Selective inhibitors of death in mutant huntingtin cells

Huntington disease (HD) is an inherited neurodegenerative disorder with unclear pathophysiology. We developed a high-throughput assay in a neuronal cell culture model of HD, screened 43,685 compounds and identified 29 novel selective inhibitors of cell death in mutant huntingtin-expressing cells. Four compounds were active in diverse HD models, which suggests a role for cell death in HD; these compounds are mechanistic probes and potential drug leads for HD.     

Atg4b-Dependent Autophagic Flux Alleviates Huntington’s Disease Progression

The accumulation of aggregated mutant huntingtin (mHtt) inclusion bodies is involved in Huntigton’s disease (HD) progression. Medium sized-spiny neurons (MSNs) in the corpus striatum are highly vulnerable to mHtt aggregate accumulation and degeneration, but the mechanisms and pathways involved remain elusive. Here we have developed a new model to study MSNs degeneration in the context of HD. We produced organotypic cortico-striatal slice cultures (CStS) from HD transgenic mice mimicking specific features of HD progression. We then show that induction of autophagy using catalytic inhibitors of mTOR prevents MSNs degeneration in HD CStS. Furthermore, disrupting autophagic flux by overexpressing Atg4b in neurons and slice cultures, accelerated mHtt aggregation and neuronal death, suggesting that Atg4b-dependent autophagic flux influences HD progression. Under these circumstances induction of autophagy using catalytic inhibitors of mTOR was inefficient and did not affect mHtt aggregate accumulation and toxicity, indicating that mTOR inhibition alleviates HD progression by inducing Atg4b-dependent autophagic flux. These results establish modulators of Atg4b-dependent autophagic flux as new potential targets in the treatment of HD.     

The discovery of substituted 4-(3-hydroxyanilino)-quinolines as potent RET kinase inhibitors

Substituted 4-(3-hydroxyanilino)-quinoline compounds, initially identified as small-molecule inhibitors of src family kinases, have been evaluated as potential inhibitors of RET kinase. Three compounds, 38, 31, and 40, had K(i)'s of 3, 25, and 50 nM in an in vitro kinase assay; while a cell based kinase assay showed K(i)'s of 300, 100, and 45 nM, respectively. These compounds represent potential new leads for the treatment of medullary and papillary thyroid cancer.     

5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives as inhibitors of full-length RORγt

Retinoid-related orphan receptor gamma-t (RORγt) belongs to the nuclear receptor superfamily that takes vital roles in the development and maturation of T-helper 17 cell (Th17) and lymph-node genesis. Because Th17 cells have been proved to be major effectors in human autoimmune and inflammatory diseases, the agonists and antagonists of RORγt have been discovered as promising leads for the therapeutics of these diseases. Most of the current studies of RORγt inhibitors have been focused on ligand binding domain (LBD) of RORγt because the structure and binding pockets of LBD have been elucidated and studied in detail. Recent research elucidated that the hinge domain (HD) of RORγt was significantly involved in the SUMOylation of RORγt and thus specifically affecting T cell development but not lymph-node genesis. These discoveries highlighted the potential of HD of RORγt as the target of RORγt inhibitors that could specifically inhibit Th17-related activities without affecting lymph-node genesis. In this study, we utilized a screening system with full-length RORγt including DBD, HD and LBD to evaluate the activities of a synthesized library of tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives. We identified a potent lead compound (28) that effectively inhibited Th17 cell differentiation. Docking and structure–activity relationship (SAR) studies showed that compound 28 may not bind in the binding pocket as most of the known inhibitors, but may bind in the pocket closed to Gln223 and Leu244 in HD. Our studies showed evidence that the HD of RORγt could afford a binding pocket for Th17 specific inhibitors and this domain should be further studied to discover potent and specific RORγt inhibitors.     

High-throughput identification of inhibitors of human mitochondrial peptide deformylase

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid-based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)-based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent.     

A profiling platform for the identification of selective metalloprotease inhibitors

Although proteases represent an estimated 5% to 10% of potential drug targets, inhibitors for metalloproteases (MPs) account for only a small proportion of all approved drugs, failures of which have typically been associated with lack of selectivity. In this study, the authors describe a novel and universal binding assay based on an actinonin derivative and show its binding activities for several MPs and its lack of activity toward all the non-MPs tested. This newly developed assay would allow for the rapid screening for inhibitors of a given MP and for the selectivity profiling of the resulting hits. The assay has successfully enabled for the first time simultaneous profiling of 8 well-known inhibitors against a panel of selected MPs. Previously published activities for these inhibitors were confirmed, and the authors have also discovered new molecular targets for some of them. The authors conclude that their profiling platform provides a generic assay solution for the identification of novel metalloprotease inhibitors as well as their selectivity profiling using a simple and homogeneous assay.     

New fluorogenic substrate for the first continuous steroid sulfatase assay

The screening for new inhibitors of steroid sulfatase requires an efficient test system. To overcome the shortcomings of the available discontinuous fluorimetric assay, several coumarin-type compounds were investigated as potential new substrates. 3,4-Benzocoumarin 7-O-sulfate was found to have appropriate substrate properties for the establishment of the first direct continuous assay of steroid sulfatase.     

The effect of the potential PhoQ histidine kinase inhibitors on Shigella flexneri virulence

PhoQ/PhoP is an important two-component system that regulates Shigella virulence. We explored whether the PhoQ/PhoP system is a promising target for new antibiotics against S. flexneri infection. By using a high-throughput screen and enzymatic activity coupled assay, four compounds were found as potential PhoQ inhibitors. These compounds not only inhibited the activity of SF-PhoQc autophosphorylation but also displayed high binding affinities to the SF-PhoQc protein in the Surface Plasmon Resonance response. A S. flexneri cell invasion assay showed that three of these potential PhoQ inhibitors inhibit the invasion of HeLa cells by S. flexneri 9380. In a Mouse Sereny test, mice inoculated with S. flexneri 9380 pre-treated with the potential PhoQ inhibitors 1, 2, 3 or 4 displayed no inflammation, whereas mice inoculated with S. flexneri 9380 alone displayed severe keratoconjunctival inflammation. All four potential PhoQ inhibitors showed no significant cytotoxicity or hemolytic activity. These data suggest that the four potential PhoQ inhibitors inhibited the virulence of S. flexneri and that PhoQ/PhoP is a promising target for the development of drugs against S. flexneri infection.     

Novel and highly sensitive fluorescent assay for leucine aminopeptidases

l-Leucine aminopeptidases (LAPs) are implicated in the progress of many pathological disorders and play some regulatory roles in tumor cell proliferation, invasion, and/or angiogenesis. Thus, LAPs not only could become new diagnostic or prognostic biomarkers but also may have potential as novel molecular targets for the treatment of several cancers. Highly sensitive assays are critical for early detection of changes in LAP activity and for screening potent LAP inhibitors. In this study, we developed a novel and highly sensitive fluorescent assay for LAPs based on substituted aminopyridines as fluorescent reporters. This assay was at least 100- and 20-fold more sensitive than commercial colorimetric and fluorescent LAP substrates, respectively. We also showed that this assay was a useful tool for monitoring LAP activities in extracts from cancer cell lines, as well as for the high-throughput screening of inhibitors, which could lead to new cancer treatments.     

A novel inhibitor of the insulin/IGF signaling pathway protects from age‐onset,neurodegeneration‐linked proteotoxicity

Aging manipulation is an emerging strategy aimed to postpone the manifestation of late‐onset neurodegenerative disorders such as Alzheimer's (AD) and Huntington's diseases (HD) and to slow their progression once emerged. Reducing the activity of the insulin/IGF signaling cascade (IIS), a prominent aging‐regulating pathway, protects worms from proteotoxicity of various aggregative proteins, including the AD‐associated peptide, Aβ‐ and the HD‐linked peptide, polyQ40. Similarly, IGF1 signaling reduction protects mice from AD‐like disease. These discoveries suggest that IIS inhibitors can serve as new drugs for the treatment of neurodegenerative maladies including AD and HD. Here, we report that NT219, a novel IIS inhibitor, mediates a long‐lasting, highly efficient inhibition of this signaling cascade by a dual mechanism; it reduces the autophosphorylation of the IGF1 receptor and directs the insulin receptor substrates 1 and 2 (IRS 1/2) for degradation. NT219 treatment promotes stress resistance and protects nematodes from AD‐ and HD‐associated proteotoxicity without affecting lifespan. Our discoveries strengthen the theme that IIS inhibition has a therapeutic potential as a cure for neurodegenerative maladies and point at NT219 as a promising compound for the treatment of these disorders through a selective manipulation of aging.     

Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies

Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent alpha-cyanoester and alpha-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC(50) values for a series of known inhibitors. The new IC(50) values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC(50) values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure-activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay.     

Increased prothrombin, apolipoprotein A-IV, and haptoglobin in the cerebrospinal fluid of patients with Huntington's disease

Huntington's disease (HD) is a progressive neurodegenerative disease caused by an unstable CAG trinucleotide repeat expansion. The need for biomarkers of onset and progression in HD is imperative, since currently reliable outcome measures are lacking. We used two-dimensional electrophoresis and mass spectrometry to analyze the proteome profiles in cerebrospinal fluid (CSF) of 6 pairs of HD patients and controls. Prothrombin, apolipoprotein A-IV (Apo A-IV) and haptoglobin were elevated in CSF of the HD patients in comparison with the controls. We used western blot as a semi-quantified measurement for prothrombin and Apo A-IV, as well as enzyme linked immunosorbent assay (ELISA) for measurement of haptoglobin, in 9 HD patients and 9 controls. The albumin quotient (Qalb), a marker of blood-brain barrier (BBB) function, was not different between the HD patients and the controls. The ratios of CSF prothrombin/albumin (prothrombin/Alb) and Apo A-IV/albumin (Apo A-IV/Alb), and haptoglobin level were significantly elevated in HD. The ratio of CSF prothrombin/Alb significantly correlated with the disease severity assessed by Unified Huntington's Disease Rating Scale (UHDRS). The results implicate that increased CSF prothrombin, Apo A-IV, and haptoglobin may be involved in pathogenesis of HD and may serve as potential biomarkers for HD.     

Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay

ABSTRACT: BACKGROUND: There is an urgent need for the discovery of new anti-malarial drugs. Thus, it is essential to explore different potential new targets that are unique to the parasite or that are required for its viability in order to develop new interventions for treating the disease. Plasmodium serine hydroxymethyltransferase (SHMT), an enzyme in the dTMP synthesis cycle, is a potential target for such new drugs, but convenient methods for producing and assaying the enzyme are still lacking, hampering the ability to screen inhibitors. METHODS: Production of recombinant Plasmodium falciparum SHMT (PfSHMT) and Plasmodium vivax SHMT (PvSHMT), using auto-induction media, were compared to those using the conventional Luria Bertani medium with isopropyl thio-beta-D-galactoside (LB-IPTG) induction media. Plasmodium SHMT activity, kinetic parameters, and response to inhibitors were measured spectrophotometrically by coupling the reaction to that of 5,10-ethylenetetrahydrofolate dehydrogenase (MTHFD). The identity of the intermediate formed upon inactivation of Plasmodium SHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). The active site environment of Plasmodium SHMT was probed based on changes in the fluorescence emission spectrum upon addition of amino acids and folate. RESULTS: Auto-induction media resulted in a two to three-fold higher yield of Pf- and PvSHMT (7.38 and 29.29 mg/L) compared to that produced in cells induced in LB-IPTG media. A convenient spectrophotometric activity assay coupling Plasmodium SHMT and MTHFD gave similar kinetic parameters to those previously obtained from the anaerobic assay coupling SHMT and 5,10-methylenetetrahydrofolate reductase (MTHFR); thus demonstrating the validity of the new assay procedure. The improved method was adopted to screen for Plasmodium SHMT inhibitors, of which some were originally designed as inhibitors of malarial dihydrofolate reductase. Plasmodium SHMT was slowly inactivated by thiosemicarbazide and formed a covalent intermediate, PLP-thiosemicarbazone. CONCLUSIONS: Auto-induction media offers a cost-effective method for the production of Plasmodium SHMTs and should be applicable for other Plasmodium enzymes. The SHMT-MTHFD coupled assay is equivalent to the SHMT-MTHFR coupled assay, but is more convenient for inhibitor screening and other studies of the enzyme. In addition to inhibitors of malarial SHMT, the development of species-specific, anti-SHMT inhibitors is plausible due to the presence of differential active sites on the Plasmodium enzymes.     

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC₅₀ for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.     

Relative responses of an X-ray-resistant hybrid cell-line and its parent line to X-irradiation, ultraviolet light, actinomycin D and cordycepin.

Using colony formation as an assay, a rat-mouse hybrid cell-line (HD1) and one of its parent lines (H4) have been studied as to their abilities to survive exposure to ionizing radiation, ultraviolet light, and the drugs actinomycin D and cordycepin. HD1 cells are more resistant than H4 to ionizing radiation, actinomycin D and cordycepin. Both cell lines respond similarly to ultraviolet light. When both cell-lines were co-treated with actinomycin D or cordycepin, the toxic effect of ionizing radiation was enhanced, whereas that of ultraviolet light (U.V.L.) was unchanged. The data suggest that RNA synthesis is more important immediately after irradiation with X-rays than with U.V.L. and that cells resistant to the toxic effect of ionizing radiation are also resistant to the toxicity induced by inhibitors of RNA synthesis.     

Mutant Huntingtin induces activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein (BNip3)

Huntington''s disease (HD) is a neurodegenerative disorder characterized by progressive neuronal death in the basal ganglia and cortex. Although increasing evidence supports a pivotal role of mitochondrial dysfunction in the death of patients'' neurons, the molecular bases for mitochondrial impairment have not been elucidated. We provide the first evidence of an abnormal activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNip3) in cells expressing mutant Huntingtin. In this study, we show an abnormal accumulation and dimerization of BNip3 in the mitochondria extracted from human HD muscle cells, HD model cell cultures and brain tissues from HD model mice. Importantly, we have shown that blocking BNip3 expression and dimerization restores normal mitochondrial potential in human HD muscle cells. Our data shed light on the molecular mechanisms underlying mitochondrial dysfunction in HD and point to BNip3 as a new potential target for neuroprotective therapy in HD.     

The time-dependent effect of 2,2′-dichlorodiethyl sulfide (sulfur mustard, HD, 1,1′-thiobis [2-chloroethane]) on the lymphocyte viability and the kinetics of protection by poly(ADP-ribose) polymerase inhibitors

2,2-Dichlorodiethyl sulfide (sulfur mustard, HD, 1,1-thiobis [2-chloroethane]) is a potent vesicant which can cause severe lesions to skin, lung, and eyes. Due to the high number of debilitating exposures during the Iran — Iraq war to the alkylating agent, HD, there is an increased interest in its mechanism of action and in the development of therapeutic interventions to prevent HD-induced lesions. Recently we reported anin vitro assay using human mononuclear leukocytes for studying HD-induced pathology. To study the time dependence of HD-induced mononuclear leukocyte cell death and to determine the parameters of any potential therapeutic intervention, an assay was developed and automated using a flow cytometer to measure propidium iodide exclusion by mononuclear cells. This assay demonstrated that HD-initiated cell death did not begin before 4 h post-exposlure, but after 4 h proceeded in a concentration-dependent manner. In this assay both niacinamide and 3-aminobenzamide, poly(ADP-ribose) polymerase inhibitors, were shown to be effective in blocking HD-induced cell death when added to the cultures during the first 4 h post-exposure. They offered partial protection when added between 6 and 12 h and were of no benefit when added after 12 h post-exposure.Abbreviations ADP adenosine diphosphate - DNA deoxyribonucleic acid - HD 2,2-dichlorodiethyl sulfide - NAD nicotinamide-adenine dinucleotide - PI propidium iodide - SD standard deviation     

Discovery of a potent angiotensin converting enzyme inhibitor via virtual screening

Prompted by the prominent role of angiotensin converting enzyme (ACE) in hypertension, heart failures, myocardial infarction and diabetic nephropathy, we have attempted to discover novel ACE inhibitors through ligand-based virtual screening. Molecular docking method and rigorously validated model was utilized to search a natural compounds database. Finally, 36 compounds were randomly selected and subjected to in vitro ACE kinase inhibitory assay using fluorescence assays method. The results showed that three compounds (Licochalcone A, Echinatin and EGCG) have strong potential to be developed as a new class of ACE inhibitors. Further chemical modification via fragment modifications guided by structure and ligand-based computational methodologies can lead to discover better agents as potential clinical candidates.