Publications by authors named "Scott D Larsen"

80 Publications

Physicochemical properties and formulation development of a novel compound inhibiting Staphylococcus aureus biofilm formation.

PLoS One 2021 8;16(2):e0246408. Epub 2021 Feb 8.

Division of Cardiovascular Medicine, Department of Medicine, University of Missouri, Columbia, Missouri, United States of America.

The emergence of antibiotic resistance over the past several decades has given urgency to new antibacterial strategies that apply less selective pressure. A new class of anti-virulence compounds were developed that are active against methicillin-resistant Staphylococcus aureus (MRSA), by inhibiting bacterial virulence without hindering their growth to reduce the selective pressure for resistance development. One of the compounds CCG-211790 has demonstrated potent anti-biofilm activity against MRSA. This new class of anti-virulence compounds inhibited the gene expression of virulence factors involved in biofilm formation and disrupted the biofilm structures. In this study, the physicochemical properties of CCG-211790, including morphology, solubility in pure water or in water containing sodium dodecyl sulfate, solubility in organic solvents, and stability with respect to pH were investigated for the first time. Furthermore, a topical formulation was developed to enhance the therapeutic potential of the compound. The formulation demonstrated acceptable properties for drug release, viscosity, pH, cosmetic elegance and stability of over nine months.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246408PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870075PMC
February 2021

Development of 2,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one inhibitors of aldehyde dehydrogenase 1A (ALDH1A) as potential adjuncts to ovarian cancer chemotherapy.

Eur J Med Chem 2021 Feb 3;211:113060. Epub 2020 Dec 3.

Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, 48109, USA. Electronic address:

There is strong evidence that inhibition of one or more Aldehyde Dehydrogenase 1A (ALDH1A) isoforms may be beneficial in chemotherapy-resistant ovarian cancer and other tumor types. While many previous efforts have focused on development of ALDH1A1 selective inhibitors, the most deadly ovarian cancer subtype, high-grade serous (HGSOC), exhibits elevated expression of ALDH1A3. Herein, we report continued development of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in this critical tumor subtype. Optimization of the CM39 scaffold, aided by metabolite ID and several new ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular ALDH inhibition in HGSOC cell lines, and substantial improvements in microsomal stability culminating in orally bioavailable compounds. We demonstrate that two compounds 68 and 69 are able to synergize with chemotherapy in a resistant cell line and patient-derived HGSOC tumor spheroids, indicating their suitability for future in vivo proof of concept experiments.
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http://dx.doi.org/10.1016/j.ejmech.2020.113060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855449PMC
February 2021

Optimization of Eliglustat-Based Glucosylceramide Synthase Inhibitors as Substrate Reduction Therapy for Gaucher Disease Type 3.

ACS Chem Neurosci 2020 10 9;11(20):3464-3473. Epub 2020 Oct 9.

Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States.

There remain no approved therapies for rare but devastating neuronopathic glyocosphingolipid storage diseases, such as Sandhoff, Tay-Sachs, and Gaucher disease type 3. We previously reported initial optimization of the scaffold of eliglustat, an approved therapy for the peripheral symptoms of Gaucher disease type 1, to afford , which effected modest reductions in brain glucosylceramide (GlcCer) in normal mice at 60 mg/kg. The relatively poor pharmacokinetic properties and high Pgp-mediated efflux of prompted further optimization of the scaffold. With a general objective of reducing topological polar surface area, and guided by multiple metabolite identification studies, we were successful at identifying (CCG-222628), which achieves remarkably greater brain exposure in mice than . After demonstrating an over 60-fold improvement in potency over at reducing brain GlcCer in normal mice, we compared with Sanofi clinical candidate venglustat (Genz-682452) in the CBE mouse model of Gaucher disease type 3. At doses of 10 mg/kg, and venglustat effected comparable reductions in both brain GlcCer and glucosylsphingosine. Importantly, achieved these equivalent pharmacodynamic effects at significantly lower brain exposure than venglustat.
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http://dx.doi.org/10.1021/acschemneuro.0c00558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919060PMC
October 2020

A New Paroxetine-Based GRK2 Inhibitor Reduces Internalization of the -Opioid Receptor.

Mol Pharmacol 2020 06 31;97(6):392-401. Epub 2020 Mar 31.

Life Sciences Institute (R.A.B., H.V.W.), Departments of Medicinal Chemistry (H.V.W., S.D.L.) and Pharmacology (R.A.B., Z.Y.W., M.A.P.), and Vahlteich Medicinal Chemistry Core, College of Pharmacy (H.V.W., S.D.L.), University of Michigan, Ann Arbor, Michigan; and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology (Y.-C.Y., J.J.G.T.), Purdue University, West Lafayette, Indiana

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or scaffold. A new inhibitor, , which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. and the closely related compound also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or scaffold to block internalization of the -opioid receptor (MOR). showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on , which explains why -based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, , which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that blocked the internalization of the -opioid receptor most efficaciously because it has the ability to cross cell membranes.
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http://dx.doi.org/10.1124/mol.119.118661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237867PMC
June 2020

Identification of Pirin as a Molecular Target of the CCG-1423/CCG-203971 Series of Antifibrotic and Antimetastatic Compounds.

ACS Pharmacol Transl Sci 2019 Apr 18;2(2):92-100. Epub 2019 Mar 18.

Department of Pharmacology & Toxicology and Michigan State University, East Lansing, Michigan, 48824, United States.

A series of compounds (including CCG-1423 and CCG-203971) discovered through an MRTF/SRF-dependent luciferase screen has shown remarkable efficacy in a variety of and models, including significant reduction of melanoma metastasis and bleomycin- induced fibrosis. Although these compounds are efficacious in these disease models, the molecular target is unknown. Here, we describe affinity isolation-based target identification efforts which yielded pirin, an iron-dependent cotranscription factor, as a target of this series of compounds. Using biophysical techniques including isothermal titration calorimetry and X-ray crystallography, we verify that pirin binds these compounds We also show with genetic approaches that pirin modulates MRTF- dependent luciferase reporter activity. Finally, using both siRNA and a previously validated pirin inhibitor, we show a role for pirin in TGF-- induced gene expression in primary dermal fibroblasts. A recently developed analog, CCG-257081, which co crystallizes with pirin, is also effective in the prevention of bleomycin-induced dermal fibrosis.
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http://dx.doi.org/10.1021/acsptsci.8b00048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006939PMC
April 2019

Discovery and Optimization of Triazine Nitrile Inhibitors of Cathepsin L for the Potential Treatment of Chronic Toxoplasmosis in the CNS.

ACS Chem Neurosci 2020 08 18;11(16):2450-2463. Epub 2020 Feb 18.

Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States.

With roughly 2 billion people infected, the neurotropic protozoan remains one of the most pervasive and infectious parasites. infection is the second leading cause of death due to foodborne illness in the United States, causes severe disease in immunocompromised patients, and is correlated with several cognitive and neurological disorders. Currently, no therapies exist that are capable of eliminating the persistent infection in the central nervous system (CNS). In this study we report the identification of triazine nitrile inhibitors of cathepsin L (CPL) from a high throughput screen and their subsequent optimization. Through rational design, we improved inhibitor potency to as low as 5 nM, identified pharmacophore features that can be exploited for isoform selectivity (up to 7-fold for CPL versus human isoform), and improved metabolic stability ( > 60 min in mouse liver microsomes) guided by a metabolite ID study. We demonstrated that this class of compounds is capable of crossing the blood-brain barrier in mice (1:1 brain/plasma at 2 h). Importantly, we also show for the first time that treatment of bradyzoite cysts in vitro with triazine nitrile inhibitors reduces parasite viability with efficacy equivalent to a CPL genetic knockout.
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http://dx.doi.org/10.1021/acschemneuro.9b00674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431380PMC
August 2020

Structure-Based Design of Selective, Covalent G Protein-Coupled Receptor Kinase 5 Inhibitors.

ACS Med Chem Lett 2019 Dec 12;10(12):1628-1634. Epub 2019 Nov 12.

University of Michigan, Vahlteich Medicinal Chemistry Core, College of Pharmacy, 428 Church Street, Ann Arbor, Michigan 48109, United States.

The ability of G protein-coupled receptor (GPCR) kinases (GRKs) to regulate desensitization of GPCRs has made GRK2 and GRK5 attractive targets for treating heart failure and other diseases such as cancer. Although advances have been made toward developing inhibitors that are selective for GRK2, there have been far fewer reports of GRK5 selective compounds. Herein, we describe the development of GRK5 subfamily selective inhibitors, and that covalently interact with a nonconserved cysteine (Cys474) unique to this subfamily. Compounds and feature a highly amenable pyrrolopyrimidine scaffold that affords high nanomolar to low micromolar activity that can be easily modified with Michael acceptors with various reactivities and geometries. Our work thereby establishes a new pathway toward further development of subfamily selective GRK inhibitors and establishes Cys474 as a new and useful covalent handle in GRK5 drug discovery.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912873PMC
December 2019

5-Aryl-1,3,4-oxadiazol-2-ylthioalkanoic Acids: A Highly Potent New Class of Inhibitors of Rho/Myocardin-Related Transcription Factor (MRTF)/Serum Response Factor (SRF)-Mediated Gene Transcription as Potential Antifibrotic Agents for Scleroderma.

J Med Chem 2019 05 18;62(9):4350-4369. Epub 2019 Apr 18.

Department of Pharmacology and Toxicology , Michigan State University , East Lansing , Michigan 48824 , United States.

Through a phenotypic high-throughput screen using a serum response element luciferase promoter, we identified a novel 5-aryl-1,3,4-oxadiazol-2-ylthiopropionic acid lead inhibitor of Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediated gene transcription with good potency (IC = 180 nM). We were able to rapidly improve the cellular potency by 5 orders of magnitude guided by sharply defined and synergistic SAR. The remarkable potency and depth of the SAR, as well as the relatively low molecular weight of the series, suggests, but does not prove, that binding to the unknown molecular target may be occurring through a covalent mechanism. The series nevertheless has no observable cytotoxicity up to 100 μM. Ensuing pharmacokinetic optimization resulted in the development of two potent and orally bioavailable anti-fibrotic agents that were capable of dose-dependently reducing connective tissue growth factor gene expression in vitro as well as significantly reducing the development of bleomycin-induced dermal fibrosis in mice in vivo.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590913PMC
May 2019

A Pan-ALDH1A Inhibitor Induces Necroptosis in Ovarian Cancer Stem-like Cells.

Cell Rep 2019 03;26(11):3061-3075.e6

Division of Hematology-Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Division of Gynecology-Oncology, Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA; Division of Hematology-Oncology, Department of Internal Medicine, Division of Gynecology-Oncology, Department of Obstetrics and Gynecology, and UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA; Magee-Womens Research Institute, Pittsburgh, PA, USA. Electronic address:

Ovarian cancer is typified by the development of chemotherapy resistance. Chemotherapy resistance is associated with high aldehyde dehydrogenase (ALDH) enzymatic activity, increased cancer "stemness," and expression of the stem cell marker CD133. As such, ALDH activity has been proposed as a therapeutic target. Although it remains controversial which of the 19 ALDH family members drive chemotherapy resistance, ALDH1A family members have been primarily linked with chemotherapy resistant and stemness. We identified two ALDH1A family selective inhibitors (ALDH1Ai). ALDH1Ai preferentially kills CD133 ovarian cancer stem-like cells (CSCs). ALDH1Ai induce necroptotic CSC death, mediated, in part, by the induction of mitochondrial uncoupling proteins and reduction in oxidative phosphorylation. ALDH1Ai is highly synergistic with chemotherapy, reducing tumor initiation capacity and increasing tumor eradication in vivo. These studies link ALDH1A with necroptosis and confirm the family as a critical therapeutic target to overcome chemotherapy resistance and improve patient outcomes.
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http://dx.doi.org/10.1016/j.celrep.2019.02.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061440PMC
March 2019

Synthesis of deuterium-labelled amlexanox and its metabolic stability against mouse, rat, and human microsomes.

J Labelled Comp Radiopharm 2019 05 3;62(5):202-208. Epub 2019 Apr 3.

Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, USA.

As part of a program toward making analogues of amlexanox (1), currently under clinical investigation for the treatment of type 2 diabetes and obesity, we have synthesized derivative 5 in which deuterium has been introduced into two sites of metabolism on the C-7 isopropyl function of amlexanox. The synthesis of 5 was completed in an efficient three-step process utilizing reduction of key olefin 7b to 8 by Wilkinson's catalyst to provide specific incorporation of di-deuterium across the double bond. Compound 5 displayed nearly equivalent potency to amlexanox (IC , 1.1μM vs 0.6μM, respectively) against recombinant human TBK1. When incubated with human, rat, and mouse liver microsomes, amlexanox (1) and d -amlexanox (5) were stable (t  > 60 minutes) with 1 showing marginally greater stability relative to 5 except for rat liver microsomes. These data show that incorporating deuterium into two sites of metabolism does not majorly suppress Cyp-mediated metabolism relative to amlexanox.
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http://dx.doi.org/10.1002/jlcr.3716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525014PMC
May 2019

Dual inhibition of Kif15 by oxindole and quinazolinedione chemical probes.

Bioorg Med Chem Lett 2019 01 4;29(2):148-154. Epub 2018 Dec 4.

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, United States; Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, United States. Electronic address:

The mitotic spindle is a microtubule-based machine that segregates a replicated set of chromosomes during cell division. Many cancer drugs alter or disrupt the microtubules that form the mitotic spindle. Microtubule-dependent molecular motors that function during mitosis are logical alternative mitotic targets for drug development. Eg5 (Kinesin-5) and Kif15 (Kinesin-12), in particular, are an attractive pair of motor proteins, as they work in concert to drive centrosome separation and promote spindle bipolarity. Furthermore, we hypothesize that the clinical failure of Eg5 inhibitors may be (in part) due to compensation by Kif15. In order to test this idea, we screened a small library of kinase inhibitors and identified GW108X, an oxindole that inhibits Kif15 in vitro. We show that GW108X has a distinct mechanism of action compared with a commercially available Kif15 inhibitor, Kif15-IN-1 and may serve as a lead with which to further develop Kif15 inhibitors as clinically relevant agents.
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http://dx.doi.org/10.1016/j.bmcl.2018.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681659PMC
January 2019

In vitro and in vivo delivery of a sustained release nanocarrier-based formulation of an MRTF/SRF inhibitor in conjunctival fibrosis.

J Nanobiotechnology 2018 Nov 27;16(1):97. Epub 2018 Nov 27.

Department of Ophthalmology, King's College London, Westminster Bridge Road, London, SE1 7EH, UK.

Background: Sustained drug delivery is a large unmet clinical need in glaucoma. Here, we incorporated a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor, CCG-222740, into slow release large unilamellar vesicles derived from the liposomes DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and tested their effects in vitro and in vivo.

Results: The vesicles were spherical particles of around 130 nm and were strongly cationic. A large amount of inhibitor could be incorporated into the vesicles. We showed that the nanocarrier CCG-222740 formulation gradually released the inhibitor over 14 days using high performance liquid chromatography. Nanocarrier CCG-222740 significantly decreased ACTA2 gene expression and was not cytotoxic in human conjunctival fibroblasts. In vivo, nanocarrier CCG-222740 doubled the bleb survival from 11.0 ± 0.6 days to 22.0 ± 1.3 days (p = 0.001), decreased conjunctival scarring and did not have any local or systemic adverse effects in a rabbit model of glaucoma filtration surgery.

Conclusions: Our study demonstrates proof-of-concept that a nanocarrier-based formulation efficiently achieves a sustained release of a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor and prevents conjunctival fibrosis in an established rabbit model of glaucoma filtration surgery.
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http://dx.doi.org/10.1186/s12951-018-0425-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258153PMC
November 2018

Structure-Based Optimization of a Novel Class of Aldehyde Dehydrogenase 1A (ALDH1A) Subfamily-Selective Inhibitors as Potential Adjuncts to Ovarian Cancer Chemotherapy.

J Med Chem 2018 10 28;61(19):8754-8773. Epub 2018 Sep 28.

Department of Biochemistry and Molecular Biology , Indiana University School of Medicine , Indianapolis , Indiana 46202 , United States.

Aldehyde dehydrogenase (ALDH) activity is commonly used as a marker to identify cancer stem-like cells. The three ALDH1A isoforms have all been individually implicated in cancer stem-like cells and in chemoresistance; however, which isoform is preferentially expressed varies between cell lines. We sought to explore the structural determinants of ALDH1A isoform selectivity in a series of small-molecule inhibitors in support of research into the role of ALDH1A in cancer stem cells. An SAR campaign guided by a cocrystal structure of the HTS hit CM39 (7) with ALDH1A1 afforded first-in-class inhibitors of the ALDH1A subfamily with excellent selectivity over the homologous ALDH2 isoform. We also discovered the first reported modestly selective single isoform 1A2 and 1A3 inhibitors. Two compounds, 13g and 13h, depleted the CD133 putative cancer stem cell pool, synergized with cisplatin, and achieved efficacious concentrations in vivo following IP administration. Compound 13h additionally synergized with cisplatin in a patient-derived ovarian cancer spheroid model.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477540PMC
October 2018

Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability.

Bioorg Med Chem Lett 2018 06 9;28(10):1972-1980. Epub 2018 Mar 9.

Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, United States; Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, United States. Electronic address:

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2 μM to as low as 110 nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5 brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5938124PMC
June 2018

Utilizing a structure-based docking approach to develop potent G protein-coupled receptor kinase (GRK) 2 and 5 inhibitors.

Bioorg Med Chem Lett 2018 05 30;28(9):1507-1515. Epub 2018 Mar 30.

Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, United States; Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI, United States. Electronic address:

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the desensitization and internalization of GPCRs. Two of these, GRK2 and GRK5, are upregulated in heart failure and are promising targets for heart failure treatment. Although there have been several reports of potent and selective inhibitors of GRK2 there are few for GRK5. Herein, we describe a ligand docking approach utilizing the crystal structures of the GRK2-Gβγ·GSK180736A and GRK5·CCG215022 complexes to search for amide substituents predicted to confer GRK2 and/or GRK5 potency and selectivity. From this campaign, we successfully generated two new potent GRK5 inhibitors, although neither exhibited selectivity over GRK2.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916850PMC
May 2018

Gedunin- and Khivorin-Derivatives Are Small-Molecule Partial Agonists for Adhesion G Protein-Coupled Receptors GPR56/ADGRG1 and GPR114/ADGRG5.

Mol Pharmacol 2018 05 23;93(5):477-488. Epub 2018 Feb 23.

Departments of Pharmacology (H.M.S., A.V.S., G.G.T.) and Medicinal Chemistry (S.D.L.), University of Michigan, Ann Arbor, Michigan

Adhesion G protein-coupled receptors (aGPCRs) have emerged as potential therapeutic targets in multiple cancers and in neurologic diseases. However, there are few modulatory compounds that act on these receptors. The majority of aGPCRs are orphans and a general activation mechanism has only recently been defined: aGPCRs are activated by a tethered agonist. aGPCRs constitutively cleave themselves during biosynthesis to generated two-part receptors comprising an extracellular domain (ECD) and a 7-transmembrane spanning domain (7TM). ECD dissociation reveals the tethered agonist initiating G protein signaling. Synthetic peptides that mimic the tethered agonist region can activate aGPCRs. We hypothesized that small molecules could act in the same way as peptide agonists. High throughput screening of the 2000-compound Spectrum Collection library using the serum response element luciferase gene reporter assay revealed two related classes of small molecules that could activate the aGPCR GPR56/ADGRG1. The most potent compound identified was 3--acetoxydihydrodeoxygedunin, or 3--DOG. 3--DOG activated engineered, low-activity GPR56 7TM in independent biochemical and cell-based assays with an EC of ∼5 M. The compound also activated a subset of aGPCRs but not two class A GPCRs tested. The mode of 3--DOG-mediated receptor activation is that of partial agonist. 3--DOG activated GPR56 less efficaciously than peptide agonist and could antagonize both the peptide agonist and the endogenous tethered agonist, which are pharmacological hallmarks of partial agonists. Taken together, we have uncovered a novel group of aGPCR partial agonists that will serve as invaluable resources for understanding this unique class receptors.
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http://dx.doi.org/10.1124/mol.117.111476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5878671PMC
May 2018

Structural Determinants Influencing the Potency and Selectivity of Indazole-Paroxetine Hybrid G Protein-Coupled Receptor Kinase 2 Inhibitors.

Mol Pharmacol 2017 12 25;92(6):707-717. Epub 2017 Oct 25.

Life Sciences Institute (R.B., H.V.W., M.C.C., J.J.G.T.), Departments of Medicinal Chemistry (H.V.W., S.D.L., J.J.G.T.), Pharmacology (R.B., J.J.G.T.), Biological Chemistry (M.C.C., J.J.G.T.), and Vahlteich Medicinal Chemistry Core, College of Pharmacy (H.V.W., S.D.L.), University of Michigan, Ann Arbor, Michigan; Department of Chemistry, Georgia State University, Atlanta, Georgia (X.-Q.Y.); Center for Translational Medicine, Temple University, Philadelphia, Pennsylvania (A.C., J.S., J.Y.C, W.J.K.); and Department of Biological Sciences, Purdue University, West Lafayette Indiana (J.J.G.T.)

G protein-coupled receptor kinases (GRKs) phosphorylate activated receptors to promote arrestin binding, decoupling from heterotrimeric G proteins, and internalization. GRK2 and GRK5 are overexpressed in the failing heart and thus have become therapeutic targets. Previously, we discovered two classes of GRK2-selective inhibitors, one stemming from GSK180736A, a Rho-associated coiled-coil containing kinase 1 (ROCK1) inhibitor, the other from paroxetine, a selective serotonin-reuptake inhibitor. These two classes of compounds bind to the GRK2 active site in a similar configuration but contain different hinge-binding "warheads": indazole and benzodioxole, respectively. We surmised from our prior studies that an indazole would be the stronger hinge binder and would impart increased potency when substituted for benzodioxole in paroxetine derivatives. To test this hypothesis, we synthesized a series of hybrid compounds that allowed us to compare the effects of inhibitors that differ only in the identity of the warhead. The indazole-paroxetine analogs were indeed more potent than their respective benzodioxole derivatives but lost selectivity. To investigate how these two warheads dictate selectivity, we determined the crystal structures of three of the indazole hybrid compounds (CCG224061, CCG257284, and CCG258748) in complex with GRK2-G Comparison of these structures with those of analogous benzodioxole-containing complexes confirmed that the indazole-paroxetine hybrids form stronger interactions with the hinge of the kinase but also stabilize a distinct conformation of the kinase domain of GRK2 compared with previous complexes with paroxetine analogs. This conformation is analogous to one that can be assumed by GRK5, at least partially explaining the loss in selectivity.
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http://dx.doi.org/10.1124/mol.117.110130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691592PMC
December 2017

Minor Structural Variations of Small Molecules Tune Regulatory Activities toward Pathological Factors in Alzheimer's Disease.

ChemMedChem 2017 11 9;12(22):1828-1838. Epub 2017 Oct 9.

Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, Republic of Korea.

Chemical tools have been valuable for establishing a better understanding of the relationships between metal ion dyshomeostasis, the abnormal aggregation and accumulation of amyloid-β (Aβ), and oxidative stress in Alzheimer's disease (AD). Still, very little information is available to correlate the structures of chemical tools with specific reactivities used to uncover such relationships. Recently, slight structural variations to the framework of a chemical tool were found to drastically determine the tool's reactivities toward multiple pathological facets to various extents. Herein, we report our rational design and characterization of a structural series to illustrate the extent to which the reactivities of small molecules vary toward different targets as a result of minor structural modifications. These compounds were rationally and systematically modified based on consideration of properties, including ionization potentials and metal binding, to afford their desired reactivities with metal-free or metal-bound Aβ, reactive oxygen species (ROS), and free organic radicals. Our results show that although small molecules are structurally similar, they can interact with multiple factors associated with AD pathogenesis and alleviate their reactivities to different degrees. Together, our studies demonstrate the rational structure-directed design that can be used to develop chemical tools capable of regulating individual or interrelated pathological features in AD.
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http://dx.doi.org/10.1002/cmdc.201700456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828035PMC
November 2017

Local delivery of novel MRTF/SRF inhibitors prevents scar tissue formation in a preclinical model of fibrosis.

Sci Rep 2017 03 31;7(1):518. Epub 2017 Mar 31.

National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK.

The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway represents a promising therapeutic target to prevent fibrosis. We have tested the effects of new pharmacological inhibitors of MRTF/SRF signalling in a preclinical model of fibrosis. CCG-222740, a novel MRTF/SRF inhibitor, markedly decreased SRF reporter gene activity and showed a greater inhibitory effect on MRTF/SRF target genes than the previously described MRTF-A inhibitor CCG-203971. CCG-222740 was also five times more potent, with an IC of 5 μM, in a fibroblast-mediated collagen contraction assay, was less cytotoxic, and a more potent inhibitor of alpha-smooth muscle actin protein expression than CCG-203971. Local delivery of CCG-222740 and CCG-203971 in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery increased the long-term success of the surgery by 67% (P < 0.0005) and 33% (P < 0.01), respectively, and significantly decreased fibrosis and scarring histologically. Unlike mitomycin-C, neither CCG-222740 nor CCG-203971 caused any detectable epithelial toxicity or systemic side effects with very low drug levels measured in the aqueous, vitreous, and serum. We conclude that inhibitors of MRTF/SRF-regulated gene transcription such as CCG-222740, potentially represent a new therapeutic strategy to prevent scar tissue formation in the eye and other tissues.
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http://dx.doi.org/10.1038/s41598-017-00212-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428058PMC
March 2017

Structure-Based Design of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors Based on Paroxetine.

J Med Chem 2017 04 29;60(7):3052-3069. Epub 2017 Mar 29.

Department of Medicinal Chemistry, College of Pharmacy, ‡Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, §Ph.D. Program in Chemical Biology, ⊥Vahlteich Medicinal Chemistry Core, University of Michigan , Ann Arbor, Michigan 48109, United States.

In heart failure, the β-adrenergic receptors (βARs) become desensitized and uncoupled from heterotrimeric G proteins. This process is initiated by G protein-coupled receptor kinases (GRKs), some of which are upregulated in the failing heart, making them desirable therapeutic targets. The selective serotonin reuptake inhibitor, paroxetine, was previously identified as a GRK2 inhibitor. Utilizing a structure-based drug design approach, we modified paroxetine to generate a small compound library. Included in this series is a highly potent and selective GRK2 inhibitor, 14as, with an IC of 30 nM against GRK2 and greater than 230-fold selectivity over other GRKs and kinases. Furthermore, 14as showed a 100-fold improvement in cardiomyocyte contractility assays over paroxetine and a plasma concentration higher than its IC for over 7 h. Three of these inhibitors, including 14as, were additionally crystallized in complex with GRK2 to give insights into the structural determinants of potency and selectivity of these inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641445PMC
April 2017

Pharmacokinetic optimitzation of CCG-203971: Novel inhibitors of the Rho/MRTF/SRF transcriptional pathway as potential antifibrotic therapeutics for systemic scleroderma.

Bioorg Med Chem Lett 2017 04 10;27(8):1744-1749. Epub 2017 Mar 10.

Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.
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http://dx.doi.org/10.1016/j.bmcl.2017.02.070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395305PMC
April 2017

Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence.

Nat Chem Biol 2017 02 19;13(2):218-225. Epub 2016 Dec 19.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.

The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a ∼540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme-artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.
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http://dx.doi.org/10.1038/nchembio.2259DOI Listing
February 2017

Optimisation of Intestinal Fibrosis and Survival in the Mouse S. Typhimurium Model for Anti-fibrotic Drug Discovery and Preclinical Applications.

J Crohns Colitis 2017 Jun;11(6):724-736

Division of Gastroenterology, University of Michigan, Ann Arbor, MI, USA.

Background And Aims: Intestinal fibrosis is a frequent complication in Crohn's disease [CD]. The mouse Salmonella typhimurium model, due to its simplicity, reproducibility, manipulability, and penetrance, is an established fibrosis model for drug discovery and preclinical trials. However, the severity of fibrosis and mortality are host- and bacterial strain-dependent, thus limiting the original model. We re-evaluated the S. typhimurium model to optimise fibrosis and survival, using commercially available mouse strains.

Methods: Fibrotic and inflammatory markers were evaluated across S. typhimurium ΔaroA:C57bl/6 studies performed in our laboratory. A model optimisation study was performed using three commercially available mouse strains [CBA/J, DBA/J, and 129S1/SvImJ] infected with either SL1344 or ΔaroA S. typhimurium. Fibrotic penetrance was determined by histopathology, gene expression, and αSMA protein expression. Fibrosis severity, penetrance, and survival were analysed across subsequent CBA studies.

Results: Fibrosis severity and survival are both host- and bacterial strain-dependent. Marked tissue fibrosis and 100% survival occurred in the CBA/J strain infected with SL1344. Subsequent experiments demonstrated that CBA/J mice develop extensive intestinal fibrosis, characterised by transmural tissue fibrosis, a Th1/Th17 cytokine response, and induction of pro-fibrotic genes and extracellular matrix proteins. A meta-analysis of subsequent SL1344:CBA/J studies demonstrated that intestinal fibrosis is consistent and highly penetrant across histological, protein, and gene expression markers. As proof-of-concept, we tested the utility of the SL1344:CBA/J fibrosis model to evaluate efficacy of CCG-203971, a novel anti-fibrotic drug.

Conclusion: The S. typhimurium SL1344:CBA/J model is an optimised model for the study of intestinal fibrosis.
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http://dx.doi.org/10.1093/ecco-jcc/jjw210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881735PMC
June 2017

Pharmacological Inhibition of Myocardin-related Transcription Factor Pathway Blocks Lung Metastases of RhoC-Overexpressing Melanoma.

Mol Cancer Ther 2017 01 11;16(1):193-204. Epub 2016 Nov 11.

Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan.

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF transcription, we use a previously developed small-molecule inhibitor, CCG-203971, which at low micromolar concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971-mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes G cell-cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared with the low RhoC-expressing SK-Mel-19. Furthermore, pharmacologic inhibition of the MRTF pathway reduced both the number and size of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a novel approach to melanoma therapeutics. Mol Cancer Ther; 16(1); 193-204. ©2016 AACR.
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555736PMC
January 2017

Structure-mechanism-based engineering of chemical regulators targeting distinct pathological factors in Alzheimer's disease.

Nat Commun 2016 10 13;7:13115. Epub 2016 Oct 13.

Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.

The absence of effective therapeutics against Alzheimer's disease (AD) is a result of the limited understanding of its multifaceted aetiology. Because of the lack of chemical tools to identify pathological factors, investigations into AD pathogenesis have also been insubstantial. Here we report chemical regulators that demonstrate distinct specificity towards targets linked to AD pathology, including metals, amyloid-β (Aβ), metal-Aβ, reactive oxygen species, and free organic radicals. We obtained these chemical regulators through a rational structure-mechanism-based design strategy. We performed structural variations of small molecules for fine-tuning their electronic properties, such as ionization potentials and mechanistic pathways for reactivity towards different targets. We established in vitro and/or in vivo efficacies of the regulators for modulating their targets' reactivities, ameliorating toxicity, reducing amyloid pathology, and improving cognitive deficits. Our chemical tools show promise for deciphering AD pathogenesis and discovering effective drugs.
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http://dx.doi.org/10.1038/ncomms13115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065625PMC
October 2016

CARP-1 functional mimetics are novel inhibitors of drug-resistant triple negative breast cancers.

Oncotarget 2016 Nov;7(45):73370-73388

John D. Dingell VA Medical Center, Wayne State University, Detroit, MI, 48201 USA.

Doxorubicin and Cisplatin are the frontline therapeutics for treatment of the triple negative breast cancers (TNBCs). Emergence of drug-resistance often contributes to failure of drugs and poor prognosis, and thus necessitates development of new and improved modalities to treat TNBCs. We generated and characterized chemotherapy-resistant TNBC cells following their culture in chronic presence of Doxorubicin or Cisplatin, and tested whether their viabilities were inhibited by a novel class of CARP- 1 functional mimetic (CFM) compounds. Analogs of parent compound CFM-4 were obtained through structure-activity based medicinal chemistry studies. CFM-4.16, a novel analog of CFM-4, caused superior inhibition of viability of TNBC cells when used in combination with doxorubicin. Doxorubicin and cisplatin inhibited viabilities of parental cells with GI50 dose of 0.02-0.1 μM and 1.65 μM, respectively. The GI50 dose of doxorubicin for doxorubicin-resistant TNBC cells was ≥ 10.0 μM. For Cisplatin-resistant cells, the GI50 dose of Cisplatin was ≥ 6-15.0 μM for MDA-MB-468 sublines and ≥ 150.0 μM for MDA-MB-231 sublines. CFM-4.16 inhibited viability of chemotherapy-resistant TNBC cells, in part by inhibiting oncogenic cMet activation and expression, stimulating CARP-1 expression, caspase-8 cleavage and apoptosis. CFM-4.16 pretreatment enhanced anti-TNBC efficacies of inhibitors of cMET (Tevatinib) or cSrc (Dasatinib). CFM-4.16 suppressed growth of resistant TNBC cells in soft agar as well as in three-dimensional suspension cultures derived from enriched, stem-like cells. Finally, a nanolipid formulation of CFM-4.16 in combination with doxorubicin had superior efficacy in inhibiting TNBC xenograft growth. Our findings collectively demonstrate therapeutic potential of CFM-4.16 for parental and drug-resistant TNBCs.
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http://dx.doi.org/10.18632/oncotarget.12333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341985PMC
November 2016

Blockade of the renin-angiotensin system prevents acute and immunologically relevant colitis in murine models.

Pediatr Surg Int 2016 Dec 26;32(12):1103-1114. Epub 2016 Sep 26.

Section of Pediatric Surgery, Department of Surgery, The University of Michigan Medical School, Mott Children's Hospital, F3970, Ann Arbor, MI, 48109-0245, USA.

Background: Blockade of the renin-angiotensin system (RAS) has been shown to alleviate inflammatory processes in the gastrointestinal tract. The aim of this study was to determine if blockade of the RAS would be effective in an immunologically relevant colitis model, and to compare outcome with an acute colitis model.

Methods: A losartan analog, CCG-203025 (CHClNOS) containing a highly polar sulfonic acid moiety that we expected would allow localized mucosal antagonism with minimal systemic absorption was selected as an angiotensin II type 1a receptor antagonist (AT1aR-A). Two colitis models were studied: (1) Acute colitis was induced in 8- to 10-week-old C57BL/6J mice by 2.5 % dextran sodium sulfate (DSS, in drinking water) for 7 days. (2) IL10-/-colitis Piroxicam (200 ppm) was administered orally in feed to 5-week-old IL-10-/-mice (C57BL/6J background) for 14 days followed by enalaprilat (ACE-I), CCG-203025 or PBS administered transanally for 14 days.

Results: In the DSS model, weight loss and histologic score for CCG-203025 were better than with placebo. In the IL10-/-model, ACE-I suppressed histologic damage better than CCG-203025. Both ACE-I and CCG-203025 reduced pro-inflammatory cytokines and chemokines.

Conclusions: This study demonstrated the therapeutic efficacy of both ACE-I and AT1aR-A for preventing the development of both acute and immunologically relevant colitis.
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http://dx.doi.org/10.1007/s00383-016-3965-3DOI Listing
December 2016

Occludin S471 Phosphorylation Contributes to Epithelial Monolayer Maturation.

Mol Cell Biol 2016 08 14;36(15):2051-66. Epub 2016 Jul 14.

Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, USA Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA

Multiple organ systems require epithelial barriers for normal function, and barrier loss is a hallmark of diseases ranging from inflammation to epithelial cancers. However, the molecular processes regulating epithelial barrier maturation are not fully elucidated. After contact, epithelial cells undergo size-reductive proliferation and differentiate, creating a dense, highly ordered monolayer with high resistance barriers. We provide evidence that the tight junction protein occludin contributes to the regulation of epithelial cell maturation upon phosphorylation of S471 in its coiled-coil domain. Overexpression of a phosphoinhibitory occludin S471A mutant prevents size-reductive proliferation and subsequent tight junction maturation in a dominant manner. Inhibition of cell proliferation in cell-contacted but immature monolayers recapitulated this phenotype. A kinase screen identified G-protein-coupled receptor kinases (GRKs) targeting S471, and GRK inhibitors delayed epithelial packing and junction maturation. We conclude that occludin contributes to the regulation of size-reductive proliferation and epithelial cell maturation in a phosphorylation-dependent manner.
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http://dx.doi.org/10.1128/MCB.00053-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946429PMC
August 2016