scholarly journals Steady-state enzyme kinetics

2021 ◽  
Author(s):  
Matthew D. Lloyd
Keyword(s):  
Steady State ◽  
Enzyme Kinetics ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Enzyme Inhibitors ◽  
Physiological Role ◽  
Hiv Protease ◽  
First Year ◽  
Hiv Protease Inhibitors ◽  
Compound Collection

Steady-state enzyme kinetics is a cornerstone technique of biochemistry and related sciences since it allows the characterization and quantification of enzyme behaviour. Enzyme kinetics is widely used to investigate the physiological role of enzymes, determine the effects of mutations and characterize enzyme inhibitors. Well-known examples of enzyme inhibitors used to treat diseases include anti-infectives (e.g., penicillin, clavulanic acid and HIV protease inhibitors); anti-inflammatories (e.g., aspirin and ibuprofen); cholesterol-lowering statins; tyrosine kinase inhibitors used to treat cancer; and Viagra. Commonly, new disease treatments are discovered by using enzyme kinetics to identify the few active compounds residing within a large compound collection (‘high-throughput screening’). The subject of enzyme kinetics is typically introduced to first-year undergraduates with a mathematical description of behaviour. This Beginners Guide will give a brief overview of experimental enzyme kinetics and the characterization of enzyme inhibitors. Colorimetric assays using a microtitre plate will be considered, although most principles also apply to other assays.

scholarly journals Effect of Rifampin on Steady-State Pharmacokinetics of Atazanavir with Ritonavir in Healthy Volunteers

2006 ◽  
Vol 50 (10) ◽  
pp. 3336-3342 ◽  
Author(s):  
D. M. Burger ◽  
S. Agarwala ◽  
M. Child ◽  
A. Been-Tiktak ◽  
Y. Wang ◽  
...  
Keyword(s):  
Steady State ◽  
Standard Of Care ◽  
Hiv Protease ◽  
Control Group ◽  
Hiv Protease Inhibitors ◽  
Time Curve ◽  
Concentration Time ◽  
Immunodeficiency Virus ◽  
Dosing Regimens ◽  
Grade 3

ABSTRACT Mycobacterium tuberculosis is a concern in patients with human immunodeficiency virus (HIV) infection. Rifampin (RIF), an agent used against M. tuberculosis, is contraindicated with most HIV protease inhibitors. Atazanavir (ATV) has clinical efficacy comparable to a standard of care regimen in naive patients and, when dosed with low-dose ritonavir (RTV), also in treatment-experienced patients. We evaluated here the safety and pharmacokinetics of ATV, resulting from three regimens of ATV, RTV, and RIF in 71 healthy subjects. The pharmacokinetics for ATV and RTV were assessed after 6 and 10 days of dosing with ATV 400 mg (n = 53) and with ATV-RTV at 300 and 100 mg (ATV/RTV 300/100; n = 52), respectively. Steady-state pharmacokinetics for ATV, RTV, RIF, and desacetyl-rifampin (des-RIF) were measured after 10 days of dosing of ATV/RTV/RIF 300/100/600 (n = 17), ATV/RTV/RIF 300/200/600 (n = 17), or ATV/RTV/RIF 400/200/600 (n = 14). An RIF 600-alone arm was enrolled as a control group (n = 18). With ATV/RTV/RIF 400/200/600, ATV area under the concentration-time curve values were comparable, but the C min values were lower relative to ATV 400 alone. ATV exposures were substantially reduced for the other RIF-containing regimens relative to ATV 400 alone and for all regimens relative to ATV/RTV 300/100 alone. RIF and des-RIF exposures were 1.6- to 2.5-fold higher than with RIF 600 alone. The incidence of grade 3/4 alanine aminotransferase/aspartate aminotransferase values was limited to 1 subject each in both the ATV/RTV/RIF 300/200/600 and the ATV/RTV/RIF 400/200/600 treatments. Coadministration of ATV with RIF was safe and generally well tolerated. Since ATV exposures were reduced in all regimens, ATV and RIF should not be coadministered at the dosing regimens studied.

Proteases as drug targets

2003 ◽  
Vol 70 ◽  
pp. 147-161 ◽  
Author(s):  
Andy J. Docherty ◽  
Tom Crabbe ◽  
James P. O'Connell ◽  
Colin R. Groom
Keyword(s):  
Rheumatoid Arthritis ◽  
Drug Targets ◽  
Enzyme Inhibitors ◽  
Proteolytic Enzymes ◽  
Angiotensin Converting Enzyme Inhibitors ◽  
Hiv Protease ◽  
Hiv Protease Inhibitors ◽  
Converting Enzyme Inhibitors ◽  
Recent Success ◽  
The Impact

The effective management of AIDS with HIV protease inhibitors, or the use of angiotensin-converting enzyme inhibitors to treat hypertension, indicates that proteases do make good drug targets. On the other hand, matrix metalloproteinase (MMP) inhibitors from several companies have failed in both cancer and rheumatoid arthritis clinical trials. Mindful of the MMP inhibitor experience, this chapter explores how tractable proteases are as drug targets from a chemistry perspective. It examines the recent success of other classes of drug for the treatment of rheumatoid arthritis, and highlights the need to consider where putative targets lie on pathophysiological pathways--regardless of what kind of therapeutic entity would be required to target them. With genome research yielding many possible new drug targets, it explores the likelihood of discovering proteolytic enzymes that are causally responsible for disease processes and that might therefore make better targets, especially if they lead to the development of drugs that can be administered orally. It also considers the impact that biologics are having on drug discovery, and in particular whether biologically derived therapeutics such as antibodies are likely to significantly alter the way we view proteases as targets and the methods used to discover therapeutic inhibitors.

Determining and Overcoming Resistance to HIV Protease Inhibitors

2004 ◽  
Vol 4 (2) ◽  
pp. 137-152 ◽  
Author(s):  
Jana Prejdova ◽  
Milan Soucek ◽  
Jan Konvalinka
Keyword(s):  
Protease Inhibitors ◽  
Hiv Protease ◽  
Hiv Protease Inhibitors

A QSAR Study of HIV Protease Inhibitors Using Theoretical Descriptors

2010 ◽  
Vol 6 (4) ◽  
pp. 269-282 ◽  
Author(s):  
Subhash C. Basak ◽  
Denise Mills ◽  
Rajni Garg ◽  
Barun Bhhatarai
Keyword(s):  
Protease Inhibitors ◽  
Hiv Protease ◽  
Hiv Protease Inhibitors ◽  
Qsar Study

scholarly journals Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 576
Author(s):  
Sofia Giacosa ◽  
Catherine Pillet ◽  
Irinka Séraudie ◽  
Laurent Guyon ◽  
Yann Wallez ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Renal Carcinoma ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Cancer Therapeutics ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Cell Renal Cell Carcinoma ◽  
Von Hippel Lindau ◽  
Renal Carcinoma Cells

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL− cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug–gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

scholarly journals High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chialin Cheng ◽  
Surya A. Reis ◽  
Emily T. Adams ◽  
Daniel M. Fass ◽  
Steven P. Angus ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Neural Progenitor Cells ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Clinical Investigation ◽  
Neuronal Model ◽  
Tau Phosphorylation ◽  
Neuronal Cultures ◽  
Proteomic Profiling ◽  
Induced Pluripotent

AbstractMutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.

scholarly journals Development of a Robust High-Throughput Screening Platform for Inhibitors of the Striatal-Enriched Tyrosine Phosphatase (STEP)

2021 ◽  
Vol 22 (9) ◽  
pp. 4417
Author(s):  
Lester J Lambert ◽  
Stefan Grotegut ◽  
Maria Celeridad ◽  
Palak Gosalia ◽  
Laurent JS De Backer ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Synaptic Function ◽  
Label Free ◽  
Protein Tyrosine

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.

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