scholarly journals A 3D primary human cell-based in vitro model of non-alcoholic steatohepatitis for efficacy testing of clinical drug candidates

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Simon Ströbel ◽  
Radina Kostadinova ◽  
Katia Fiaschetti-Egli ◽  
Jana Rupp ◽  
Manuela Bieri ◽  
...  
Keyword(s):  
Complex Disease ◽  
Therapeutic Potential ◽  
Expression Patterns ◽  
Therapeutic Interventions ◽  
Type I ◽  
Drug Candidates ◽  
Procollagen Type ◽  
Alcoholic Steatohepatitis ◽  
Inflammatory Stimuli

AbstractNon-alcoholic steatohepatitis (NASH) is a progressive and severe liver disease, characterized by lipid accumulation, inflammation, and downstream fibrosis. Despite its increasing prevalence, there is no approved treatment yet available for patients. This has been at least partially due to the lack of predictive preclinical models for studying this complex disease. Here, we present a 3D in vitro microtissue model that uses spheroidal, scaffold free co-culture of primary human hepatocytes, Kupffer cells, liver endothelial cells and hepatic stellate cells. Upon exposure to defined and clinically relevant lipotoxic and inflammatory stimuli, these microtissues develop key pathophysiological features of NASH within 10 days, including an increase of intracellular triglyceride content and lipids, and release of pro-inflammatory cytokines. Furthermore, fibrosis was evident through release of procollagen type I, and increased deposition of extracellular collagen fibers. Whole transcriptome analysis revealed changes in the regulation of pathways associated with NASH, such as lipid metabolism, inflammation and collagen processing. Importantly, treatment with anti-NASH drug candidates (Selonsertib and Firsocostat) decreased the measured specific disease parameter, in accordance with clinical observations. These drug treatments also significantly changed the gene expression patterns of the microtissues, thus providing mechanisms of action and revealing therapeutic potential. In summary, this human NASH model represents a promising drug discovery tool for understanding the underlying complex mechanisms in NASH, evaluating efficacy of anti-NASH drug candidates and identifying new approaches for therapeutic interventions.

scholarly journals A 3D primary human cell-based in vitro model of non-alcoholic steatohepatitis for efficacy testing of clinical drug candidates

2021 ◽  
Author(s):  
Simon Ströbel ◽  
Radina Kostadinova ◽  
Katia Fiaschetti-Egli ◽  
Jana Rupp ◽  
Manuela Bieri ◽  
...  
Keyword(s):  
Complex Disease ◽  
Therapeutic Potential ◽  
Expression Patterns ◽  
Therapeutic Interventions ◽  
Type I ◽  
Drug Candidates ◽  
Procollagen Type ◽  
Alcoholic Steatohepatitis ◽  
Inflammatory Stimuli

Abstract Non-alcoholic steatohepatitis (NASH) is a progressive and severe liver disease, characterized by lipid accumulation, inflammation, and downstream fibrosis. Despite its increasing prevalence, there is no approved treatment yet available for patients. This has been at least partially due to the lack of predictive preclinical models for studying this complex disease. Here, we present a 3D in vitro microtissue model that uses spheroidal, scaffold free co-culture of primary human hepatocytes, Kupffer cells, liver endothelial cells and hepatic stellate cells. Upon exposure to defined and clinically relevant lipotoxic and inflammatory stimuli, these microtissues develop key pathophysiological features of NASH within 10 days, including an increase of intracellular triglyceride content and lipids, and release of pro-inflammatory cytokines. Furthermore, fibrosis was evident through release of procollagen type I, and increased deposition of extracellular collagen fibers. Whole transcriptome analysis revealed changes in the regulation of pathways associated with NASH, such as lipid metabolism, inflammation and collagen processing. Importantly, treatment with anti-NASH drug candidates (Selonsertib and Firsocostat) decreased the measured specific disease parameter, in accordance with clinical observations. These drug treatments also significantly changed the gene expression patterns of the microtissues, thus providing mechanisms of action and revealing therapeutic potential. In summary, this human NASH model represents a promising drug discovery tool for understanding the underlying complex mechanisms in NASH, evaluating efficacy of anti-NASH drug candidates and identifying new approaches for therapeutic interventions.

scholarly journals PolypharmDB, a Deep Learning-Based Resource, Quickly Identifies Repurposed Drug Candidates for COVID-19

2020 ◽  
Author(s):  
Dar'ya S. Redka ◽  
Stephen S. MacKinnon ◽  
Melissa Landon ◽  
Andreas Windemuth ◽  
Naheed Kurji ◽  
...  
Keyword(s):  
Deep Learning ◽  
Small Molecule ◽  
Drug Targets ◽  
Therapeutic Potential ◽  
Host Cells ◽  
Therapeutic Interventions ◽  
Clinical Environment ◽  
Drug Candidates ◽  
Small Molecule Drugs

<p>There is an immediate need to discover treatments for COVID-19, the pandemic caused by the SARS-CoV-2 virus. Standard small molecule drug discovery workflows that start with library screens are an impractical path forward given the timelines to discover, develop, and test clinically. To accelerate the time to patient testing, here we explored the therapeutic potential of small molecule drugs that have been tested to some degree in a clinical environment, including approved medications, as possible therapeutic interventions for COVID-19. Motivating our process is a concept termed polypharmacology, i.e. off-target interactions that may hold therapeutic potential. In this work, we used Ligand Design, our deep learning drug design platform, to interrogate the polypharmacological profiles of an internal collection of small molecule drugs with federal approval or going through clinical trials, with the goal of identifying molecules predicted to modulate targets relevant for COVID-19 treatment. Resulting from our efforts is PolypharmDB, a resource of drugs and their predicted binding of protein targets across the human proteome. Mining PolypharmDB yielded molecules predicted to interact with human and viral drug targets for COVID-19, including host proteins linked to viral entry and proliferation and key viral proteins associated with the virus life-cycle. Further, we assembled a collection of prioritized approved drugs for two specific host-targets, TMPRSS2 and cathepsin B, whose joint inhibition was recently shown to block SARS-CoV-2 virus entry into host cells. Overall, we demonstrate that our approach facilitates rapid response, identifying 30 prioritized candidates for testing for their possible use as anti-COVID drugs. Using the PolypharmDB resource, it is possible to identify repurposed drug candidates for newly discovered targets within a single work day. We are making a complete list of the molecules we identified available at no cost to partners with the ability to test them for efficacy, in vitro and/or clinically.</p><div><br></div>

scholarly journals PolypharmDB, a Deep Learning-Based Resource, Quickly Identifies Repurposed Drug Candidates for COVID-19

2020 ◽  
Author(s):  
Dar'ya S. Redka ◽  
Stephen S. MacKinnon ◽  
Melissa Landon ◽  
Andreas Windemuth ◽  
Naheed Kurji ◽  
...  
Keyword(s):  
Deep Learning ◽  
Small Molecule ◽  
Drug Targets ◽  
Therapeutic Potential ◽  
Host Cells ◽  
Therapeutic Interventions ◽  
Clinical Environment ◽  
Drug Candidates ◽  
Small Molecule Drugs

<p>There is an immediate need to discover treatments for COVID-19, the pandemic caused by the SARS-CoV-2 virus. Standard small molecule drug discovery workflows that start with library screens are an impractical path forward given the timelines to discover, develop, and test clinically. To accelerate the time to patient testing, here we explored the therapeutic potential of small molecule drugs that have been tested to some degree in a clinical environment, including approved medications, as possible therapeutic interventions for COVID-19. Motivating our process is a concept termed polypharmacology, i.e. off-target interactions that may hold therapeutic potential. In this work, we used Ligand Design, our deep learning drug design platform, to interrogate the polypharmacological profiles of an internal collection of small molecule drugs with federal approval or going through clinical trials, with the goal of identifying molecules predicted to modulate targets relevant for COVID-19 treatment. Resulting from our efforts is PolypharmDB, a resource of drugs and their predicted binding of protein targets across the human proteome. Mining PolypharmDB yielded molecules predicted to interact with human and viral drug targets for COVID-19, including host proteins linked to viral entry and proliferation and key viral proteins associated with the virus life-cycle. Further, we assembled a collection of prioritized approved drugs for two specific host-targets, TMPRSS2 and cathepsin B, whose joint inhibition was recently shown to block SARS-CoV-2 virus entry into host cells. Overall, we demonstrate that our approach facilitates rapid response, identifying 30 prioritized candidates for testing for their possible use as anti-COVID drugs. Using the PolypharmDB resource, it is possible to identify repurposed drug candidates for newly discovered targets within a single work day. We are making a complete list of the molecules we identified available at no cost to partners with the ability to test them for efficacy, in vitro and/or clinically.</p><div><br></div>

Effects of fluoride on human bone cells in vitro: differences in responsiveness between stromal osteoblast precursors and mature osteoblasts

1994 ◽  
Vol 130 (4) ◽  
pp. 381-386 ◽  
Author(s):  
Moustapha Kassem ◽  
Leif Mosekilde ◽  
Erik F Eriksen
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Sodium Fluoride ◽  
Bone Cells ◽  
Human Bone ◽  
Type I ◽  
Procollagen Type ◽  
Procollagen Type I ◽  
Osteoblast Precursors

Kassem M, Mosekilde L, Eriksen EF. Effects of fluoride on human bone cells in vitro: differences in responsiveness between stromal osteoblast precursors and mature osteoblasts. Eur J Endocrinol 1994;130:381–6. ISSN 0804–4643 The cellular effects of sodium fluoride (NaF) on human bone cells in vitro have been variable and dependent on the culture system used. Variability could be attributed to differences in responsiveness to NaF among different populations of cells at various stages of differentiation in the osteoblastic lineage. In this study we compared the effects of NaF in serum-free medium on cultures of more differentiated human osteoblast-like (hOB) cells derived from trabecular bone explants and on osteoblast committed precursors derived from human bone marrow, i.e. human marrow stromal osteoblast-like (hMS(OB)) cells. Sodium fluoride (10−5 mol/l) increased proliferation of hMS(OB) cells (p<0.05, N = 10) but was not mitogenic to hOB cells (p>0.05, N= 10). Alkaline phosphatase (AP) production increased in both hMS(OB) (p<0.05, N=9) and hOB cells (p<0.05, N=9). No significant effects on procollagen type I propeptide production were obtained in either culture. In the presence of 1,25-dihydroxycholecalciferol (10−9 mol/l), NaF enhanced alkaline phosphatase (p<0.05, N=8), procollagen type I propeptide (p<0.05, N=7) and osteocalcin (p<0.05, N=7) production by hMS(OB) cells but not by hOB cells. Our results suggest that osteoblast precursors are more sensitive to NaF action than mature osteoblasts and that the in vivo effects of NaF on bone formation may be mediated by stimulating proliferation and differentiation of committed osteoblast precursors in bone marrow. M Kassem, Mayo Clinic, Endocrine Research Unit, W-Joseph 5-164, Rochester, MN 55904, USA

scholarly journals Development of Small-Molecule STING Activators for Cancer Immunotherapy

Biomedicines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 33
Author(s):  
Hee Ra Jung ◽  
Seongman Jo ◽  
Min Jae Jeon ◽  
Hyelim Lee ◽  
Yeonjeong Chu ◽  
...  
Keyword(s):  
Cancer Immunotherapy ◽  
Cyclic Gmp ◽  
Therapeutic Potential ◽  
Interferon Response ◽  
Type I ◽  
Anti Cancer ◽  
Cancer Agent ◽  
Sting Pathway

In cancer immunotherapy, the cyclic GMP–AMP synthase–stimulator of interferon genes (STING) pathway is an attractive target for switching the tumor immunophenotype from ‘cold’ to ‘hot’ through the activation of the type I interferon response. To develop a new chemical entity for STING activator to improve cyclic GMP-AMP (cGAMP)-induced innate immune response, we identified KAS-08 via the structural modification of DW2282, which was previously reported as an anti-cancer agent with an unknown mechanism. Further investigation revealed that direct STING binding or the enhanced phosphorylation of STING and downstream effectors were responsible for DW2282-or KAS-08-mediated STING activity. Furthermore, KAS-08 was validated as an effective STING pathway activator in vitro and in vivo. The synergistic effect of cGAMP-mediated immunity and efficient anti-cancer effects successfully demonstrated the therapeutic potential of KAS-08 for combination therapy in cancer treatment.

scholarly journals Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns.

1990 ◽  
Vol 111 (2) ◽  
pp. 567-580 ◽  
Author(s):  
R Moll ◽  
D L Schiller ◽  
W W Franke
Keyword(s):  
Mammalian Species ◽  
Expression Patterns ◽  
Prostate Gland ◽  
Coiled Coil ◽  
Immunological Methods ◽  
Type I ◽  
Peptide Fragments ◽  
Binding Assays ◽  
Human Intestinal Epithelium

A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.

scholarly journals Rat alveolar type I cells proliferate, express OCT-4, and exhibit phenotypic plasticity in vitro

2009 ◽  
Vol 297 (6) ◽  
pp. L1045-L1055 ◽  
Author(s):  
Robert F. Gonzalez ◽  
Lennell Allen ◽  
Leland G. Dobbs
Keyword(s):  
Phenotypic Plasticity ◽  
Expression Patterns ◽  
Clara Cell ◽  
Cell Phenotype ◽  
Alveolar Type ◽  
Type I ◽  
Aquaporin 5 ◽  
Internal Surface Area ◽  
Phenotypic Markers

Alveolar type I (TI) cells are large, squamous cells that cover 95–99% of the internal surface area of the lung. Although TI cells are believed to be terminally differentiated, incapable of either proliferation or phenotypic plasticity, TI cells in vitro both proliferate and express phenotypic markers of other differentiated cell types. Rat TI cells isolated in purities of >99% proliferate in culture, with a sixfold increase in cell number before the cells reach confluence; >50% of the cultured TI cells are Ki67+. At cell densities of 1–2 cells/well, ∼50% of the cells had the capacity to form colonies. Under the same conditions, type II cells do not proliferate. Cultured TI cells express RTI40 and aquaporin 5, phenotypic markers of the TI cell phenotype. By immunofluorescence, Western blotting, and Q-PCR, TI cells express OCT-4A (POU5F1), a transcription factor associated with maintenance of the pluripotent state in stem cells. Based on the expression patterns of various marker proteins, TI cells are distinct from either of two recently described putative pulmonary multipotent cell populations, the bronchoalveolar stem cell or the OCT-4+ stem/progenitor cell. Although TI cells in adult rat lung tissue do not express either surfactant protein C (SP-C) or CC10, respective markers of the TII and Clara cell phenotypes, in culture TI cells can be induced to express both SP-C and CC10. Together, the findings that TI cells proliferate and exhibit phenotypic plasticity in vitro raise the possibility that TI cells may have similar properties in vivo.

scholarly journals Human single-chain antibodies neutralize SARS-CoV-2 variants by engaging an essential epitope of the spike: a new weapon against COVID-19

2021 ◽  
Author(s):  
Olga Minenkova ◽  
Daniela Santapaola ◽  
Ferdinando Maria Milazzo ◽  
Annamaria Anastasi ◽  
Gianfranco Battistuzzi ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Therapeutic Interventions ◽  
Binding Motif ◽  
Single Chain ◽  
Drug Candidates ◽  
Treatment And Prevention ◽  
Variable Domains ◽  
Phage Libraries ◽  
Syncytia Formation

As of June 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency and effective therapeutic interventions for the treatment and prevention of coronavirus disease 2019 (COVID-19) are urgently needed. SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) represent a promising approach to COVID-19 therapy. However, the recently described accumulating mutations in the SARS-CoV-2 spike protein are challenging the efficacy of approved and investigational mAbs, whose widespread use is also hampered by their significant costs and possible side effects, including Antibody-Dependent Enhancement (ADE). Here we describe a cluster of SARS-CoV-2 neutralizing human single chain variable fragment antibodies, identified by phage display, sharing a common VH CDR3 sequence. Phage libraries were built by amplifying variable domains of immunoglobulin genes from cDNA derived from lymphocytes of COVID-19 convalescent subjects living in Bergamo, Italy. The scFv76-cluster antibodies (scFv76-cl Abs) exhibit high affinity for the spike receptor binding domain (RBD) of Wuhan strain and emerging variants, leading to inhibition of RBD/human ACE2 interaction. The antigenic epitope recognized by scFv76 was mapped in the receptor binding motif (RBM) of RBD at residues L455, F456, Y473, N487 and Y489. None of these residues has been to date listed among the RBD mutations of SARS-CoV-2 variants of concern (VOCs), suggesting an important role of such epitope in viral infectivity. Treatment with scFv76-cl Abs is effective against SARS-CoV-2, as determined by in vitro experiments of viral infection, replication, cytopathogenicity and spike-mediated syncytia formation. Moreover, their intranasal administration is shown to counteract infection in two independent animal models. Overall, the biochemical and biological characteristics of scFv76-cl Abs are compatible with their clinical use for COVID-19 therapy by intranasal or aerosol administration. To our knowledge, this is the first example of promising human anti-SARS-CoV-2 scFv antibodies as drug candidates for COVID-19 therapy.

scholarly journals A Novel, Nonpeptidic, Orally Active Bivalent Inhibitor of Human β-Tryptase

Pharmacology ◽  
2018 ◽  
Vol 102 (5-6) ◽  
pp. 233-243 ◽  
Author(s):  
Sarah F. Giardina ◽  
Douglas S. Werner ◽  
Maneesh Pingle ◽  
Donald E. Bergstrom ◽  
Lee D. Arnold ◽  
...  
Keyword(s):  
Active Sites ◽  
Rational Design ◽  
Therapeutic Potential ◽  
X Ray Crystallography ◽  
Inflammatory Stimuli ◽  
Invaluable Tool ◽  
Xenograft Models ◽  
Orally Bioavailable

β-Tryptase is released from mast cells upon degranulation in response to allergic and inflammatory stimuli. Human tryptase is a homotetrameric serine protease with 4 identical active sites directed toward a central pore. These active sites present an optimized scenario for the rational design of bivalent inhibitors, which bridge 2 adjacent active sites. Using (3-[1-acylpiperidin-4-yl]phenyl)methanamine as the pharmacophoric core and a disiloxane linker to span 2 active sites we have successfully produced a novel bivalent tryptase inhibitor, compound 1a, with a comparable profile to previously described inhibitors. Pharmacological properties of compound 1a were studied in a range of in vitro enzymic and cellular screening assays, and in vivo xenograft models. This non-peptide inhibitor of tryptase demonstrated superior activity (IC50 at 100 pmol/L tryptase = 1.82 nmol/L) compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and 1a demonstrated good oral bioavailability and efficacy in HMC-1 xenograft models. Furthermore, compound 1a demonstrated extremely slow off rates and high selectivity against-related proteases. This highly potent, orally bioavailable and selective inhibitor of human tryptase will be an invaluable tool in future studies to explore the therapeutic potential of attenuating the activity of this elusive target.

Sign in / Sign up

Export Citation Format

Share Document