Rational Drug Target Research Articles

Screening baccharin analogs as selective inhibitors against type 5 17β-hydroxysteroid dehydrogenase (AKR1C3)

Aldo–keto reductase 1C3 (AKR1C3), also known as type 5 17β-hydroxysteroid dehydrogenase, is a downstream steroidogenic enzyme and converts androgen precursors to the potent androgen receptor ligands: testosterone and 5α-dihydrotestosterone. Studies have shown that AKR1C3 is involved in the development of castration resistant prostate cancer (CRPC) and that it is a rational drug target for the treatment of CRPC. Baccharin, a component of Brazilian propolis, has been observed to exhibit a high inhibitory potency and selectivity for AKR1C3 over other AKR1C isoforms and is a promising lead compound for developing more potent and selective inhibitors. Here, we report the screening of fifteen baccharin analogs as selective inhibitors against AKR1C3 versus AKR1C2 (type 3 3α-hydroxysteroid dehydrogenase). Among these analogs, the inhibitory activity and selectivity of thirteen compounds were evaluated for the first time. The substitution of the 4-dihydrocinnamoyloxy group of baccharin by an acetate group displayed nanomolar inhibitory potency (IC50: 440nM) and a 102-fold selectivity over AKR1C2. By contrast, when the cinnamic acid group of baccharin was esterified, there was a dramatic decrease in potency and selectivity for AKR1C3 in comparison to baccharin. Low or sub-micromolar inhibition was observed when the 3-prenyl group of baccharin was removed, and the selectivity over AKR1C2 was low. Although unsubstituted baccharin was still the most potent (IC50: 100nM) and selective inhibitor for AKR1C3, these data provide structure–activity relationships required for the optimization of new baccharin analogs. They suggest that the carboxylate group on cinnamic acid, the prenyl group, and either retention of 4-dihydrocinnamoyloxy group or acetate substituent on cinnamic acid are important to maintain the high potency and selectivity for AKR1C3.

Neurodegenerative Diseases: Phenome to Genome Analysis

Genome-Wide Association Studies (GWAS) tools are an attractive starting point for disease target discovery. Genetic association evidence provides a strong link to disease phenotypes across diverse human DNAs. The National Center for Biotechnology Information (NCBI) Phenome-Genome Integrator tool (PheGenI) was used to identify genes associated with neurodegenerative disease phenotypes (NeuroGenes). Four hundred ninty-nine known proteins and 30 uncharacterized proteins were found to be associated with diverse neurological diseases including Alzeheimer’s, amyotrophic lateral sclerosis (ALS), epilepsy, multiple scelerosis, stroke and Parkinson’s. The NeuroGenes also were associated with other diseases and associated disorders. Using diverse bioinformatics and proteomics tools, an atlas of the NeuroGenes was created to facilitate rational biomarker and drug target discovery. NeuroGenes protein expression was detected in diverse body fluids. The NeuroGenes were categorized into functional classes of proteins using protein motif and domains analysis tools. Expression Quantitative Trait Loci (eQTL) analysis identified single nucleotide polymorphisms (SNPs) associated with mRNA expression across diverse sets of samples from Alzheimer’s disease, Parkinson’s disease, brain infarction, multiple sclerosis, schizophrenia, stroke, mental retardation and neurological cancers. Thirty uncharacterized proteins encompassing structural protein, translation initiation factor, GDP/GTP exchange factor (GEF), transmembrane proteins, tubulin/histonebinding and a lysosomal hydrolase were identified as putative proteins for dementia, Parkinson’s, neurobehavior, multiple scelerosis and ALS. These results may provide new biomarkers for neurological diseases.

Untangling the ATR-CHEK1 network for prognostication, prediction and therapeutic target validation in breast cancer

ATR-CHEK1 signalling is critical for genomic stability. ATR-CHEK1 signalling may be deregulated in breast cancer and have prognostic, predictive and therapeutic significance. We investigated ATR, CHEK1 and phosphorylated CHEK1 Ser345 protein (pCHEK1) levels in 1712 breast cancers. ATR and CHEK1 mRNA expression was evaluated in 1950 breast cancers. Pre-clinically, biological consequences of ATR gene knock down or ATR inhibition by the small molecule inhibitor (VE-821) were investigated in MCF7 and MDA-MB-231 breast cancer cell lines and in non-tumorigenic breast epithelial cells (MCF10A). High ATR and high cytoplasmic pCHEK1 levels were significantly associated with higher tumour stage, higher mitotic index, pleomorphism and lymphovascular invasion. In univariate analyses, high ATR and high cytoplasmic pCHEK1 levels were associated with poor breast cancer specific survival (BCSS). In multivariate analysis, high ATR level remains an independent predictor of adverse outcome. At the mRNA level, high CHEK1 remains associated with aggressive phenotypes including lymph node positivity, high grade, Her-2 overexpression, triple negative, aggressive molecular phenotypes and adverse BCSS. Pre-clinically, CHEK1 phosphorylation at serine345 following replication stress was impaired in ATR knock down and in VE-821 treated breast cancer cells. Doxycycline inducible knockdown of ATR suppressed growth, which was restored when ATR was re-expressed. Similarly, VE-821 treatment resulted in a dose dependent suppression of cancer cell growth and survival (MCF7 and MDA-MB-231) but was less toxic in non-tumorigenic breast epithelial cells (MCF10A). We provide evidence that ATR and CHEK1 are promising biomarkers and rational drug targets for personalized therapy in breast cancer.

NTRK1 Fusion in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, yet with no targeted therapy with substantial survival benefit. Recent studies on solid tumors showed that fusion genes often play driver roles and are promising targets for pharmaceutical intervention. To survey potential fusion genes in GBMs, we analysed RNA-Seq data from 162 GBM patients available through The Cancer Genome Atlas (TCGA), and found that 3′ exons of neurotrophic tyrosine kinase receptor type 1 (NTRK1, encoding TrkA) are fused to 5′ exons of the genes that are highly expressed in neuronal tissues, neurofascin (NFASC) and brevican (BCAN). The fusions preserved both the transmembrane and kinase domains of NTRK1 in frame. NTRK1 is a mediator of the pro-survival signaling of nerve growth factor (NGF) and is a known oncogene, found commonly altered in human cancer. While GBMs largely lacked NTRK1 expression, the fusion-positive GBMs expressed fusion transcripts in high abundance, and showed elevated NTRK1-pathway activity. Lentiviral transduction of the NFASC-NTRK1 fusion gene in NIH 3T3 cells increased proliferation in vitro, colony formation in soft agar, and tumor formation in mice, suggesting the possibility that the fusion contributed to the initiation or maintenance of the fusion-positive GBMs, and therefore may be a rational drug target.

Abstract LB-293: Phase II study of the oral AKT inhibitor, MK-2206, for acute myeloid leukemia (AML) in second relapse.

Abstract Outcomes of adults with AML remain unsatisfactory. To explore the role of the AKT signaling pathway in AML, we examined samples from AML patients (pts) utilizing reverse phase protein arrays (RPPAs). High levels of the phosphorylated Ser473 form of AKT were associated with shorter remission (p=0.03) and shorter overall survival (p=0.09) in 92 newly diagnosed AML pats with intermediate-risk cytogenetics undergoing induction chemotherapy, identifying AKT as rational drug target. We studied the effects of MK-2206, an investigative oral, highly specific allosteric inhibitor of the 3 isoforms of human AKT, in human AML. MK-2206 dephosphorylated AKT and inhibited growth of AML cell lines and primary AML blasts at low-micromolar concentrations. We then conducted a phase 2 trial of MK-2206 (200 mg once weekly) in adult pats who had failed 1 other therapy for persistent AML or relapsed after a prior CR <12 months, a population with expected CR rates of <5%. Nineteen pats (18 evaluable) with median age 70 (31-86) years were enrolled, one of whom had isolated extramedullary relapse. Nine pats had prior MDS; 5 had normal cytogenetics, 5 had complex cytogenetics or del7, 3 trisomy 8, and 6 had other cytogenetic changes; no patients had FLT3 mutations. Median number of cycles was 2; 4 pats completed ≥ 3 cycles. One response (a CRp) was observed (95% CI: 0-17%). This pat was a 77-yo man with a normal karyotype AML and prior history of CMML, who failed 2 prior regimens including high-dose cytarabine, and continued on MK-2206 for a total of 11 cycles after which he progressed. The most common grade 3/4 adverse event (AE) related to MK-2206 was pruritic rash occurring in 6/18 pats (30%). Other frequent MK-2206-related AEs were rash (n=6; grade 1/2), hyperglycemia (n=12; all grade 1/2 except for one grade 4), QTC prolongation (n=3; all grade 1). We used RPPAs to assess target inhibition in peripheral blood (PB) in 8 pats prior to and 24 hours after the first MK-2206 dose, and in bone marrow (BM) prior to and after 28 days in 5 pats. We examined (a) the direct target, (b) immediate downstream targets (20 total), and (c) all 157 proteins assayed. Considered alone, the decrease in Ser473 AKT was significant (t-test p=0.018). The larger sets show no significant changes after adjusting for multiple testing. Decrease in Ser473 AKT (median 28%; range, 12-45%) was seen in 5/8 PB and 3/5 BM samples; and in Thr308 pAKT in 5/8 PB and 4/5 BM samples. In turn, changes in AKT targets were less prevalent (downregulation of pFOX3A in 3/5, pPRAS40 in 2/5, pS6K in 4/5 and p4EBP1 in 2/5 PB samples), possibly due to incomplete AKT inhibition. Our results indicate single-agent MK-2206 has limited activity in this pat population, with rash being the dose-limiting toxicity precluding further dose escalation. The modest inhibitory effects on AKT and its downstream targets in our RPPA studies suggest that other approaches to block this pathway should be explored in the AML setting. Citation Format: Marina Y. Konopleva, Roland B. Walter, Stefan Faderl, Elias Jabbour, Zhihong Zeng, Gautam Borthakur, Peter Ruvolo, Xuelin Huang, Tapan Kadia, Jennie Feliu, Jan A. Burger, Michael Andreeff, Wenbin Liu, Steven M. Kornblau, Keith Baggerly, Elihu Estey, Hagop Kantarjian. Phase II study of the oral AKT inhibitor, MK-2206, for acute myeloid leukemia (AML) in second relapse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-293. doi:10.1158/1538-7445.AM2013-LB-293

Safety and efficacy of CYT387, a JAK1 and JAK2 inhibitor, in myelofibrosis

JAK-STAT is a rational drug target in myelofibrosis (MF) given its association with JAK2/MPL mutations and aberrant inflammatory cytokine expression. We conducted a Phase 1/2 trial of CYT387, a potent JAK1/2 inhibitor, in patients with high- or intermediate-risk primary or post-polycythemia vera/essential thrombocythemia MF. Pre-planned safety and efficacy analysis has been completed for the initial 60 patients. In the dose-escalation phase (n=21), the maximum-tolerated dose was 300 mg/day based on reversible grade 3 headache and asymptomatic hyperlipasemia. Twenty-one and 18 additional patients were accrued at two biologically effective doses, 300 mg/day and 150 mg/day, respectively. Anemia and spleen responses, per International Working Group criteria, were 59% and 48%, respectively. Among 33 patients who were red cell-transfused in the month prior to study entry, 70% achieved a minimum 12-week period without transfusions (range 4.7–>18.3 months). Most patients experienced constitutional symptoms improvement. Grade 3/4 adverse reactions included thrombocytopenia (32%), hyperlipasemia (5%), elevated liver transaminases (3%) and headache (3%). New-onset treatment-related peripheral neuropathy was observed in 22% of patients (sensory symptoms, grade 1). CYT387 is well tolerated and produces significant anemia, spleen and symptom responses in MF patients. Plasma cytokine and gene expression studies suggested a broad anticytokine drug effect.

Abstract PR8: Network modeling of epithelial-to-mesenchymal transition in liver cancer metastasis

Abstract Hepatocellular carcinoma (HCC) is the third most common cause of cancer death in the world and its incidence is rising in the United States. Over 90% of cancer deaths occur due to tumor metastasis. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to leave the primary tumor site and establish distant metastases. Cells undergoing EMT lose cell-cell adhesion properties and change cell morphology in order to migrate into the blood stream. Loss of expression of E-cadherin, a cell adhesion protein, is considered the hallmark of EMT, and molecular analyses have revealed complex signaling pathways regulating E-cadherin expression. The hepatocyte growth factor (HGF)/C-met receptor axis is a major EMT inducer and is dysregulated in 40% of HCC. TGF-beta signaling, another important EMT inducer, is dysregulated in 20% of HCC. To systematically understand signaling components that regulate HCC metastasis, we constructed an EMT network (74 nodes, 143 edges) by integrating the signaling pathways involved in developmental EMT and known dysregulations in metastatic HCC. This network was subsequently translated into a predictive, discrete, dynamic model. Using HGF and TGF-beta1 as our EMT inducers and E-cadherin expression as our EMT marker, we reveal that the EMT process, although robust, appears to be targetable through inhibition of a small subset of critical (EMT “driver”) nodes. Our model suggests the following: 1) A critical eight-node transcriptional network that is downstream of major growth signals is necessary for EMT induction. 2) Cross-talk with TGF-beta and Notch signaling pathways exists and coordinated inhibition of both pathways will inhibit HGF-induced EMT. 3) Of the 135 nodes that did not block EMT transmission (termed “passenger” nodes), when combined with other EMT “passengers,” 11 combination knockouts were able to inhibit transmission of the EMT signal. 5) Analysis of the states in the basin of attraction of the “EMT negative” steady states has revealed a critical subnetwork (a strongly connected component of the larger EMT network) for EMT transmission. Initial testing has revealed that Snail1 inhibition blocks TGF-Beta1 induced EMT. Further predictions for nodes that inhibit EMT are currently being tested with a novel in vitro EMT screen. These results reveal network modeling as an important tool for identifying critical mediators in biological processes. Furthermore, we propose network modeling as a tool for rational drug targeting of disease pathways, specifically in liver cancer metastasis. This proffered talk is also presented as Poster A12. Citation Format: Steven N. Steinway, Hien Dang, Wei Ding, Carl B. Rountree, Reka Albert. Network modeling of epithelial-to-mesenchymal transition in liver cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr PR8.

Rapid rational drug targeting of Merkel cell polyomavirus (MCV)-positive Merkel cell carcinoma (MCC) using the survivin inhibitor YM155.

8577 Background: MCC is an aggressive, chemoresistant skin cancer causing more deaths each year than chronic myelogenous leukemia. We discovered a new virus, Merkel cell polyomavirus (MCV), clonally integrated into ~80% of primary and metastatic MCC in 2008. To find therapeutic targets for this cancer, we examined cellular genes perturbed by MCV infection. Methods: Digital transcriptome subtraction was used to discover MCV and also to reveal survivin gene (BIRC5) upregulation in virus-positive tumors. MCV T antigen knockdown studies in seven MCC lines and large T (LT) transduction into BJ fibroblasts were used to confirm this. Drug screening was performed in vitro using Cell-Titer Glo assays in a two stage analysis. In vivo screening used an MKL-1 (MCV+) MCC NOD-SCIDg mouse xenograft model with a single three-week treatment round. Results: MCV large T oncoprotein induces survivin transcription through retinoblastoma protein sequestration by the LT LXCXE motif. MCV T antigen knockdown results in nonapoptotic MCC cell death and loss of survivin expression. YM155, a phase II survivin transcription inhibitor, causes MCV+ MCC cell necroptosis associated with autophagy at 1-12 nM EC50. Of 1359 other drugs from LOPAC and NCI Oncology Set II libraries, only bortezomib had in vitro potency comparable to YM155. In MKL-1 xenograft studies, mice were treated with saline, bortezomib or YM155 for three weeks using standard dosings. Bortezomib did not significantly improve mouse survival (33%) over saline (24%) during treatment. In contrast, all YM155-treated mice survived (100%, p<0.001) the 3 week treatment period. Tumors resumed growth once YM155 treatment was stopped suggesting that YM155 is cytostatic in vivo rather than cytotoxic. Conclusions: Survivin expression is induced by MCV LT and is critical to MCV+ MCC survival. A survivin inhibitor, YM155 was nontoxic to mice and cytostatic for MCV+ MCC xenografts. Using genomic technologies, in less than four years, the primary viral cause for most MCC was discovered, new diagnostic tests developed and a promising rational drug candidate identified. A cooperative group trial (E1611) for YM155 and bortezomib in MCC patients is currently planned.

Con: The Case for Expanded Lung Cancer Research Support

Con: The Case for Expanded Lung Cancer Research Support

What if Gut Hormones Aren't Really Hormones: DPP-4 Inhibition and Local Action of GLP-1 in the Gastrointestinal Tract

It is increasingly clear that the gastrointestinal (GI) tract plays a major role in glucose regulation after meal ingestion. A prominent mechanism of GI influence over blood glucose is through the secretion of glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 (GLP-1), together termed incretins because of their actions to stimulate internal secretions (i.e., insulin). Indeed, common estimates are that 70% of β-cell release after meals is attributable to the incretins, and both of these peptides are necessary for normal glucose tolerance (1). Moreover, iv administration of GLP-1, and to a much lesser degree glucose-dependent insulinotropic polypeptide, enhances insulin secretion and improves glucose disposal in persons with type 2 diabetes. This has led to the development of two new classes of medication for diabetes, GLP-1 receptor agonists, which mimic the effects of endogenous GLP-1, and dipeptidyl peptidase IV (DPP-4) inhibitors, which prevent metabolism of the native peptide and extend its activity (2).

The novel JAK inhibitor CYT387 suppresses multiple signalling pathways, prevents proliferation and induces apoptosis in phenotypically diverse myeloma cells

Janus kinases (JAKs) are involved in various signalling pathways exploited by malignant cells. In multiple myeloma (MM), the interleukin-6/JAK/signal transducers and activators of transcription (IL-6/JAK/STAT) pathway has been the focus of research for a number of years and IL-6 has an established role in MM drug resistance. JAKs therefore make a rational drug target for anti-MM therapy. CYT387 is a novel, orally bioavailable JAK1/2 inhibitor, which has recently been described. This preclinical evaluation of CYT387 for treatment of MM demonstrated that CYT387 was able to prevent IL-6-induced phosphorylation of STAT3 and greatly decrease IL-6- and insulin-like growth factor-1-induced phosphorylation of AKT and extracellular signal-regulated kinase in human myeloma cell lines (HMCL). CYT387 inhibited MM proliferation in a time- and dose-dependent manner in 6/8 HMCL, and this was not abrogated by the addition of exogenous IL-6 (3/3 HMCL). Cell cycling was inhibited with a G(2)/M accumulation of cells, and apoptosis was induced by CYT387 in all HMCL tested (3/3). CYT387 synergised in killing HMCL when used in combination with the conventional anti-MM therapies melphalan and bortezomib. Importantly, apoptosis was also induced in primary patient MM cells (n=6) with CYT387 as a single agent, and again synergy was seen when combined with conventional therapies.

Pilot and feasibility study: prospective proteomic profiling of mammary epithelial cells from high-risk women provides evidence of activation of pro-survival pathways

Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.

Cloning, expression, purification and kinetics of trehalose-6-phosphate phosphatase of filarial parasite Brugia malayi

The pleiotropic functions of disaccharide trehalose in the biology of nematodes and its absence from mammalian cells suggest that its biosynthesis may provide a useful target for developing novel nematicidal drugs. The trehalose-6-phosphate phosphatase (TPP), one of the enzymes of trehalose metabolism has not been characterized so far in nematodes except the free living nematode Caenorhabditis elegans where it's silencing results into lethal outcomes. This prompted us to clone and characterize Brugia malayi TPP in order to discover novel antifilarial drug target. The recombinant protein (Bm-TPP) was purified with apparent homogeneity on a metal ion column and it was found to possess high phosphatase activity with robust specificity for the substrate trehalose-6-phosphate. Bm-TPP was found to be a member of the HAD-like hydrolase super family II based on the conserved motifs required for catalytic reaction. The Km for substrate trehalose-6-phosphate was around 0.42mM with pH optimum ∼7.0 and the enzyme showed an almost absolute requirement for Mg2+ as a metal ion. Bm-TPP was expressed in all the life-stages of B. malayi. In the absence of an effective macrofilaricidal agent and validated antifilarial drug target, Bm-TPP bodes well as a rational drug target against lymphatic filariasis.

Targeting peripheral afferent nerve terminals for cough and dyspnea

Chronic unproductive coughing and dyspnea are symptoms that severely diminish the quality of life in a substantial proportion of the population. There are presently few if any drugs that effectively treat these symptoms. Rational drug targets for cough and dyspnea have emerged over the recent years based on developments in our understanding of the innervation of the respiratory tract. These drug targets can be subcategorized into those that target the vagal afferent nerve endings, and those that target neural activity within the CNS. This review focuses on targets presumed to be in the peripheral terminals of afferent nerves within the airways. Conceptually, the activity of peripheral afferent nerves involved with unwanted urge-to-cough or dyspnea sensations can be inhibited by limiting the intensity of the stimulus, inhibiting the amplitude of the stimulus-induced generator potential, or inhibiting the transduction between the generator potential and action potential discharge and conduction. These mechanisms reveal many therapeutic strategies for anti-tussive and anti-dyspnea drug development with peripheral sites of action.

In silico genome‐scale modeling and analysis for identifying anti‐tubercular drug targets

AbstractMycobacterium tuberculosis is the deadly pathogen responsible for causing tuberculosis in humans, continuing to infect and kill millions of people globally. Despite the availability of a number of anti‐tuberculosis drugs and advances in high‐throughput drug discovery technology there is an urgent need for designing novel anti‐tubercular treatments due to growing parasite resistance and compromised immune systems in some patients. Therefore, it is highly necessary to develop systematic approaches that can facilitate the drug discovery by identification of drug targets in effective and efficient ways. In this sense, with the availability of whole genome sequence, application of genome‐scale modeling is becoming increasingly important for deriving rational drug target identification. This approach is indeed powerful in unraveling the metabolic behavior of pathogens and helps in identifying most relevant metabolites/genes as drug targets, which are experimentally testable. Herein, we present a review on the application of genome‐scale modeling and analysis in the context of identification of anti‐tubercular drug targets. Drug Dev Res 72: 121–129, 2011. © 2010 Wiley‐Liss, Inc.

Molecular Chaperones as Rational Drug Targets for Parkinsons Disease Therapeutics

Parkinson's disease is a neurodegenerative movement disorder that is caused, in part, by the loss of dopaminergic neurons within the substantia nigra pars compacta of the basal ganglia. The presence of intracellular protein aggregates, known as Lewy bodies and Lewy neurites, within the surviving nigral neurons is the defining neuropathological feature of the disease. Accordingly, the identification of specific genes mutated in families with Parkinson's disease and of genetic susceptibility variants for idiopathic Parkinson's disease has implicated abnormalities in proteostasis, or the handling and elimination of misfolded proteins, in the pathogenesis of this neurodegenerative disorder. Protein folding and the refolding of misfolded proteins are regulated by a network of interactive molecules, known as the chaperone system, which is composed of molecular chaperones and co-chaperones. The chaperone system is intimately associated with the ubiquitin-proteasome system and the autophagy-lysosomal pathway which are responsible for elimination of misfolded proteins and protein quality control. In addition to their role in proteostasis, some chaperone molecules are involved in the regulation of cell death pathways. Here we review the role of the molecular chaperones Hsp70 and Hsp90, and the cochaperones Hsp40, BAG family members such as BAG5, CHIP and Hip in modulating neuronal death with a focus on dopaminergic neurodegeneration in Parkinson's disease. We also review current progress in preclinical studies aimed at targetting the chaperone system to prevent neurodegeneration. Finally, we discuss potential future chaperone-based therapeutics for the symptomatic treatment and possible disease modification of Parkinson's disease.

BACE: Therapeutic target and potential biomarker for Alzheimer's disease

β-Site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease involved in the production of Alzheimer's disease (AD) Aβ amyloid peptides. This enzyme is ubiquitously expressed, with highest levels in the brain and pancreas. Its cellular trafficking is tightly controlled as it recycles between endosomes and trans-Golgi network. BACE expression increases in response to aging and various stress stimuli. It is elevated in the brain cortex of AD sufferers, and increased levels of BACE in the cerebrospinal fluid of patients with mild cognitive impairment may provide an early biomarker of AD. BACE is considered as a rational drug target for AD therapy, and inhibitors are under development. Anomalies in the behaviour and biochemistry of BACE(-/-) mice have pointed to the role this enzyme plays in the processing of neuregulin and of voltage-gated sodium channel β-subunit. A full understanding of BACE biology in health and disease is needed to establish a safe AD therapy based on BACE inhibitors.

Efficacy of S-adenosylhomocysteine hydrolase inhibitors, D-eritadenine and (S)-DHPA, against the growth of Cryptosporidium parvum in vitro

D-eritadenine and (S)-DHPA are aliphatic adenosine analogues known to target S-adenosylhomocysteine hydrolase (SAHH) and potent antiviral compounds. In the present study, we demonstrate that these two compounds also display efficacy against recombinant SAHH enzyme of the protozoan parasite Cryptosporidium parvum, as well as inhibition of parasite growth in vitro. Our data confirm that SAHH could serve as a rational drug target in cryptosporidial infection and antiviral adenosine analogues are potential candidates for drug development against cryptosporidiosis.

Epigenetic Targets in Human Neoplasms

Epigenetic silencing of tumor suppressor genes is a salient feature of tumor cells. Re-expression of epigenetically silenced genes is a feasible and achievable strategy for cancer treatment. DNA methylation is the most characterized epigenetic silencing mechanism and the reversal of DNA methylation, genetically or pharmacologically, induces gene re-expression and proliferation arrest in tumor cells. Other epigenetic targets, such as histone acetylation and methylation, are also rational drug targets, and several small-molecule modulators of histone acetylation and methylation are currently under development or already in clinical trials. Epigenetic deregulation of miRNAs induces aberrant expression of miRNAs, which have been associated with the development and progression of cancer. The reversal of DNA methylation can induce the re-expression of miRNAs, and oligonucleotides can silence aberrantly expressed miRNAs. Evaluating the combination of different epigenetic modifiers and ensuring their optimization are the next challenges towards the establishment of epigenetic therapy.

Molecular cloning and characterization of a novel immunoreactive ATPase/RNA helicase in human filarial parasite Brugia malayi

DEAD box proteins are putative RNA unwinding proteins found in organisms ranging from mammals to bacteria. We have identified a novel immunodominant cDNA clone, BmL3-helicase, encoding DEAD box RNA helicase by immunoscreening of a larval stage cDNA library of Brugia malayi. The cDNA sequence exhibited strong sequence homology to Caenorhabditis elegans and C. briggsae RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. The clone also showed similarity with RNA helicase of Wolbachia, an endosymbiotic bacterium of filarial parasite. It was overexpressed as approximately 50 kDa His-tag fusion protein, and ATP hydrolysis assay of recombinant enzyme showed that either ATP or dATP was required for the unwinding activity, indicating BmL3-helicase as an ATP/dATP-dependent RNA helicase. The recombinant protein also demonstrated cross-seroreactivity with human bancroftian sera. The presence of BmL3-helicase in various life stages of B. malayi was confirmed by immunoblotting of parasite-life-cycle extracts with polyclonal sera against the BmL3-helicase, which showed high levels of expression in microfilaria, L(3,) and adult (both male and female) stages. In the absence of an effective macrofilaricidal agent and validated anti-filarial drug targets, RNA helicases could be utilized as a rational drug target for developing agents against the human filarial parasite.