the ER with the goal of alleviating ER export disease. In addition, implementation of novel technologies that utilise normal ER function to store and release biologically active substances of therapeutic relevance are presented as a new frontier in drug delivery.
Recent progress in microbial genome sequencing, along with functional genomics technologies based on gene expression, proteomics and genetics have facilitated the identification of significant numbers of Gram-positive virulence genes. These genes represent a novel and heterogeneous class of targets for antimicrobial drug development. This review will concentrate of the contribution of two functional genomics technologies, in vivo expression technology (IVET) based on gene expression and signature-tagged mutagenesis (STM), a genetics based technology to the identification of virulence genes in Gram-positive pathogens.
Ion channels are membrane proteins that flicker open and shut to regulate the flow of ions down their electrochemical gradient across the membrane and consequently regulate cellular excitability. Every living cell expresses ion channels, as they are critical life-sustaining proteins. Ion channels are generally either activated by voltage or by ligand interaction. For each group of ion channels the channels' molecular biology and biophysics will be introduced and the pharmacology of that group of channels will be reviewed. The in vitro and in vivo literature will be reviewed and, for ion channel groups in which clinical trials have been conducted, the efficacy and therapeutic potential of the neuroprotective compounds will be reviewed. A large part of this article will deal with glutamate receptors, focusing specifically on N-methyl-D-aspartate (NMDA) receptors. Although the outcome of clinical trials for NMDA receptor antagonists as therapeutics for acute stroke is disappointing, the culmination of these failed trials was preceded by a decade of efforts to develop these agents. Sodium and calcium channel antagonists will be reviewed and the newly emerging efforts to develop therapeutics targeting potassium channels will be discussed. The future development of stroke therapeutics targeting ion channels will be discussed in the context of the failures of the last decade in hopes that this decade will yield successful stroke therapeutics.
The two most common neurodegenerative diseases are Alzheimer’s disease (AD) and Parkinson’s disease (PD). The symptoms are caused by the initially selective degeneration of neuronal subpopulations involved in memory (AD) or movement control (PD). The cause of both diseases is unknown, but ageing is an inevitable risk factor. The identification of disease-associated genes was a breakthrough for the understanding of molecular mechanisms of neurodegeneration and has provided the basis for the establishment of cell culture and animal model systems, instrumental for target validation and drug screening. Familial AD is caused by mutations in the β-amyloid precursor protein (βAPP) and in the gene products responsible for its proteolytic processing, namely the presenilins. Transgenic mice expressing these mutant genes develop characteristic AD plaques in an age-dependent manner. A reduction of plaque burden and amelioration of cognitive decline in these animals was recently achieved by vaccination with amyloid β-protein fibrils. The other hallmark lesion of AD, the neurofibrillary tangle, has been modelled recently in transgenic mice expressing mutant tau protein linked to frontotemporal dementia. PD is characterised by intraneuronal cytoplasmic deposits (Lewy bodies) of the PD-associated gene product α-synuclein. Transgenic expression of α-synuclein recreated hallmark features of PD in mice and fruit flies, establishing α-synuclein as PD-causing drug target. Moreover, environmental risk factors such as the pesticide rotenone have been used successfully to generate rodent models of PD. Lesion models of PD are being exploited for the development of experimental gene therapy and transplantation approaches.
The discovery of specific receptors for 9-tetrahydrocannabinol, the major psychoactive component of marijuana, opened new horizons for the possible therapeutic exploitation of Cannabis sativa and the cannabinoids. Endogenous ligands of cannabinoid receptors, the ‘endocannabinoids’, were found and the molecular mechanisms underlying their biological effects and the regulation of their levels are now being identified. Cause/effect relationships between alterations of cannabinoid receptor/endocannabinoid levels in tissues and the symptoms of various pathological states are starting to be revealed. These studies may open the way to the possible use of substances that manipulate endocannabinoid levels and actions, such as inhibitors of the biosynthesis and inactivation and receptor antagonists, as cannabinoid-based therapeutic agents with little or no psychotropic side effect, thus potentially fulfilling an ambition nurtured for almost two centuries.
A major hallmark of malignant disease is the capacity of cancer cells to cross tissue boundaries and spread to distant sites throughout the body. Extracellular matrix-degrading enzymes are thus required to break down structural barriers to invasion and metastasis. Extensive and compelling evidence indicates that several members of the matrix metalloproteinase (MMP) family of enzymes play a key role in this regard and forms the rationale for the development and testing of compounds that can inhibit their proteolytic activity. In addition, data now indicate that MMPs contribute to other aspects of cancer, including angiogenesis and the initial stages of tumour development, thus expanding the potential clinical utility of MMP inhibitors (MMPIs). Conversely, some MMPs may defy cancer progression and others may not participate in cancer, but undoubtedly play normal physiologic roles. These possibilities and the mechanisms underlying the influence of MMPs in cancer must be understood and considered in the design of therapeutic agents in order to optimise their efficacy and minimise their toxicity. Herein we review the biology and regulation of the MMPs, their roles in cancer development and progression, the status of preclinical and clinical efforts aimed at inhibiting their activity and the challenges and promise ahead.
Uveitis (intraocular inflammatory diseases) is the cause of approximately 10% of severe visual handicap in the US. It can be of infectious or autoimmune aetiology and can be localised primarily to the anterior chamber (anterior uveitis), the posterior segment of the eye (posterior uveitis) or involve the entire globe (panuveitis). Pathology derives from the presence of inflammatory cells in the optical axis. In the treatment of uveitic conditions, the main therapeutic goals are to downregulate the immune response, preserve the integrity of the ocular architecture and eventually eliminate the inciting uveitogenic stimuli. Current therapy is based on topical or systemic corticosteroid, with or without second line agents, such as cyclosporin A or antimetabolites. The serious adverse effects of these drugs are the impetus for development of less toxic and more specific therapies for uveitis. In this review, we present a brief discussion of ocular immunology, the pathophysiology of uveitis and emerging strategies in the treatment of these blinding diseases.
Computer simulation of biological systems for in silico target validation has the potential for increasing the efficiency of pharmaceutical R&D by expanding the number of parameters tested ‘virtually’. Only the most interesting subset of these has to then be probed in vivo. By avoiding less informative experiments and focusing on variables with the greatest influence on clinical end points, valuable drug targets can be advanced more quickly and those with little or no leverage bypassed. With many compounds failing today due to insufficient efficacy, use of in silico methods should lead to a greater percentage of compounds making the transition to clinical drug. Though biosystems modelling has great advantages, the actual adoption and implementation within each organisation will be a variable process because it has a disruptive approach to current pharma R&D practices. Some companies may have scientific personnel, technical infrastructure and a management culture that provide fertile ground for such a change; other companies will likely struggle more than once to adopt such methods. Innovators need to understand the challenges to implementation they will face and what actions are likely to improve their chances of success. Organisations which successfully implement biosystems modelling for in silico target validation should be able to design experiments and trials more tailored to each drug target and thus achieve higher yields of approved drugs from compounds tested.
