Monogenic hypercholesterolemias: New genes,new drug targets

This review is focused on recent data on structure and functions of PCSK9 proprotein convertase, a newly identified participant in cholesterol metabolism in mammalian organisms, including humans. Proprotein convertase acts as a molecular chaperone for the low density lipoprotein (LDL) receptor, targeting it to the lysosomal degradation pathway. Various mutations increasing the PCSK9 affinity toward the LDL receptor cause autosomal dominant hypercholesterolemia. In contrast, loss-of-function mutations in PCSK9 gene decrease the blood plasma cholesterol level, thus acting as a protection factor against atherosclerosis and coronary heart disease. It is supposed that pharmacological agents inhibiting the interaction between PCSK9 and LDL receptor may substantially amplify the benefits of drugs—statins and cholesterol absorption blockers—in the treatment of all types of hypercholesterolemia, including its widespread multigenic and multifactorial forms.     

Mining predicted essential genes of Brugia malayi for nematode drug targets

We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression.     

Network-based assessment of the selectivity of metabolic drug targets in Plasmodium falciparum with respect to human liver metabolism

ABSTRACT: BACKGROUND: The search for new drug targets for antibiotics against Plasmodium falciparum, a major cause of human deaths, is a pressing scientific issue, as multiple resistance strains spread rapidly. Metabolic network-based analyses may help to identify those parasite's essential enzymes whose homologous counterparts in the human host cells are either absent, non-essential or relatively less essential. RESULTS: Using the well-curated metabolic networks PlasmoNet of the parasite Plasmodium falciparum and HepatoNet1 of the human hepatocyte, the selectivity of 48 experimental antimalarial drug targets was analyzed. Applying in silico gene deletions, 24 of these drug targets were found to be perfectly selective, in that they were essential for the parasite but non-essential for the human cell. The selectivity of a subset of enzymes, that were essential in both models, was evaluated with the reduced fitness concept. It was, then, possible to quantify the reduction in functional fitness of the two networks under the progressive inhibition of the same enzymatic activity. Overall, this in silico analysis provided a selectivity ranking that was in line with numerous in vivo and in vitro observations. CONCLUSIONS: Genome-scale models can be useful to depict and quantify the effects of enzymatic inhibitions on the impaired production of biomass components. From the perspective of a host-pathogen metabolic interaction, an estimation of the drug targets-induced consequences can be beneficial for the development of a selective anti-parasitic drug.     

In silico characterization of putative drug targets in Staphylococcus saprophyticus,causing bovine mastitis

The bovine mastitis caused by coagulase negative staphylococci (CNS) has increased in many herds of urban and rural areas ofIndia. Emergence of multi drug resistant bacteria has further made its management more complex and serious. Therefore,innovation of novel specific drug for the treatment of disease caused by particular organism remained to be a challenge. Hence, inthe present study a bacterium was isolated from milk of the cow with bovine mastitis and was identified as S. saprophyticus, 44pathways of S. saprophyticus retrieved (KEGG) from web server were found to be non homologous to the host Bos taurus, out ofwhich 39 pathways were found to be in cytoplasm, 2 in cell wall and 3 in the cell membrane. The knowledge of the present studycould make the drug discovery easier which have high affinity to the target site of the causative organism.     

Design and characterization of ebolavirus GP prehairpin intermediate mimics as drug targets

Ebolaviruses are highly lethal filoviruses that cause hemorrhagic fever in humans and nonhuman primates. With no approved treatments or preventatives, the development of an anti-ebolavirus therapy to protect against natural infections and potential weaponization is an urgent global health need. Here, we describe the design, biophysical characterization, and validation of peptide mimics of the ebolavirus N-trimer, a highly conserved region of the GP2 fusion protein, to be used as targets to develop broad-spectrum inhibitors of ebolavirus entry. The N-trimer region of GP2 is 90% identical across all ebolavirus species and forms a critical part of the prehairpin intermediate that is exposed during viral entry. Specifically, we fused designed coiled coils to the N-trimer to present it as a soluble trimeric coiled coil as it appears during membrane fusion. Circular dichroism, sedimentation equilibrium, and X-ray crystallography analyses reveal the helical, trimeric structure of the designed N-trimer mimic targets. Surface plasmon resonance studies validate that the N-trimer mimic binds its native ligand, the C-peptide region of GP2. The longest N-trimer mimic also inhibits virus entry, thereby confirming binding of the C-peptide region during viral entry and the presence of a vulnerable prehairpin intermediate. Using phage display as a model system, we validate the suitability of the N-trimer mimics as drug screening targets. Finally, we describe the foundational work to use the N-trimer mimics as targets in mirror-image phage display, which will be used to identify d-peptide inhibitors of ebolavirus entry.     

Conserved fungal genes as potential targets for broad-spectrum antifungal drug discovery

The discovery of novel classes of antifungal drugs depends to a certain extent on the identification of new, unexplored targets that are essential for growth of fungal pathogens. Likewise, the broad-spectrum capacity of future antifungals requires the target gene(s) to be conserved among key fungal pathogens. Using a genome comparison (or concordance) tool, we identified 240 conserved genes as candidates for potential antifungal targets in 10 fungal genomes. To facilitate the identification of essential genes in Candida albicans, we developed a repressible C. albicans MET3 (CaMET3) promoter system capable of evaluating gene essentiality on a genome-wide scale. The CaMET3 promoter was found to be highly amenable to controlled gene expression, a prerequisite for use in target-based whole-cell screening. When the expression of the known antifungal target C. albicans ERG1 was reduced via down-regulation of the CaMET3 promoter, the CaERG1 conditional mutant strain became hypersensitive, specifically to its inhibitor, terbinafine. Furthermore, parallel screening against a small compound library using the CaERG1 conditional mutant under normal and repressed conditions uncovered several hypersensitive compound hits. This work therefore demonstrates a streamlined process for proceeding from selection and validation of candidate antifungal targets to screening for specific inhibitors.     

Herbal bioactivation, molecular targets and the toxicity relevance

There have been increasing reports on the adverse reactions associated with herbal consumption. For many of these adverse reactions, the underlying biochemical mechanisms are unknown, but bioactivation of herbal compounds to generate reactive intermediates have been implicated. This minireview updates our knowledge on metabolic activation of herbal compounds, molecular targets and the toxicity relevance. A number of studies have documented that some herbal compounds can be converted to toxic or even carcinogenic metabolites by Phase I [e.g. cytochrome P450s (CYPs)] and less frequently by Phase II enzymes. For example, aristolochic acids (AAs) in Aristolochia spp, which undergo reduction of the nitro group by hepatic CYP1A1/2 or peroxidases in extrahepatic tissues to generate highly reactive cyclic nitrenium ions. The latter can react with macromolecules (DNA and protein), resulting in activation of H-ras and myc oncogenes and gene mutation in renal cells and finally carcinogenesis of the kidneys. Teucrin A and teuchamaedryn A, two diterpenoids found in germander (Teuchrium chamaedrys) used as an adjuvant to slimming herbal supplements that caused severe hepatotoxicity, are converted by CYP3A4 to reactive epoxide which reacts with proteins such as CYP3A and epoxide hydrolase and inactivate them. Some naturally occurring alkenylbenzenes (e.g. safrole, methyleugenol and estragole) and flavonoids (e.g. quercetin) can undergo bioactivation by sequential 1-hydroxylation and sulfation, resulting in reactive intermediates capable of forming DNA adducts. Extensive pulegone metabolism generated p-cresol that is a glutathione depletory. The hepatotoxicity of kava is possibly due to intracellular glutathione depletion and/or quinone formation. Moreover, several herbal compounds including capsaicin from chili peppers, dially sulfone in garlic, methysticin and dihydromethysticin in kava, oleuropein in olive oil, and resveratrol found in grape seeds are mechanism-based (suicide) inhibitors of various CYPs. Together with advances of proteomics, metabolomics and toxicogenomics, an integrated systems toxicological approach may provide deep insights into mechanistic aspects of herb-induced toxicities, and contribute to bridging the relationships between herbal bioactivation, protein/DNA adduct formation and the toxicological consequences.     

Mitotic drug targets

Mitosis is the key event of the cell cycle during which the sister chromatids are segregated onto two daughter cells. It is well established that abrogation of the normal mitotic progression is a highly efficient concept for anti‐cancer treatment. In fact, various drugs that target microtubules and thus interfere with the function of the mitotic spindle are in clinical use for the treatment of various human malignancies for many years. However, since microtubule inhibitors not only target proliferating cells severe side effects limit their use. Therefore, the identification of novel mitotic drug targets other than microtubules have gained recently much attention. This review will summarize the latest developments on the identification and clinical evaluation of novel mitotic drug targets and will introduce novel concepts for chemotherapy that are based on recent progress in our understanding how mitotic progression is regulated and how anti‐mitotic drugs induce tumor cell death. J. Cell. Biochem. 111: 258–265, 2010. © 2010 Wiley‐Liss, Inc.     

Apoptosis-based therapies and drug targets

The pathogenesis of many diseases is most closely connected with aberrantly regulated apoptotic cell death. The past 15 years have witnessed an explosion in the basic knowledge of mechanisms that regulate apoptosis and the mediators that either trigger or inhibit cell death. Consequently, great interest has emerged in devising therapeutic strategies for modulating the key molecules of life-and-death decisions. Numerous novel approaches are currently being followed employing gene therapy and antisense strategies, recombinant biologics or classical organic and combinatorial chemistry in order to target specific apoptotic regulators. Although drug development is still in its infancy, several therapeutics have progressed to clinical testing or have even been approved in record time. This review outlines the recent advances in the field of apoptosis-based therapies and explores some highlights of a very active field of drug development.     

Predicting conserved essential genes in bacteria: in silico identification of putative drug targets

Many genes have been listed as putatively essential for bacterial viability in the Database of Essential Genomes (DEG), although few have been experimentally validated. By prioritising targets according to the criteria suggested by the Research and Training in Tropical Diseases (TDR) Targets database, we have developed a modified down-selection tool to identify essential genes conserved across diverse genera. Using this approach we identified 52 proteins conserved to 7 or more of the 14 genomes in DEG. We confirmed the validity of the down-selection by attempting to make mutants of 8 of these targets in a model organism, Yersinia pseudotuberculosis, which is not closely related to any of the bacteria in DEG. Mutants were recovered for only one of the 8 targets, suggesting that the other 7 were essential in Y. pseudotuberculosis, an impressive success rate compared to other approaches of identification for such targets. Identification of essential proteins common in diverse bacterial genera can then be used to facilitate the selection of effective targets for novel broad-spectrum antibiotics.     

Proteases as drug targets

The effective management of AIDS with HIV protease inhibitors, or the use of angiotensin-converting enzyme inhibitors to treat hypertension, indicates that proteases do make good drug targets. On the other hand, matrix metalloproteinase (MMP) inhibitors from several companies have failed in both cancer and rheumatoid arthritis clinical trials. Mindful of the MMP inhibitor experience, this chapter explores how tractable proteases are as drug targets from a chemistry perspective. It examines the recent success of other classes of drug for the treatment of rheumatoid arthritis, and highlights the need to consider where putative targets lie on pathophysiological pathways--regardless of what kind of therapeutic entity would be required to target them. With genome research yielding many possible new drug targets, it explores the likelihood of discovering proteolytic enzymes that are causally responsible for disease processes and that might therefore make better targets, especially if they lead to the development of drugs that can be administered orally. It also considers the impact that biologics are having on drug discovery, and in particular whether biologically derived therapeutics such as antibodies are likely to significantly alter the way we view proteases as targets and the methods used to discover therapeutic inhibitors.     

Population genetics and microevolution of clinical Candida glabrata reveals recombinant sequence types and hyper-variation within mitochondrial genomes,virulence genes,and drug targets

Candida glabrata is the second most common etiological cause of worldwide systemic candidiasis in adult patients. Genome analysis of 68 isolates from 8 hospitals across Scotland, together with 83 global isolates, revealed insights into the population genetics and evolution of C. glabrata. Clinical isolates of C. glabrata from across Scotland are highly genetically diverse, including at least 19 separate sequence types that have been recovered previously in globally diverse locations, and 1 newly discovered sequence type. Several sequence types had evidence for ancestral recombination, suggesting transmission between distinct geographical regions has coincided with genetic exchange arising in new clades. Three isolates were missing MATα1, potentially representing a second mating type. Signatures of positive selection were identified in every sequence type including enrichment for epithelial adhesins thought to facilitate fungal adhesin to human epithelial cells. In patent microevolution was identified from 7 sets of recurrent cases of candidiasis, revealing an enrichment for nonsynonymous and frameshift indels in cell surface proteins. Microevolution within patients also affected epithelial adhesins genes, and several genes involved in drug resistance including the ergosterol synthesis gene ERG4 and the echinocandin target FKS1/2, the latter coinciding with a marked drop in fluconazole minimum inhibitory concentration. In addition to nuclear genome diversity, the C. glabrata mitochondrial genome was particularly diverse, with reduced conserved sequence and conserved protein-encoding genes in all nonreference ST15 isolates. Together, this study highlights the genetic diversity within the C. glabrata population that may impact virulence and drug resistance, and 2 major mechanisms generating this diversity: microevolution and genetic exchange/recombination.     

Abeta toxicity in Alzheimer's disease: globular oligomers (ADDLs) as new vaccine and drug targets

Over the past several years, experiments with synthetic amyloid-beta peptide (Abeta) and animal models have strongly suggested that pathogenesis of Alzheimer's disease (AD) involves soluble assemblies of Abeta peptides (Trends Neurosci. 24 (2001) 219). These soluble neurotoxins (known as ADDLs and protofibrils) seem likely to account for the imperfect correlation between insoluble fibrillar amyloid deposits and AD progression. Recent experiments have detected the presence of ADDLs in AD-afflicted brain tissue and in transgenic-mice models of AD. The presence of high affinity ADDL binding proteins in hippocampus and frontal cortex but not cerebellum parallels the regional specificity of AD pathology and suggests involvement of a toxin receptor-mediated mechanism. The properties of ADDLs and their presence in AD-afflicted brain are consistent with their putative role even in the earliest stages of AD, including forms of mild cognitive impairment.     

抑郁症易感基因和抗抑郁药物靶点相关[]神经细胞类型及相互作用网络分析

基于抑郁症的全基因组关联分析研究(GWAS),对于获得的单核苷酸多态性位点(SNP)使用Haploreg软件进行基因注释,得到SNP注释的102个易感基因.。使用MAGMA软件对GWAS的汇总统计数据做基因水平的分析,获得了270个校正之后显著的基因,两者合并共得到320个抑郁症易感基因。通过药物数据库Drugbank获取133个抗抑郁药物靶点基因。使用EWCE包对抑郁症易感基因和抗抑郁药物靶点在三套脑组织单细胞测序数据中,分别进行神经细胞类型富集分析。结果发现大脑皮质的GABA神经元(抑制性神经元)和谷氨酸能神经元(兴奋性神经元)是抑郁症易感基因和抗抑郁药物靶点共同的神经元。这两种类型的神经细胞可能是抗抑郁药物与抑郁症易感基因相互作用的神经细胞,另外少突胶质前体细胞可能是抑郁症特有的易感神经细胞。使用Network Calculator软件构建网络并进行进行网络拓扑学参数分析。结果表明抑郁症易感基因与抗抑郁药物靶点组成了一个具有显著的相互连接的网络。本研究从单细胞层面揭示抑郁症的遗传机制,在网络层面为寻找新的抗抑郁药物靶点提供了一定的启示。     

Antimalarial drug development and new targets

The Molecular Approaches to Malaria (MAM2000) conference, Lorne, Australia, 2-5 February 2000, brought together world-class malaria research scientists. The development of new tools and technologies - transfection, DNA microarrays and proteomic analysis - and the availability of DNA sequences generated by the Malaria Genome Project, along with more classic approaches, have facilitated the identification of novel drug targets, the development of new antimalarials and the generation of a deeper understanding of the molecular mechanism(s) of drug resistance in malaria. It is hoped that combinations of these technologies could lead to strategies that enable the development of effective, efficient and affordable new drugs to overcome drug-resistant malaria, as discussed at MAM2000 and outlined here by Ian Macreadie and colleagues.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic-producing strains, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic producers, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.