Abstract 15297: Discovery and Preclinical Optimization of Selective ABCA1 Inducers as Multifunctional Therapeutic Candidates

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Cutler Lewandowski ◽  
Md. Wasim Khan ◽  
Manel Ben Aissa ◽  
Brian Layden ◽  
Gregory Thatcher
Keyword(s):  
Liver Steatosis ◽  
Acid Synthesis ◽  
Luciferase Assay ◽  
Pharmacokinetic Parameters ◽  
Lead Compound ◽  
Binding Assays ◽  
Drug Candidates ◽  
Direct Binding ◽  
Isoform Selectivity

Introduction: Reduced expression of cholesterol transporter ABCA1 is critical in pathogenesis of type 2 diabetes (T2D) and related conditions, such as cardiovascular disease (CVD) and Alzheimer’s disease (AD). Thus, increasing ABCA1 represents a novel therapeutic strategy for these conditions. However, prior drug development efforts have achieved limited success at increasing ABCA1 (controlled by liver X receptor [LXR] β) while avoiding unwanted liver triglyceride production (through LXRα via transcription factor SREBP1c). Hypothesis: We hypothesized that phenotypic screening for selective ABCA1 inducers followed by medicinal chemistry optimization would bypass the isoform selectivity issues encountered in traditional target-based drug discovery and enable development of lead therapeutic candidates with preclinical efficacy and safety. Methods/Results: We screened 20k compounds for ABCA1 and SREBP1c-linked luciferase activity, followed by qPCR to validate and prioritize selective ABCA1-inducing hits. We synthesized ~70 structural analogs of the best hit, achieving substantial EC 50 (4.5 μM to 270 nM) and E max (3.5-fold to 6.0-fold vs. vehicle) improvements in ABCA1 luciferase assay while maintaining selectivity against SREBP1c. Direct binding assays confirmed selectivity for LXRβ vs. LXRα, corroborating cell-based data. Lead compounds enhanced cellular cholesterol efflux, reduced inflammation in vitro , and attenuated high-fat diet (HFD) induced weight gain, insulin resistance, and inflammation in mice. Metabolomics analysis revealed that our lead compound corrected HFD-induced perturbations in liver glucose and fatty acid synthesis. Finally, side effects associated with published LXR agonists - liver steatosis and neutropenia - were not observed with our compound. Conclusions: We established a platform to develop selective ABCA1 inducers as drug candidates. Via this platform, we identified a safe and efficacious lead compound for T2D. Our study also represents the first report of an LXR agonist characterized by metabolomics - a powerful tool to complement biochemical readouts. Continued optimization to improve pharmacokinetic parameters, plus evaluation in CVD and AD models, is ongoing.

Novel interaction of selenium-binding protein with glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase of Arabidopsis thaliana

2006 ◽  
Vol 33 (9) ◽  
pp. 847 ◽  
Author(s):  
Adamantia Agalou ◽  
Herman P. Spaink ◽  
Andreas Roussis
Keyword(s):  
Arabidopsis Thaliana ◽  
Metabolic Regulation ◽  
Binding Protein ◽  
Binding Assays ◽  
Fructose Bisphosphate ◽  
Metabolic Role ◽  
Fructose Bisphosphate Aldolase ◽  
Direct Binding ◽  
High Degree

The metabolic role and regulation of selenium, particularly in plants, is poorly understood. One of the proteins probably involved in the metabolic regulation of this element is the selenium-binding protein (SBP) with homologues present across prokaryotic and eukaryotic species. The high degree of conservation of SBP in different organisms suggests that this protein may play a role in fundamental biological processes. In order to gain insight into the biochemical function of SBP in plants we used the yeast two-hybrid system to identify proteins that potentially interact with an Arabidopsis thaliana (L.) Heynh. homologue. Among the putative binding partners of SBP, a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a fructose-bisphosphate aldolase (FBA) were found as reliable positive candidates. The interaction of these proteins with SBP was confirmed by in vitro binding assays. Previous findings in Escherichia coli, demonstrated the direct binding of selenium to both GAPDH and aldolase. Therefore our results reveal the interaction, at least in pairs, of three proteins that are possibly linked to selenium and suggest the existence of a protein network consisting of at least SBP, GAPDH and FBA, triggered by or regulating selenium metabolism in plant cells.

scholarly journals Uptake and metabolic fate of [HisA8,HisB4,GluB10,HisB27]insulin in rat liver in vivo

1998 ◽  
Vol 332 (2) ◽  
pp. 421-430 ◽  
Author(s):  
François AUTHIER ◽  
Gianni M. Di GUGLIELMO ◽  
Gillian M. DANIELSEN ◽  
John J. M. BERGERON
Keyword(s):  
Insulin Receptor ◽  
Subcellular Fractionation ◽  
Wild Type ◽  
Binding Assays ◽  
Insulin Degrading Enzyme ◽  
Direct Binding ◽  
Temporal Interaction ◽  
Kinetics Of Uptake

Receptor-mediated endocytosis and subsequent endosomal proteolysis of [125I]TyrA14-[HisA8,HisB4,GluB10,HisB27]insulin ([125I]TyrA14-H2 analogue), an insulin analogue exhibiting a high affinity for the insulin receptor, has been studied in liver parenchymal cells by quantitative subcellular fractionation and compared with that of wild-type [125I]TyrA14-insulin. Whereas the kinetics of uptake of the H2 analogue by liver was not different from that of insulin, the H2 analogue radioactivity after the 2 min peak declined significantly more slowly. A significant retention of the H2 analogue compared with insulin in both plasma membrane and endosomal fractions was observed and corresponded to decreased processing and dissociation of the H2 analogue. Cell-free endosomes preloaded in vivo with radiolabelled ligands and incubated in vitro processed insulin and extraluminally released insulin intermediates at a 2–3-fold higher rate than the H2 analogue. In vitro proteolysis of both non-radiolabelled and monoiodinated molecules by endosomal lysates showed a decreased response to the endosomal proteolytic machinery for the H2 analogue. However, in cross-linking and competition studies the H2 analogue exhibited an affinity for insulin-degrading enzyme identical with that of wild-type insulin. Brij-35-permeabilized endosomes revealed a 2-fold higher rate of dissociation of insulin from internalized receptors compared with the H2 analogue. After the administration of a saturating dose of both ligands, a rapid and reversible ligand-induced translocation of insulin receptor was observed, but without receptor loss. The H2 analogue induced a higher receptor concentration and tyrosine autophosphorylation of the receptor β subunit in endosomes. Moreover, a prolonged temporal interaction of the in vivo injected H2 analogue with receptor was observed by direct binding assays performed on freshly prepared subcellular fractions. These results indicate that endosomal proteolysis for the H2 analogue is slowed as a result of an increased residence time of the analogue on the insulin receptor and a low affinity of endosomal acidic insulinase for the dissociated H2 molecule.

Role of the scaffold protein RACK1 in apical expression of CFTR

2007 ◽  
Vol 293 (1) ◽  
pp. C294-C304 ◽  
Author(s):  
Michael Auerbach ◽  
Carole M. Liedtke
Keyword(s):  
Surface Proteins ◽  
Apical Plasma Membrane ◽  
Epithelial Cell Line ◽  
Spectrometric Analysis ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Binding Assays ◽  
Direct Binding

Previous studies from this laboratory demonstrated a role for protein kinase C (PKC)ε in the regulation of cAMP-dependent cystic fibrosis transmembrane regulator (CFTR) Cl channel function via binding of PKCε to RACK1, a receptor for activated C kinase, and of RACK1 to human Na+/H+ exchanger regulatory factor (NHERF1). In the present study, we investigated the role of RACK1 in regulating CFTR function in a Calu-3 airway epithelial cell line. Confocal microscopy and biotinylation of apical surface proteins demonstrate apical localization of RACK1 independent of actin. Mass spectrometric analysis of NHERF1 revealed copurification of tubulin, which, in in vitro binding assays, selectively binds to NHERF1, but not RACK1, via a PDZ1 domain. In binding and pulldown assays, we show direct binding of a PDZ2 domain to NHERF1, pulldown of endogenous NHERF1 by a PDZ2 domain, and inhibition of NHERF1-tubulin binding by a PDZ1 domain. Downregulation of RACK1 using double-stranded silencing RNA reduced the amount of RACK1 by 77.5% and apical expression of biotinylated CFTR by 87.4%. Expression of CFTR, NHERF1, and actin were not altered by treatment with siRACK1 or by nontargeting control silencing RNA, which, in addition, did not affect RACK1 expression. On the basis of these results, we model a RACK1 proteome consisting of PKCε-RACK1-NHERF1-NHERF1-tubulin with a role in stable expression of CFTR in the apical plasma membrane of epithelial cells.

P0009EVALUATION OF THE POTENTIAL FOR DRUG INTERACTIONS WITH VEVERIMER

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Jun Shao ◽  
Dawn Parsell ◽  
Robert Guttendorf ◽  
Yick Sen Wu ◽  
Li Tsao ◽  
...  
Keyword(s):  
Drug Interactions ◽  
Healthy Subjects ◽  
Gastric Ph ◽  
Pharmacokinetic Parameters ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Direct Binding ◽  
Ph Dependent

Abstract Background and Aims Veverimer is an investigational drug being developed as an orally administered hydrochloric acid binder for the treatment of metabolic acidosis associated with chronic kidney disease (CKD). In clinical studies, treatment with veverimer safely and effectively increased serum bicarbonate and improved objective and subjective measures of physical functioning1-3. Veverimer, a free-amine polymer, is not systemically absorbed; therefore, its potential for drug-drug interactions (DDIs) is limited to those that occur in the gastrointestinal (GI) tract (i.e., direct binding or indirect effects resulting from transient increases in gastric pH). We assessed the potential for DDIs with veverimer both in vitro and in vivo in healthy subjects. Methods In vitro binding to veverimer was evaluated with 16 drugs of varying molecular weight and charge. In a separate study, the effect of veverimer on gastric pH was measured continuously in vivo in healthy subjects using a microelectrode pH probe placed in the gastric compartment. Human DDI studies were conducted with 4 orally administered drugs, including those that demonstrated the most in vitro binding to veverimer and those with pH-dependent solubility (furosemide, aspirin, warfarin, dabigatran). Results Veverimer did not bind to any of the positively charged, neutral or zwitterionic drugs tested in vitro. It bound to 3 small (MW <332 Da), negatively charged drugs (aspirin, ethacrynic acid, furosemide); these interactions were reduced or eliminated in the presence of physiologically relevant concentrations of chloride. Neither furosemide nor aspirin showed clinically meaningful changes in pharmacokinetic parameters when coadministered with veverimer in human DDI studies (Figure 1). Veverimer increased gastric pH by ∼3.0 and 1.5 pH units in fasted and fed subjects, respectively. The increase in gastric pH was short-lived, with a peak within 1 hour after dosing and a return to baseline after ∼1.5 hours and ∼3 hours under fasting and fed conditions, respectively. The effect of veverimer on gastric pH was similar in the presence and absence of omeprazole. No clinically meaningful changes in systemic exposure, as indicated by Cmax and AUC, were observed when 3 drugs with pH-dependent solubility were coadministered with veverimer in human DDI studies (Figure 1). Conclusions In human DDI studies, we observed: a) no effect of veverimer on the bioavailability of drugs with physicochemical characteristics most susceptible to direct binding to the polymer; b) small, short-lived effects of veverimer on gastric pH; and c) no effect of veverimer on the bioavailability of drugs with pH-sensitive solubility. Therefore, it is concluded that there is a negligible risk of veverimer involvement in clinically significant DDIs.

scholarly journals Plectin 1f scaffolding at the sarcolemma of dystrophic (mdx) muscle fibers through multiple interactions with β-dystroglycan

2007 ◽  
Vol 176 (7) ◽  
pp. 965-977 ◽  
Author(s):  
Günther A. Rezniczek ◽  
Patryk Konieczny ◽  
Branislav Nikolic ◽  
Siegfried Reipert ◽  
Doris Schneller ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Protein Isoforms ◽  
Mdx Mice ◽  
Binding Assays ◽  
Protein Fragments ◽  
Glycoprotein Complex ◽  
Dystrophin Glycoprotein Complex ◽  
Direct Binding

In skeletal muscle, the cytolinker plectin is prominently expressed at Z-disks and the sarcolemma. Alternative splicing of plectin transcripts gives rise to more than eight protein isoforms differing only in small N-terminal sequences (5–180 residues), four of which (plectins 1, 1b, 1d, and 1f) are found at substantial levels in muscle tissue. Using plectin isoform–specific antibodies and isoform expression constructs, we show the differential regulation of plectin isoforms during myotube differentiation and their localization to different compartments of muscle fibers, identifying plectins 1 and 1f as sarcolemma-associated isoforms, whereas plectin 1d localizes exclusively to Z-disks. Coimmunoprecipitation and in vitro binding assays using recombinant protein fragments revealed the direct binding of plectin to dystrophin (utrophin) and β-dystroglycan, the key components of the dystrophin–glycoprotein complex. We propose a model in which plectin acts as a universal mediator of desmin intermediate filament anchorage at the sarcolemma and Z-disks. It also explains the plectin phenotype observed in dystrophic skeletal muscle of mdx mice and Duchenne muscular dystrophy patients.

scholarly journals In vitro antineurodegenerative activity and in silico predictions of blood-brain barrier penetration of Helichrysum plicatum flower extract

2020 ◽  
pp. 45-51
Author(s):  
Miloš Jovanović ◽  
Zorica Drinić ◽  
Dubravka Bigović ◽  
Ana Alimpić-Aradski ◽  
Sonja Duletić-Laušević ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Blood Brain Barrier ◽  
In Silico ◽  
Brain Barrier ◽  
Pharmacokinetic Parameters ◽  
Drug Candidates ◽  
Flower Extract ◽  
Blood Brain ◽  
Pharmacokinetic Properties

This study aimed to assess the antineurodegenerative and antioxidant activity of Helichrysum plicatum flower extract, as well as to identify extract ingredients with acceptable pharmacokinetic parameters such as gastrointestinal absorption, blood-brain barrier permeation, and P-glycoprotein-mediated effusion for optimal therapeutic brain exposure. Antioxidant activity was evaluated by ABTS, FRAP, and b-carotene bleaching assays, while antineurodegenerative activity was tested using acetylcholinesterase (AChE) and tyrosinase (TYR) inhibitory activity assays. In the ABTS test, the dry extract at the highest applied concentration (500 µg/mL) showed better or similar antioxidant activity compared to the standards. In the b-carotene assay, all applied concentrations of the extract showed significantly higher activity than vitamin C. No concentration-dependent activity was observed in the AChE assay, while in the TYR assay the lowest extract concentration (100 µg/mL) showed the highest percentage of inhibition (27.92 %). Pharmacokinetic parameters of compounds were predicted by in silico SwissADME online tool in accordance by the rules of drug-likeness. According to the pharmacokinetic properties, we concluded that pentoxymethoxylated flavones may represent CNS drug candidates for further studies.

scholarly journals AtHDA15 Attenuates COP1 via Transcriptional Quiescence, Direct Binding, and Sub-Compartmentalization During Photomorphogenesis

2021 ◽  
Author(s):  
Malona Velasco Alinsug ◽  
Custer C. Deocaris
Keyword(s):  
Molecular Mechanisms ◽  
Signaling Network ◽  
Light Signaling ◽  
Binding Assays ◽  
Epigenetic Machinery ◽  
Direct Binding ◽  
Regulated Gene Expression ◽  
Mutant Lines

Abstract Light is an essential environmental cue that determines the overall growth and development of plants. However, the molecular mechanisms underpinning the light signaling network are obscured by the epigenetic machinery where reversible acetylation and deacetylation play crucial roles in modulating light-regulated gene expression. In this paper, we demonstrate that HDA15 represses COP1, the master switch in the light signaling network, by deacetylation, protein interaction, and sub-compartmentalization. hda15 T-DNA mutant lines exhibited light hyposensitivity with significantly reduced HY5 and PIF3 transcript levels leading to long-hypocotyl phenotypes in the dark while its overexpression exhibited elevated HY5 transcripts and short hypocotyl phenotypes. In vivo and in vitro binding assays further show that HDA15 directly interacts with COP1 inside the nucleus modulating COP1’s repressive activities. Crossing hda15-t27 with cop1-4 mutants resulted in short-hypocotyl and dwarfed phenotypes, reminiscent of cop1-4 mutants suggesting COP1 is epistatic to HDA15. Although light signals the nucleocytoplasmic shuttling of HDA15, the presence of COP1 triggers its nuclear localization. A working model is presented elucidating the concerted interplay between HDA15 and COP1 under light and dark conditions.

scholarly journals AtHDA15 attenuates COP1 via transcriptional quiescence, direct binding, and sub-compartmentalization during photomorphogenesis

2020 ◽  
Author(s):  
Malona V. Alinsug ◽  
Custer C. Deocaris
Keyword(s):  
Molecular Mechanisms ◽  
Signaling Network ◽  
Light Signaling ◽  
Binding Assays ◽  
Epigenetic Machinery ◽  
Direct Binding ◽  
Regulated Gene Expression ◽  
Mutant Lines

AbstractLight is an essential environmental cue that determines the overall growth and development of plants. However, the molecular mechanisms underpinning the light signaling network are obscured by the epigenetic machinery where reversible acetylation and deacetylation play crucial roles in modulating light-regulated gene expression. In this paper, we demonstrate that HDA15 represses COP1, the master switch in the light signaling network, by deacetylation, protein interaction, and sub-compartmentalization. hda15 T-DNA mutant lines exhibited light hyposensitivity with significantly reduced HY5 and PIF3 transcript levels leading to long-hypocotyl phenotypes in the dark while its overexpression exhibited elevated HY5 transcripts and short hypocotyl phenotypes. In vivo and in vitro binding assays further show that HDA15 directly interacts with COP1 inside the nucleus modulating COP1’s repressive activities. Crossing hda15-t27 with cop1-4 mutants resulted in short-hypocotyl and dwarfed phenotypes, reminiscent of cop1-4 mutants suggesting COP1 is epistatic to HDA15. Although light signals the nucleocytoplasmic shuttling of HDA15, the presence of COP1 triggers its nuclear localization. A working model is presented elucidating the concerted interplay between HDA15 and COP1 under light and dark conditions.

Flavonoids as P-gp Inhibitors: A Systematic Review of SARs

2019 ◽  
Vol 26 (25) ◽  
pp. 4799-4831 ◽  
Author(s):  
Jiahua Cui ◽  
Xiaoyang Liu ◽  
Larry M.C. Chow
Keyword(s):  
Molecular Mechanisms ◽  
Metastatic Cancer ◽  
Drug Candidates ◽  
Chemical Structures ◽  
Transporter Family ◽  
Promising Area ◽  
New Generation ◽  
Nontoxic Inhibitors

P-glycoprotein, also known as ABCB1 in the ABC transporter family, confers the simultaneous resistance of metastatic cancer cells towards various anticancer drugs with different targets and diverse chemical structures. The exploration of safe and specific inhibitors of this pump has always been the pursuit of scientists for the past four decades. Naturally occurring flavonoids as benzopyrone derivatives were recognized as a class of nontoxic inhibitors of P-gp. The recent advent of synthetic flavonoid dimer FD18, as a potent P-gp modulator in reversing multidrug resistance both in vitro and in vivo, specifically targeted the pseudodimeric structure of the drug transporter and represented a new generation of inhibitors with high transporter binding affinity and low toxicity. This review concerned the recent updates on the structure-activity relationships of flavonoids as P-gp inhibitors, the molecular mechanisms of their action and their ability to overcome P-gp-mediated MDR in preclinical studies. It had crucial implications on the discovery of new drug candidates that modulated the efflux of ABC transporters and also provided some clues for the future development in this promising area.

Current Approaches to Drug Discovery for Chagas Disease: Methodological Advances

2019 ◽  
Vol 22 (8) ◽  
pp. 509-520
Author(s):  
Cauê B. Scarim ◽  
Chung M. Chin
Keyword(s):  
Drug Discovery ◽  
Chagas Disease ◽  
Drug Candidates ◽  
Lead Compounds ◽  
New Drug ◽  
Compound Libraries ◽  
Screening Assays ◽  
Cruzi Infection

Background: In recent years, there has been an improvement in the in vitro and in vivo methodology for the screening of anti-chagasic compounds. Millions of compounds can now have their activity evaluated (in large compound libraries) by means of high throughput in vitro screening assays. Objective: Current approaches to drug discovery for Chagas disease. Method: This review article examines the contribution of these methodological advances in medicinal chemistry in the last four years, focusing on Trypanosoma cruzi infection, obtained from the PubMed, Web of Science, and Scopus databases. Results: Here, we have shown that the promise is increasing each year for more lead compounds for the development of a new drug against Chagas disease. Conclusion: There is increased optimism among those working with the objective to find new drug candidates for optimal treatments against Chagas disease.

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