scholarly journals Predictability of Peripheral Lymphocyte Reduction of Novel S1P1 Agonists by In Vitro GPCR Signaling Profile

2013 ◽  
Vol 18 (9) ◽  
pp. 997-1007 ◽  
Author(s):  
Han Xu ◽  
Michele McElvain ◽  
Mike Fiorino ◽  
Brad Henkle ◽  
Lisa Sherman ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Adenylyl Cyclase ◽  
Peripheral Lymphocyte ◽  
Receptor Internalization ◽  
In Vitro Assays ◽  
Vitro Assay ◽  
In Vivo Efficacy ◽  
Erk Phosphorylation

Surrogate readouts of G-protein–coupled receptor signaling pathways using highly engineered systems are often employed in the drug discovery process. However, accumulating data have demonstrated the importance of selecting relevant biological activity rather than technically facile assays to support high-throughout screening and subsequent structure-activity relationship studies. Here we report a case study using sphingosine-1-phosphate receptor 1 (S1P1) as the model system to compare compound activity in six different in vitro assays with their ability to predict in vivo efficacy. S1P1 has long been validated as a therapeutic target for autoimmune diseases. In this article, in vivo and in vitro studies on 19 S1P1 agonists are reported. In vitro activities of these S1P1 agonists, together with S1P and FTY720p, on Ca2+ mobilization, adenylyl cyclase inhibition, extracellular signal-related kinase (ERK) phosphorylation, β-arrestin recruitment, and receptor internalization, were determined. The in vitro potency of these compounds was correlated with their ability to induce peripheral lymphocyte reduction. The results revealed that inhibition of adenylyl cyclase and induction of β-arrestin recruitment and receptor internalization are good indicators to predict in vivo efficacy, whereas induction of Ca2+ mobilization through Gqi/5 coupling and ERK phosphorylation is irrelevant. This study demonstrated the importance of identifying an appropriate in vitro assay to predict in vivo activity based on the biological relevance in the drug discovery setting.

scholarly journals 1341. Development of a Series of High-Throughput Screens to Identify Leads for Nontuberculous Mycobacteria Drug Design

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S485-S485
Author(s):  
Sarah McGuffin ◽  
Steven Mullen ◽  
Julie Early ◽  
Tanya Parish
Keyword(s):  
Drug Discovery ◽  
Drug Development ◽  
High Throughput ◽  
Nontuberculous Mycobacteria ◽  
Human Infection ◽  
Attrition Rate ◽  
In Vitro Assays ◽  
Vitro Assay

Abstract Background Nontuberculous mycobacteria (NTM), particularly Mycobacterium avium complex and Mycobacterium abscessus complex, cause significant morbidity and mortality in patients with impaired host immunity or pre-existing structural lung conditions. NTM infections are increasing at an alarming rate worldwide and there is a dearth of progress in regard to the development of efficacious and tolerable drugs to treat such infections. Traditional drug discovery screens do not account for the diverse physiological conditions, microenvironments, and compartments that the bacilli encounter during human infection. In order to help populate the NTM drug pipeline, and explore the disconnect between in vitro activity, in vivo activity, and clinical outcomes, we are developing a high throughput in vitro assay platform that will more closely model the unique infection-relevant conditions encountered by NTM. Methods We are developing and validating a suite of in vitro assays that screen compounds for activity against extracellular planktonic bacteria, extracellular bacteria within biofilms, intracellular bacteria, and nutrient-starved non-replicating bacteria. Results We are using both the smooth and rough morphotypes of M. abscessus and M. avium. We have validated high throughput assays to pharmaceutical standards for replicating and non-replicating M. abscessus. We have also tested a panel of 18 known anti-mycobacterial compounds. Assay development is currently underway to test compounds for activity against NTM in biofilm and inside macrophages as well. Conclusion To enhance hit identification for scaffolds to use as starting points for NTM drug development, focused libraries of compounds that have undergone significant preclinical profiling and/or compounds with known activity against M. tuberculosis (TB) will be screened. Such a “piggyback” approach usurps advances made in TB drug development and leverages them for NTM drug discovery. This will help expedite novel drug development, reduce attrition rate, and offer a shorter route to clinical use as it exploits the prior investment in medicinal chemistry, pharmacology, and toxicology. Disclosures All authors: No reported disclosures.

scholarly journals Leucine-Rich Diet Modulates the Metabolomic and Proteomic Profile of Skeletal Muscle during Cancer Cachexia

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1880 ◽  
Author(s):  
Bread Cruz ◽  
André Oliveira ◽  
Lais Rosa Viana ◽  
Leisa Lopes-Aguiar ◽  
Rafael Canevarolo ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cancer Cachexia ◽  
Energy Generation ◽  
In Vitro Assays ◽  
Proteomic Profile ◽  
Adult Rats ◽  
Vitro Assay ◽  
Tumour Bearing

Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle imbalance. Alternative therapy, as nutritional supplementation with leucine, shows a modulatory effect over tumour damage in vivo and in vitro. Method: Adult rats distributed into Control (C), Walker tumour-bearing (W), control fed a leucine-rich diet (L), and tumour-bearing fed a leucine-rich diet (WL) groups had the gastrocnemius muscle metabolomic and proteomic assays performed in parallel to in vitro assays. Results: W group presented an affected muscle metabolomic and proteomic profile mainly related to energy generation and carbohydrates catabolic processes, but leucine-supplemented group (WL) recovered the energy production. In vitro assay showed that cell proliferation, mitochondria number and oxygen consumption were higher under leucine effect than the tumour influence. Muscle proteomics results showed that the main affected cell component was mitochondria, leading to an impacted energy generation, including impairment in proteins of the tricarboxylic cycle and carbohydrates catabolic processes, which were modulated and improved by leucine treatment. Conclusion: In summary, we showed a beneficial effect of leucine upon mitochondria, providing information about the muscle glycolytic pathways used by this amino acid, where it can be associated with the preservation of morphometric parameters and consequent protection against the effects of cachexia.

Assessment of Inhibition of Bovine Hepatic Cytochrome P450 by 43 Commercial Bovine Medicines Using a Combination of In Vitro Assays and Pharmacokinetic Data from the Literature

2020 ◽  
Vol 13 (2) ◽  
pp. 123-131
Author(s):  
Steven X. Hu ◽  
Chase A. Mazur ◽  
Kenneth L. Feenstra
Keyword(s):  
Liver Microsomes ◽  
In Vitro Assay ◽  
In Vitro Assays ◽  
Pharmacokinetic Studies ◽  
Pharmacokinetic Data ◽  
Vitro Assay ◽  
Administration Routes ◽  
Ic50 Values

Background: There has been a lack of information about the inhibition of bovine medicines on bovine hepatic CYP450 at their commercial doses and dosing routes. Objective: The aim of this work was to assess the inhibition of 43 bovine medicines on bovine hepatic CYP450 using a combination of in vitro assay and Cmax values from pharmacokinetic studies with their commercial doses and dosing routes in the literature. Methods: Those drugs were first evaluated through a single point inhibitory assay at 3 μM in bovine liver microsomes for six specific CYP450 metabolisms, phenacetin o-deethylation, coumarin 7- hydroxylation, tolbutamide 4-hydroxylation, bufuralol 1-hydroxylation, chlorzoxazone 6-hydroxylation and midazolam 1’-hydroxylation. When the inhibition was greater than 20% in the assay, IC50 values were then determined. The potential in vivo bovine hepatic CYP450 inhibition by those drugs was assessed using a combination of the IC50 values and in vivo Cmax values from pharmacokinetic studies at their commercial doses and administration routes in the literature. Results: Fifteen bovine medicines or metabolites showed in vitro inhibition on one or more bovine hepatic CYP450 metabolisms with different IC50 values. Desfuroylceftiour (active metabolite of ceftiofur), nitroxinil and flunixin have the potential to inhibit one of the bovine hepatic CYP450 isoforms in vivo at their commercial doses and administration routes. The rest of the bovine medicines had low risks of in vivo bovine hepatic CYP450 inhibition. Conclusion: This combination of in vitro assay and in vivo Cmax data provides a good approach to assess the inhibition of bovine medicines on bovine hepatic CYP450.

In vitro activity and in vivo efficacy of fexinidazole against New World Leishmania species

2019 ◽  
Vol 74 (8) ◽  
pp. 2318-2325 ◽  
Author(s):  
Eliane de Morais-Teixeira ◽  
Ana Rabello ◽  
Marta Marques Gontijo Aguiar
Keyword(s):  
Cutaneous Leishmaniasis ◽  
New World ◽  
Leishmania Species ◽  
Vitro Activity ◽  
In Vitro Assays ◽  
In Vitro Activity ◽  
In Vivo Evaluation ◽  
In Vivo Efficacy

Abstract Objectives To evaluate the in vitro activity and in vivo efficacy of fexinidazole against the main species that cause visceral and cutaneous New World leishmaniasis. Methods The inhibitory concentrations of fexinidazole against Leishmania (Leishmania) infantum chagasi, Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis in amastigotes were determined by in vitro activity assays. For the in vivo evaluation, animals were infected with L. (L.) infantum chagasi, L. (L.) amazonensis, L. (V.) braziliensis or Leishmania (Viannia) guyanensis and divided into groups: (i) control; and (ii) treated with oral fexinidazole, from 50 to 300 mg/kg/day. For cutaneous leishmaniasis, the size of the lesion was determined weekly after the beginning of the treatment. Upon completion, parasites were recovered from the spleen and liver, or skin lesion and spleen, and evaluated by a limiting dilution assay. Results All Leishmania isolates were susceptible to fexinidazole in the in vitro assays. The viable parasites in the liver and spleen were reduced with 100 and 300 mg/kg/day, respectively, for L. (L.) infantum chagasi. For the species causing cutaneous leishmaniasis, the viable parasites in lesions and the size of the lesions were reduced, starting from 200 mg/kg/day. The viable parasites in the spleen were also reduced with 200 and 300 mg/kg/day for L. (V.) braziliensis and L. (L.) amazonensis. Conclusions Considering the defined parameters, fexinidazole showed in vitro and in vivo activity against all tested species. This drug may represent an alternative treatment for the New World species.

scholarly journals In Vitro Assays for Phototoxicity

1998 ◽  
Vol 17 (5) ◽  
pp. 567-570
Author(s):  
A. Kornhauser ◽  
R. R. Wei ◽  
W. G. Warner
Keyword(s):  
In Vitro Assay ◽  
In Vitro Assays ◽  
Vitro Assay ◽  
Cellular Targets ◽  
Photooxidative Damage ◽  
Us Government ◽  
Promising Area ◽  
Rna And Dna

This paper summarizes a few in vitro methods to assess photodamage in cells irradiated with UV of various wavelengths in the presence of a number of photo-sensitizers. A single in vitro assay for phototoxicity (photoirritation) is not likely to be predictive because of different mechanisms of phototoxicity and diverse cellular targets for injury. A number of methods have to be combined to provide a better prediction of these phenomena. Measurement of mechanistically relevant biomarkers also represents a promising area of in vitro testing for phototoxicity, and it is also briefly reviewed in this paper. Photodynamic sensitizers, representing a large class of phototoxic agents, can now be identified by sensitive measurement of photooxidative damage to cellular RNA and DNA. Currently, US government agencies have not identified a single in vitro assay for phototoxicity which would be acceptable for replacing an in vivo assay for regulatory purposes.

scholarly journals In vitro Assays and Imaging Methods for Drug Discovery for Cardiac Fibrosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Giorgia Palano ◽  
Ariana Foinquinos ◽  
Erik Müllers
Keyword(s):  
Image Analysis ◽  
Drug Discovery ◽  
High Throughput Screening ◽  
Large Scale ◽  
Cardiac Fibrosis ◽  
3D Models ◽  
In Vitro Assays ◽  
Stress Injury

As a result of stress, injury, or aging, cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components resulting in pathological remodeling, tissue stiffening, ventricular dilatation, and cardiac dysfunction that contribute to heart failure (HF) and eventually death. Currently, there are no effective therapies specifically targeting cardiac fibrosis, partially due to limited understanding of the pathological mechanisms and the lack of predictive in vitro models for high-throughput screening of antifibrotic compounds. The use of more relevant cell models, three-dimensional (3D) models, and coculture systems, together with high-content imaging (HCI) and machine learning (ML)-based image analysis, is expected to improve predictivity and throughput of in vitro models for cardiac fibrosis. In this review, we present an overview of available in vitro assays for cardiac fibrosis. We highlight the potential of more physiological 3D cardiac organoids and coculture systems and discuss HCI and automated artificial intelligence (AI)-based image analysis as key methods able to capture the complexity of cardiac fibrosis in vitro. As 3D and coculture models will soon be sufficiently mature for application in large-scale preclinical drug discovery, we expect the combination of more relevant models and high-content analysis to greatly increase translation from in vitro to in vivo models and facilitate the discovery of novel targets and drugs against cardiac fibrosis.

The Use of On-line and Off-line Chromatographic Extraction Techniques coupled with Mass Spectrometry for Support of In Vivo and In Vitro Assays in Drug Discovery

2001 ◽  
Vol 1 (5) ◽  
pp. 463-471 ◽  
Author(s):  
John A. Masucci ◽  
Gary W. Caldwell ◽  
William J. Jones ◽  
Stephen J. Juzwin ◽  
Patrick J. Sasso ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
In Vitro Assays ◽  
Extraction Techniques ◽  
On Line ◽  
Chromatographic Extraction

scholarly journals Cell adhesion-dependent inactivation of a soluble protein kinase during fertilization in Chlamydomonas.

1996 ◽  
Vol 7 (4) ◽  
pp. 515-527 ◽  
Author(s):  
Y Zhang ◽  
Y Luo ◽  
K Emmett ◽  
W J Snell
Keyword(s):  
Protein Kinase ◽  
Adenylyl Cyclase ◽  
Protein Tyrosine Kinase ◽  
Tyrosine Phosphatase ◽  
Protein Kinase Activity ◽  
Vitro Assay ◽  
Protein Tyrosine ◽  
Flagellar Protein

Within seconds after the flagella of mt+ and mt- Chlamydomonas gametes adhere during fertilization, their flagellar adenylyl cyclase is activated several fold and preparation for cell fusion is initiated. Our previous studies indicated that early events in this pathway, including control of adenylyl cyclase, are regulated by phosphorylation and dephosphorylation. Here, we describe a soluble, flagellar protein kinase activity that is regulated by flagellar adhesion. A 48-kDa, soluble flagellar protein was consistently phosphorylated in an in vitro assay in flagella isolated from nonadhering mt+ and mt- gametes, but not in flagella isolated from mt+ and mt- gametes that had been adhering for 1 min. Although the 48-kDa protein was present in the flagella isolated from adhering gametes, we demonstrate that its protein kinase was inactivated by flagellar adhesion. Immunoblot analysis and inhibitor studies indicate that the 48-kDa protein in nonadhering gametes is phosphorylated by a protein tyrosine kinase. In vivo experiments showing that the protein tyrosine phosphatase inhibitor sodium orthovanadate inhibits fertilization suggest that protein dephosphorylation may be required for signal transduction. The 48-kDa protein and its protein kinase may be among the first elements of a novel signalling pathway that couples interaction of flagellar adhesion molecules to gamete activation.

scholarly journals Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

1996 ◽  
Vol 7 (3) ◽  
pp. 355-367 ◽  
Author(s):  
D J Spiro ◽  
W Boll ◽  
T Kirchhausen ◽  
M Wessling-Resnick
Keyword(s):  
Transferrin Receptor ◽  
Kinase Inhibitor ◽  
Morphological Changes ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Phosphatidylinositol 3 Kinase ◽  
Vitro Assay ◽  
Phosphatidylinositol 3

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

Inhibition of nitric oxide synthesis improves detoxication in inflammatory liver dysfunction in vivo

1997 ◽  
Vol 273 (2) ◽  
pp. G530-G536 ◽  
Author(s):  
A. Veihelmann ◽  
T. Brill ◽  
M. Blobner ◽  
I. Scheller ◽  
B. Mayer ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
In Vitro Assays ◽  
Serum Concentrations ◽  
Aminopyrine Breath Test ◽  
Vitro Assay ◽  
Synthase Inhibitor ◽  
Cytochrome P 450 ◽  
Stimulation Of

Inflammatory stimulation of the liver induces nitric oxide (NO) biosynthesis and suppression of detoxication. In this study the effect of NO biosynthesis on cytochrome P-450 (CYP) enzyme activity was investigated by comparing in vivo and in vitro assays. To establish liver inflammation, CD rats were injected with Corynebacterium parvum (C. parvum) suspension. After 5 days NO biosynthesis was highly induced as indicated by increased NO2- plus NO3- serum concentrations. At the same time the aminopyrine breath test (ABT), measuring CYP activity in vivo, was reduced to 42% and the in vitro assay of aminopyrine turnover was suppressed to 12% of NaCl- injected controls. When C. parvum-injected animals were treated with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), CYP activities significantly improved with an ABT of 76% and an in vitro aminopyrine turnover of 47% of controls. Neither C. parvum injections nor L-NMMA treatment resulted in a significant change of CYP protein concentrations. These data indicate that suppression of xenobiotic metabolism can be attenuated by inhibition of NO biosynthesis during an ongoing process of inflammation.

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