A general framework to characterize inhibitors of calmodulin: Use of calmodulin inhibitors to study the interaction between calmodulin and its calmodulin binding domains

Abstract

The prominent role of Ca(2+) in cell physiology is mediated by a whole set of proteins involved in Ca(2+)-signal generation, deciphering and arrest. Among these intracellular proteins, calmodulin (CaM) known as a prototypical calcium sensor, serves as a ubiquitous carrier of the intracellular calcium signal in all eukaryotic cell types. CaM is assumed to be involved in many diseases including Parkinson, Alzheimer, and rheumatoid arthritis. Defects in some of many reaction partners of CaM might be responsible for disease symptoms. Several classes of drugs bind to CaM with unwanted side effects rather than specific therapeutic use. Thus, it may be more promising to concentrate at searching for pharmacological interferences with the CaM target proteins, in order to find tools for dissecting and investigating CaM-regulatory and modulatory functions in cells. In the present study, we have established a screening assay based on fluorescence polarization (FP) to identify a diverse set of small molecules that disrupt the regulatory function of CaM. The FP-based CaM assay consists in the competition of two fluorescent probes and a library of chemical compounds for binding to CaM. Screening of about 5300 compounds (Strasbourg Academic Library) by displacement of the probe yielded 39 compounds in a first step, from which 6 were selected. Those 6 compounds were characterized by means of calorimetry studies and by competitive displacement of two fluorescent probes interacting with CaM. Moreover, those small molecules were tested for their capability to displace 8 different CaM binding domains from CaM. Our results show that these CaM/small molecules interactions are not functionally equivalent. The strategy that has been set up for CaM is a general model for the development and validation of other CaM interactors, to decipher their mode of action, or rationally design more specific CaM antagonists. Moreover, this strategy may be used for other protein binding assays intended to screen for molecules with preferred binding activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Calcium-Binding Proteins as Determinants of Central Nervous System Neuronal Vulnerability to Disease

“…Thus, both calbindin [10] and calretinin [113] have been reported to display properties suitable for functioning as a sensor, namely substantial conformational rearrangement upon calcium binding and potential interaction with proteins involved in calcium signaling. So far, this has been mainly investigated in vitro or in non-neuronal cells, but it does open up the possibility for therapeutic targeting of undesirable signaling pathways, as is being explored for other CaBPs such as calmodulin [114].…”

Calmodulin Binding Proteins and Alzheimer’s Disease

“…Thus currently available pharmaceutically proven CaM antagonists likely would not be an option for medical treatment. However, circumstances could change with the development of new CaM antagonists (e.g., [ 135 ]). Since most CaMBPs bind to CaM in unique ways and since some have multiple types of binding sequences, based on the technologies that exist for developing CaM antagonists it should be possible to develop target-specificpharmaceuticals.…”

Oxidation of Са2+-Binding Domain of NADPH Oxidase 5 (NOX5): Toward Understanding the Mechanism of Inactivation of NOX5 by ROS

“…Identification of the NOX5 regulatory domain sensitive to oxidation in the present work opens the road for new specific NOX5 inhibitors identification. Screening of interactants of this domain according to the recently developed strategy [ 32 ] could lead to the discovery of important NOX5 inhibitors.…”