Quantifying Target Occupancy of Small Molecules Within Living Cells

2020 ◽  
Vol 89 (1) ◽  
pp. 557-581 ◽  
Author(s):  
M.B. Robers ◽  
R. Friedman-Ohana ◽  
K.V.M. Huber ◽  
L. Kilpatrick ◽  
J.D. Vasta ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Drug Efficacy ◽  
Living Cells ◽  
Drug Binding ◽  
Therapeutic Window ◽  
Target Engagement ◽  
Binding Properties ◽  
Structure Activity ◽  
Sar Analysis ◽  
Kinetics Of

The binding affinity and kinetics of target engagement are fundamental to establishing structure–activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.

scholarly journals Mechanism-based inhibitors of SIRT2: structure–activity relationship, X-ray structures, target engagement, regulation of α-tubulin acetylation and inhibition of breast cancer cell migration

2021 ◽  
Author(s):  
Alexander L. Nielsen ◽  
Nima Rajabi ◽  
Norio Kudo ◽  
Kathrine Lundø ◽  
Carlos Moreno-Yruela ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Living Cells ◽  
Cancer Cell Migration ◽  
Target Engagement ◽  
Small Peptide ◽  
X Ray ◽  
Tubulin Acetylation ◽  
Structure Activity ◽  
Lysine Residues ◽  
Chain Acyl

Sirtuin 2 (SIRT2) is a protein deacylase enzyme that removes acetyl groups and longer chain acyl groups from post-translationally modified lysine residues. Here, we developed small peptide-based inhibitors of its activity in living cells in culture.

An infrared assay of the kinetics of phosphate-release from physiological substrates in living cells

2014 ◽  
Author(s):  
Ren Zhongyuan ◽  
Do Leduy ◽  
Saida Mebarek ◽  
Nermin Keloglu ◽  
Saandia Ahamada ◽  
...  
Keyword(s):  
Living Cells ◽  
Phosphate Release ◽  
Kinetics Of ◽  
Physiological Substrates

scholarly journals The Influence of Varying Fluorination Patterns on the Thermodynamics and Kinetics of Benzenesulfonamide Binding to Human Carbonic Anhydrase II

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 509 ◽  
Author(s):  
Steffen Glöckner ◽  
Khang Ngo ◽  
Björn Wagner ◽  
Andreas Heine ◽  
Gerhard Klebe
Keyword(s):  
Carbonic Anhydrase ◽  
Carbonic Anhydrase Ii ◽  
The Novel ◽  
Binding Modes ◽  
X Ray ◽  
Structure Activity ◽  
Small Set ◽  
Novel Method ◽  
Human Carbonic Anhydrase ◽  
Kinetics Of

The fluorination of lead-like compounds is a common tool in medicinal chemistry to alter molecular properties in various ways and with different goals. We herein present a detailed study of the binding of fluorinated benzenesulfonamides to human Carbonic Anhydrase II by complementing macromolecular X-ray crystallographic observations with thermodynamic and kinetic data collected with the novel method of kinITC. Our findings comprise so far unknown alternative binding modes in the crystalline state for some of the investigated compounds as well as complex thermodynamic and kinetic structure-activity relationships. They suggest that fluorination of the benzenesulfonamide core is especially advantageous in one position with respect to the kinetic signatures of binding and that a higher degree of fluorination does not necessarily provide for a higher affinity or more favorable kinetic binding profiles. Lastly, we propose a relationship between the kinetics of binding and ligand acidity based on a small set of compounds with similar substitution patterns.

scholarly journals Thermodynamic stability and drug-binding properties of oligodeoxyribonucleotide duplexes containing 3-deazaadenine:thymine base pairs

1993 ◽  
Vol 21 (8) ◽  
pp. 1743-1746 ◽  
Author(s):  
Catherine Lever ◽  
Xiang Li ◽  
Richard Cosstick ◽  
Susanne Edel ◽  
Tom Brown
Keyword(s):  
Thermodynamic Stability ◽  
Drug Binding ◽  
Base Pairs ◽  
Binding Properties

Spectrochemical Behavior of Carcinogenic Polycyclic Aromatic Hydrocarbons in Biological Systems. Part II: A Theoretical Rate Model for BaP Metabolism in Living Cells

1996 ◽  
Vol 50 (11) ◽  
pp. 1352-1359 ◽  
Author(s):  
Ping Chiang ◽  
Kuang-Pang Li ◽  
Tong-Ming Hseu
Keyword(s):  
Rate Constant ◽  
Living Cells ◽  
Rate Model ◽  
Number Density ◽  
Order Process ◽  
Irreversible Reaction ◽  
First Order ◽  
Metabolism Rate ◽  
Polycyclic Aromatic ◽  
Kinetics Of

An idealized model for the kinetics of benzo[ a]pyrene (BaP) metabolism is established. As observed from experimental results, the BaP transfer from microcrystals to the cell membrane is definitely a first-order process. The rate constant of this process is signified as k1. We describe the surface–midplane exchange as reversible and use rate constants k2 and k3 to describe the inward and outward diffusions, respectively. The metabolism is identified as an irreversible reaction with a rate constant k4. If k2 and k3 are assumed to be fast and not rate determining, the effect of the metabolism rate, k4, on the number density of BaP in the midplane of the microsomal membrane, m3, can be estimated. If the metabolism rate is faster than or comparable to the distribution rates, k2 and k3, the BaP concentration in the membrane midplane, m3, will quickly be dissipated. But if k4 is extremely small, m3 will reach a plateau. Under conditions when k2 and k3 also play significant roles in determining the overall rate, more complicated patterns of m3 are expected.

Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay

Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 84-87 ◽  
Author(s):  
Daniel Martinez Molina ◽  
Rozbeh Jafari ◽  
Marina Ignatushchenko ◽  
Takahiro Seki ◽  
E. Andreas Larsson ◽  
...  
Keyword(s):  
Drug Target ◽  
Drug Transport ◽  
Drug Binding ◽  
Target Engagement ◽  
Tissue Samples ◽  
Target Proteins ◽  
Thermal Shift Assay ◽  
Cellular Thermal Shift Assay ◽  
Thermal Shift ◽  
In Cells

The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.

scholarly journals THE KINETICS OF EXOSMOSIS OF WATER FROM LIVING CELLS

1927 ◽  
Vol 10 (5) ◽  
pp. 659-664 ◽  
Author(s):  
Morton McCutcheon ◽  
Baldwin Lucke
Keyword(s):  
Osmotic Pressure ◽  
Salt Concentration ◽  
Driving Force ◽  
Temperature Characteristic ◽  
Living Cells ◽  
Velocity Constant ◽  
Osmotic Equilibrium ◽  
Kinetics Of

1. The rate of exosmosis of water was studied in unfertilized Arbacia eggs, in order to bring out possible differences between the kinetics of exosmosis and endosmosis. 2. Exosmosis, like endosmosis, is found to follow the equation See PDF for Equation, in which a is the total volume of water that will leave the cell before osmotic equilibrium is attained, x is the volume that has already left the cell at time t, and k is the velocity constant. 3. The velocity constants of the two processes are equal, provided the salt concentration of the medium is the same. 4. The temperature characteristic of exosmosis, as of endomosis, is high. 5. It is concluded that the kinetics of exosmosis and endosmosis of water in these cells are identical, the only difference in the processes being in the direction of the driving force of osmotic pressure.

Sign in / Sign up

Export Citation Format

Share Document