Published: April 15, 2011 r2011 American Chemical Society 3875 dx.doi.org/10.1021/jm200167g | J. Med. Chem. 2011, 54, 3875–3884 ARTICLE pubs.acs.org/jmc Development of the Fluorescent Biosensor hCalmodulin (hCaM)L39C-monobromobimane(mBBr)/V91C-mBBr, a Novel Tool for Discovering New Calmodulin Inhibitors and Detecting Calcium Martín Gonzalez-Andrade, † Jose Rivera-Chavez, † Alejandro Sosa-Peinado, ‡ Mario Figueroa, † Rogelio Rodríguez-Sotres, † and Rachel Mata* ,† † Facultad de Química, Universidad Nacional Autonoma de Mexico, Mexico DF 04510, Mexico ‡ Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico DF 04510, Mexico b S Supporting Information ’ INTRODUCTION The discovery of new chemical entities (synthetic, natural pro- duct, or biotechnological) with pharmacological properties has been a trend in pharmaceutical research in recent years. For this reason, the development of modern methods, including biosen- sors, for pharmacological screening is very important. A biosen- sor is a device that consists of a biological recognition system, often called a bioreceptor, and a physical transducer which allows the conversion of a biological response into a measurable effect, for example, electric or fluorescence signals. 1 This molecular target usually utilizes a biochemical mechanism for recognition and is the key to the specificity of a biosensor. Many bioreceptors have been used to build biosensors valuable for monitoring different types of analytes. Proteins, including enzymes, antibody, or ion channels, are often chosen as bioreceptors due to their specific binding capabilities. One promising biological molecular species useful for building biosensors is the protein calmodulin (CaM), the primary trans- ducer of calcium (Ca 2þ )-mediated signals in eukaryotes. 2 Ac- cording to a resolved crystal structure, CaM is dumbbell-shaped protein with two domains separated by a long central helix; each domain contains two EF hand Ca 2þ -binding loops, which tightly bind a Ca 2þ ion each. 2 CaM is involved in several cellular physiological processes, such as gene regulation, protein synthesis, fast axonal transport, smooth muscle contraction, secretion, growth, organelle tubula- tion, ion channel function, cell motility, and chemotaxis, to mention a few. Consequently, CaM has been also implicated in a variety of degenerative diseases processes such as inflammation, short-term memory, cancer, viral penetration, and immune response. 36 CaM controls all these processes, physiological or pathophysiological, through the modulation of at least 100 different proteins and CaM-dependent enzymes like calmodulin-sensitive cAMP phos- phodiesterase (PDE1), nitric oxide synthases, adenylate cyclase, several kinases, calcium-ATPase pumps, ion channels, phospha- tases as well as cytoskeletal structural proteins, among the most important. 7,8 Altogether, this information makes CaM an attrac- tive bioreceptor for building biosensors suitable for the discovery of CaM ligands useful as new drugs or research tools to fully understand physiological CaM mediated processes. To build a CaM biosensor, the transduction can be accom- plished via different methods. However, fluorescence-based meth- ods, including fluorescence resonance energy and extrinsic fluo- rescence, are the most popular. 9 The fluorescence-based methods are highly specific, low-cost, selective, and have short reaction times, although sometimes the preparation of the sensing element can be laborious. One of the most used procedures involves the Received: February 15, 2011 ABSTRACT: A novel, sensible, and specific fluorescent biosensor of human calmodulin (hCaM), namely hCaM L39C-mBBr/V91C-mBBr, was constructed. The biosensor was useful for detecting ligands with opposing fluorescent signals, calcium ions (Ca 2þ ) and CaM inhibitors in solution. Thus, the device was successfully applied to analyze the allosteric effect of Ca 2þ on trifluoroperazine (TFP) binding to CaM (Ca 2þ K d = 0.24 μM ( 0.03 with a stoichiometry 4.10 ( 0.15; TFP K d ∼ 5.740.53 μM depending on the degree of saturation of Ca 2þ , with a stoichiometry of 2:1). In addition, it was suitable for discovering additional xanthones (5, 6, and 8) with anti-CaM properties from the fungus Emericella 25379. The affinity of 15, 7, and 8 for the complex (Ca 2þ ) 4 -CaM was excellent because their experimental K d s were in the nM range (4498 nM). Docking analysis predicted that 18 bind to CaM at sites I, III, and IV as does TFP.