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1.
Matrix Biol ; 103-104: 37-57, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34653670

RESUMO

Heparan sulfate 3-O-sulfotransferases generate highly sulfated but rare 3-O-sulfated heparan sulfate (HS) epitopes on cell surfaces and in the extracellular matrix. Previous ex vivo experiments suggested functional redundancy exists among the family of seven enzymes but that Hs3st3a1 and Hs3st3b1 sulfated HS increases epithelial FGFR signaling and morphogenesis. Single-cell RNAseq analysis of control SMGs identifies increased expression of Hs3st3a1 and Hs3st3b1 in endbud and myoepithelial cells, both of which are progenitor cells during development and regeneration. To analyze their in vivo functions, we generated both Hs3st3a1-/- and Hs3st3b1-/- single knockout mice, which are viable and fertile. Salivary glands from both mice have impaired fetal epithelial morphogenesis when cultured with FGF10. Hs3st3b1-/- mice have reduced intact SMG branching morphogenesis and reduced 3-O-sulfated HS in the basement membrane. Analysis of HS biosynthetic enzyme transcription highlighted some compensatory changes in sulfotransferases expression early in development. The overall glycosaminoglycan composition of adult control and KO mice were similar, although HS disaccharide analysis showed increased N- and non-sulfated disaccharides in Hs3st3a1-/- HS. Analysis of adult KO gland function revealed normal secretory innervation, but without stimulation there was an increase in frequency of drinking behavior in both KO mice, suggesting basal salivary hypofunction, possibly due to myoepithelial dysfunction. Understanding how 3-O-sulfation regulates myoepithelial progenitor function will be important to manipulate HS-binding growth factors to enhance tissue function and regeneration.


Assuntos
Heparitina Sulfato , Sulfotransferases , Animais , Fatores de Crescimento de Fibroblastos , Camundongos , Morfogênese , Glândulas Salivares , Sulfotransferases/genética
2.
ACS Infect Dis ; 5(5): 750-758, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30582687

RESUMO

An estimated 240 million are chronically infected with hepatitis B virus (HBV), which can lead to liver disease, cirrhosis, and hepatocellular carcinoma. Currently, HBV treatment options include only nucleoside reverse transcriptase inhibitors and the immunomodulatory agent interferon alpha, and these treatments are generally not curative. New treatments with novel mechanisms of action, therefore, are highly desired for HBV therapy. The viral core protein (Cp) has gained attention as a possible therapeutic target because of its vital roles in the HBV life cycle. Several classes of capsid assembly effectors (CAEs) have been described in detail, and these compounds all increase capsid assembly rate but inhibit HBV replication by different mechanisms. In this study, we have developed a thermal shift-based screening method for CAE discovery and characterization, filling a much-needed gap in high-throughput screening methods for capsid-targeting molecules. Using this approach followed by cell-based screening, we identified the compound HF9C6 as a CAE with low micromolar potency against HBV replication. HF9C6 caused large multicapsid aggregates when capsids were assembled in vitro and analyzed by transmission electron microscopy. Interestingly, when HBV-expressing cells were treated with HF9C6, Cp was excluded from cell nuclei, suggesting that this compound may inhibit nuclear entry of Cp and capsids. Furthermore, mutational scanning of Cp suggested that HF9C6 binds the known CAE binding pocket, indicating that key Cp-compound interactions within this pocket have a role in determining the CAE mechanism of action.


Assuntos
Antivirais/química , Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Proteínas do Core Viral/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Células Hep G2 , Vírus da Hepatite B/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
3.
Eur J Med Chem ; 164: 179-192, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30594676

RESUMO

Chronic hepatitis B virus (HBV) infection represents a major health threat. Current FDA-approved drugs do not cure HBV. Targeting HBV core protein (Cp) provides an attractive approach toward HBV inhibition and possibly infection cure. We have previously identified and characterized a 5-amino-3-methylthiophene-2,4-dicarboxamide (ATDC) compound as a structurally novel hit for capsid assembly effectors (CAEs). We report herein hit validation through studies on absorption, distribution, metabolism and excretion (ADME) properties and pharmacokinetics (PK), and hit optimization via analogue synthesis aiming to probe the structure-activity relationship (SAR) and structure-property relationship (SPR). In the end, these medicinal chemistry efforts led to the identification of multiple analogues strongly binding to Cp, potently inhibiting HBV replication in nanomolar range without cytotoxicity, and exhibiting good oral bioavailability (F). Two of our analogues, 19o (EC50 = 0.11 µM, CC50 > 100 µM, F = 25%) and 19k (EC50 = 0.31 µM, CC50 > 100 µM, F = 46%), displayed overall lead profiles superior to reported CAEs 7-10 used in our studies.


Assuntos
Antivirais/química , Capsídeo/efeitos dos fármacos , Vírus da Hepatite B/ultraestrutura , Tiofenos/farmacologia , Montagem de Vírus/efeitos dos fármacos , Antivirais/síntese química , Disponibilidade Biológica , Capsídeo/metabolismo , Vírus da Hepatite B/metabolismo , Humanos , Ligação Proteica , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/uso terapêutico , Proteínas do Core Viral/metabolismo , Replicação Viral/efeitos dos fármacos
4.
mSphere ; 3(2)2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29669885

RESUMO

Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly in vitro have been previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cp in vitro and causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs. Interestingly, immunofluorescence analysis showed Cp aggregating in nuclear foci of Bay 38-7690-treated infected cells in a time- and dose-dependent manner. We found these foci to be associated with promyelocytic leukemia (PML) nuclear bodies (NBs), which are structures that affect many cellular functions, including DNA damage response, transcription, apoptosis, and antiviral responses. Cp aggregation is not an artifact of the cell system used, as it is observed in HBV-expressing HepAD38 cells, in HepG2 cells transfected with an HBV-expressing plasmid, and in HepG2-NTCP cells infected with HBV. Use of a Cp overexpression vector without HBV sequences shows that aggregation is independent of viral replication, and use of an HBV-expressing plasmid harboring a HAP resistance mutation in Cp abrogated the aggregation, demonstrating that the effect is due to direct compound-Cp interactions. These studies provide novel insight into the effects of HAP-based treatment at a single-cell level.IMPORTANCE Despite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and stability, capsid formation, and host immune modulators, with the ultimate goal of an HBV cure. Understanding the mechanisms by which novel antiviral agents act will be imperative for the development of curative HBV therapies.


Assuntos
Antivirais/farmacologia , Proteínas do Capsídeo/química , Vírus da Hepatite B/efeitos dos fármacos , Corpos de Inclusão Viral/química , Agregados Proteicos/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Capsídeo/química , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/genética , Imunofluorescência , Células Hep G2 , Hepatite B/tratamento farmacológico , Vírus da Hepatite B/fisiologia , Humanos , Proteínas Recombinantes/química , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
5.
Matrix Biol ; 57-58: 311-323, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27609403

RESUMO

Branching morphogenesis is a fundamental process in the development of diverse epithelial organs such as the lung, kidney, liver, pancreas, prostate, salivary, lacrimal and mammary glands. A unifying theme during organogenesis is the importance of epithelial cell interactions with the extracellular matrix (ECM) and growth factors (GFs). The diverse developmental mechanisms giving rise to these epithelial organs involve many organ-specific GFs, but a unifying paradigm during organogenesis is the regulation of GF activity by heparan sulfates (HS) on the cell surface and in the ECM. This primarily involves the interactions of GFs with the sulfated side-chains of HS proteoglycans. HS is one of the most diverse biopolymers and modulates GF binding and signaling at the cell surface and in the ECM of all tissues. Here, we review what is known about how HS regulates branching morphogenesis of epithelial organs with emphasis on the developing salivary gland, which is a classic model to investigate epithelial-ECM interactions. We also address the structure, biosynthesis, turnover and function of HS during organogenesis. Understanding the regulatory mechanisms that control HS dynamics may aid in the development of therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine.


Assuntos
Matriz Extracelular/fisiologia , Heparitina Sulfato/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Organogênese/fisiologia , Proteoglicanas/fisiologia , Animais , Células Epiteliais/química , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Glândulas Exócrinas/crescimento & desenvolvimento , Glândulas Exócrinas/metabolismo , Glândulas Exócrinas/ultraestrutura , Matriz Extracelular/química , Feminino , Heparitina Sulfato/química , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Rim/crescimento & desenvolvimento , Rim/metabolismo , Rim/ultraestrutura , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Fígado/ultraestrutura , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Pulmão/ultraestrutura , Masculino , Pâncreas/crescimento & desenvolvimento , Pâncreas/metabolismo , Pâncreas/ultraestrutura , Próstata/crescimento & desenvolvimento , Próstata/metabolismo , Próstata/ultraestrutura , Proteoglicanas/química , Transdução de Sinais
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