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PubMed2006

SAGE and related approaches for cancer target identification.

Comprehensive genetic, epigenetic and transcriptional analyses of normal and cancerous tissues and cells have yielded many candidate diagnostic, predictive, and prognostic markers and therapeutic targets in human cancer. This article provides a brief overview of SAGE and SAGE-like techniques, highlighting their utility and advantages relative to other genomic technologies for the discovery of drug targets. We also summarize the results of recent comprehensive profiling studies that utilize these methods to provide insights into mechanisms of tumor initiation and progression, to improve our molecular understanding of the tumor microenvironment and to reveal new targets and avenues for therapeutic interventions.

DNA Methylation↗
PubMed2004

Targeted identification of glucocorticoid-attenuated response genes: in vitro and in vivo models.

Glucocorticoids attenuate the induction of numerous inflammatory mediators. We hypothesized that a targeted screening for genes with these regulatory characteristics, called glucocorticoid-attenuated response genes (GARGs), would be an efficient way to identify genes participating in glucocorticoid-sensitive inflammatory processes. An initial application of this idea, using an in vitro model, identified 12 cDNAs induced by LPS and attenuated by dexamethasone, including a new chemokine designated LIX. In vivo studies demonstrated that endotoxemia-induced lung mRNA expression of LIX, but not of two related chemokines, is markedly enhanced by adrenalectomy and attenuated by dexamethasone. This work provided the basis for an in vivo screening project that identified 36 GARG cDNAs induced in the lung during endotoxemia. The majority represent genes of unknown function, or genes not previously implicated in the pulmonary response to inflammation. Four encode previously undescribed proteins, including a chemokine, a member of a family of guanylate-binding proteins, a 2'-5' oligoadenylate synthetase-like protein, and a novel lung-inducible Neuralized-related C3HC4 RING protein (LINCR). Our results indicate that glucocorticoid-attenuated response genes are much more diverse than originally anticipated. Future studies using microarrays in this and other inflammation models may identify many additional glucocorticoid-regulated genes potentially important in inflammatory diseases.

Adrenalectomy↗
PubMed1993

Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal.

We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.

Amino Acid Sequence↗
PubMed2005

From target identification to drug screening assays for neurodegenerative diseases.

Treatment of neurodegenerative diseases represents a major challenge for the pharmaceutical industry. Key to developing novel and efficacious therapeutics is the discovery of new druggable targets. Toward this aim, the current drug discovery process is strongly relying on the improved understanding of disease mechanisms and on a synergistic approach with chemistry, molecular biology and robotics. In this scenario, we present the case of a newly discovered molecular mechanism that may be of interest for drug discovery programmes in Huntington's disease and other neurodegenerative diseases.

Animals↗
PubMed2001

Pharmaceutical target identification by gene expression analysis.

The majority of newly-identified genes in the human genome show no significant sequence similarity to genes whose function is known, so they are not easily recognized as potential drug targets. Expression analysis is an alternative method to suggest possible functions of genes. We review statistical methods for gene expression analysis to identify potential pharmaceutical targets. Specifically, we illustrate the analysis of differential gene expression (using discriminant analysis, t-tests, and analysis of variance) and co-expression (using correlation, clustering, and chi-square). We present an example of the use of expression analysis to identify co-expressed cardiomyopathy-associated genes.

Analysis of Variance↗
PubMed2004

Synthesis and target identification of hymenialdisine analogs.

Hymenialdisine (HMD) is a sponge-derived natural product kinase inhibitor with nanomolar activity against CDKs, Mek1, GSK3beta, and CK1 and micromolar activity against Chk1. In order to explore the broader application of the pyrrolo[2,3-c]azepine skeleton of HMD as a general kinase inhibitory scaffold, we searched for additional protein targets using affinity chromatography in conjunction with the synthesis of diverse HMD analogs and profiled HMD against a panel of 60 recombinant enzymes. This effort has led to nanomolar to micromolar inhibitors of 11 new targets including p90RSK, KDR, c-Kit, Fes, MAPK1, PAK2, PDK1, PKCtheta, PKD2, Rsk1, and SGK. The synthesis of HMD analogs has resulted in the identification of compounds with enhanced and/or dramatically altered selectivities relative to HMD (28n) and in molecules with antiproliferative activities 30-fold higher than HMD (28p).

Amino Acid Sequence↗
PubMed2006

In silico-guided target identification of a scaffold-focused library: 1,3,5-triazepan-2,6-diones as novel phospholipase A2 inhibitors.

A collection of 2150 druggable active sites from the Protein Data Bank was screened by high-throughput docking to identify putative targets for five representative molecules of a combinatorial library sharing a 1,3,5-triazepan-2,6-dione scaffold. Five targets were prioritized for experimental evaluation by computing enrichment in individual protein entries among the top 2% scoring targets. Out of the five proposed proteins, secreted phospholipase A2 (sPLA2) was shown to be a true target for a panel of 1,3,5-triazepan-2,6-diones which exhibited micromolar affinities toward two human sPLA2 members.

Azepines↗
PubMed2024

An integrated in-silico approach for drug target identification in human pathogen Shigella dysenteriae.

Shigella dysenteriae, is a Gram-negative bacterium that emerged as the second most significant cause of bacillary dysentery. Antibiotic treatment is vital in lowering Shigella infection rates, yet the growing global resistance to broad-spectrum antibiotics poses a significant challenge. The persistent multidrug resistance of S. dysenteriae complicates its management and control. Hence, there is an urgent requirement to discover novel therapeutic targets and potent medications to prevent and treat this disease. Therefore, the integration of bioinformatics methods such as subtractive and comparative analysis provides a pathway to compute the pan-genome of S. dysenteriae. In our study, we analysed a dataset comprising 27 whole genomes. The S. dysenteriae strain SD197 was used as the reference for determining the core genome. Initially, our focus was directed towards the identification of the proteome of the core genome. Moreover, several filters were applied to the core genome, including assessments for non-host homology, protein essentiality, and virulence, in order to prioritize potential drug targets. Among these targets were Integration host factor subunit alpha and Tyrosine recombinase XerC. Furthermore, four drug-like compounds showing potential inhibitory effects against both target proteins were identified. Subsequently, molecular docking analysis was conducted involving these targets and the compounds. This initial study provides the list of novel targets against S. dysenteriae. Conclusively, future in vitro investigations could validate our in-silico findings and uncover potential therapeutic drugs for combating bacillary dysentery infection.

Shigella dysenteriae↗
PubMed1998

INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1.

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13-amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11-amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1(Hsalpha). Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.

Anaphase↗
PubMed2005

Target identification of the novel antiobesity agent tungstate in adipose tissue from obese rats.

Adipose tissue plays an active role in the development of obesity, and thus characterization of the molecular changes related to obesity in this tissue is a priority. Recently, we identified tungstate as a potent body weight reducing agent in obese animals, adipose tissue being one of the targets of its action. In this study a proteomics approach combining 2-DE and MS was used to identify proteins associated with obesity and targets of tungstate in white adipose tissue. Twenty-nine proteins were found differentially expressed between lean and diet-induced obese rats. Expression changes in transferrin, vimentin, vinculin, peroxiredoxins, Rho-GTP dissociation inhibitor, grifin, guanine deaminase and 3-phosphoglycerate dehydrogenase were associated here for the first time with obesity. Furthermore, tungstate treatment of obese rats reverted expression changes of 70% of the proteins modulated by obesity and another ten proteins were regulated by tungstate independently of the body weight reduction. The results suggest that the tungstate antiobesity effect can be mediated by the modulation of cellular structure, metabolism, redox state and signalling processes in adipose tissue. These findings open new avenues for the study of the aetiology of obesity and its treatment.

Adipose Tissue↗
PubMed2006

Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study.

Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation.

Animals↗
PubMed2000

Mechanism-based target identification and drug discovery in cancer research.

Cancer as a disease in the human population is becoming a larger health problem, and the medicines used as treatments have clear limitations. In the past 20 years, there has been a tremendous increase in our knowledge of the molecular mechanisms and pathophysiology of human cancer. Many of these mechanisms have been exploited as new targets for drug development in the hope that they will have greater antitumor activity with less toxicity to the patient than is seen with currently used medicines. The fruition of these efforts in the clinic is just now being realized with a few encouraging results.

Antineoplastic Agents↗
PubMed2000

New approaches for cell-specific targeting: identification of cell-selective peptides from combinatorial libraries.

Peptides that recognize specific cell types promise to be valuable tools both in research and clinical applications. Cell-specific peptides can be useful as drug delivery vehicles, diagnostic agents, affinity reagents for cell purification, gene therapy delivery agents, and research tools to probe the nature of a cell's surface. Recently, cell-specific targeting-peptides have been identified by phage-display selections against purified cell-surface markers, whole cells in tissue culture, and even tissues within live animals. These methods for identifying cell-targeting peptides will certainly increase the tools available to the scientist for cell-specific targeting.

Animals↗
PubMed2005

Target phase: an extra dimension for fish and plankton target identification.

The acoustic signal backscattered from a fish in water, Lbs(f), at a frequency, f, differs from the incident signal in both magnitude, /Lbs(f)/, and phase, arg[Lbs(f)], and it has been common practice for many years to use the backscatter magnitude from individual fish as an aid to species identification. However, very little use has been made of the phase of the backscattered signal relative to that of the incident acoustic pulse. If the gross phase changes due to propagation through water are compensated for, the residual phase signature is found to contain useful target-specific information. The phase signature can be characterized by estimating the rate at which the echo phase changes, relative to the transmitted pulse, during the echo from a fish. Clear groups are produced when single fish targets from in situ data are plotted in complex target space (target strength versus target rate of change of phase) and this is explored here by computing the acoustic backscatter, in complex target space, from a series of simple large and small model fish targets, both with and without gas-filled swimbladders, using a Monte Carlo technique. It is shown that all the features found in the in situ data can be explained in terms of the size and attitude of the fish. The modeling was carried out at the frequency of 38 kHz, which pertained to the in situ data, and the specific results only apply to this frequency. However, the complex target approach is generally applicable to fish target strength analysis independently of frequency.

Acoustics↗
PubMed2006

Mechanisms of drug action and drug resistance in Leishmania as basis for therapeutic target identification and design of antileishmanial modulators.

The control of Leishmania infections relies primarily on chemotherapy. The arsenal of drugs available against Leishmania infections is limited and includes pentavalent antimonials, pentamidine, amphotericin B, miltefosine, paromomycin, allopurinol, and few other drugs at various stages of their development process. Knowledge about action and resistance mechanisms involved may allow the development of new drugs that minimise or circumvent drug resistance or may identify new targets for drug development. The aim of this review is to propose some chemical topics to design new modulators from the mechanisms of action of drugs and resistance mechanisms to drugs used in the clinic against Leishmania infections. Thus, different classes of ABC transporters extrude antimonials in Leishmania resulting in drug-resistant phenotypes. Compounds interfering with thiol and polyamine metabolism could be designed to inhibit the antimonial detoxication and therefore, such compounds could be used in combination with antimonials. New diamidines could be synthesized in regard to their ability to inhibit topoisomerase II. The challenge for amphotericin B is to be absorbed by oral route requiring labile physico-chemical modifications. New sesquiterpens and flavonoids have to be developed as reversant of antimonial resistance. Although some studies have focused on developing inhibitors against these resistant phenotypes, new efficient modulators that are able to inhibit drug efflux are needed.

ATP-Binding Cassette Transporters↗
PubMed1994

Symmetry and asymmetry in the function of Escherichia coli integration host factor: implications for target identification by DNA-binding proteins.

BACKGROUND: Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in a wide variety of biochemical functions. In many of its activities, IHF appears to act as an architectural element, dramatically distorting the conformation of bound DNA. IHF is a dimer of non-identical subunits, each about 90 amino acids long. One dimer interacts specifically with a 30 base pair (bp) target, but well-conserved sequences are found in only half of this binding site. Thus, the IHF-DNA system has long been viewed as a paradigm of asymmetry in a protein-DNA interaction. RESULTS: We have isolated the subunits of IHF and show that either subunit is capable of specifically recognizing natural IHF-binding sites and supporting lambda site-specific recombination in vitro. Mobility shift and footprinting data indicate that the isolated subunits interact with DNA as homodimers. We also describe the design of symmetric duplexes to which heterodimeric and homodimeric IHFs can bind by recognizing specific sequences. CONCLUSIONS: Our in vitro manipulation of the IHF system demonstrates that binding and bending of target DNA can be accomplished symmetrically. The prevalence of asymmetry found for this system in nature suggests that additional selective forces may operate. We suggest that these follow from the disparity between the size of the DNA that IHF protects (30 bp) and the length of DNA that the protein can initially contact (10 bp). This disparity implies that an IHF target is recognized in stages and may dispose the part of the protein-DNA system used for initial recognition to evolve distinctly from the remainder of the interaction surface. We suggest that a limitation in the length of DNA that can be initially contacted is a general property of DNA-binding proteins. In that case, many proteins can be expected to identify complex targets by step-wise, rather than simultaneous, contact between sequence elements and DNA-binding domains.

Bacterial Proteins↗
PubMed2002

Small molecule affinity fingerprinting. A tool for enzyme family subclassification, target identification, and inhibitor design.

Classifying proteins into functionally distinct families based only on primary sequence information remains a difficult task. We describe here a method to generate a large data set of small molecule affinity fingerprints for a group of closely related enzymes, the papain family of cysteine proteases. Binding data was generated for a library of inhibitors based on the ability of each compound to block active-site labeling of the target proteases by a covalent activity based probe (ABP). Clustering algorithms were used to automatically classify a reference group of proteases into subfamilies based on their small molecule affinity fingerprints. This approach was also used to identify cysteine protease targets modified by the ABP in complex proteomes by direct comparison of target affinity fingerprints with those of the reference library of proteases. Finally, experimental data were used to guide the development of a computational method that predicts small molecule inhibitors based on reported crystal structures. This method could ultimately be used with large enzyme families to aid in the design of selective inhibitors of targets based on limited structural/function information.

Algorithms↗