Characterization Of Small Molecule Inhibitors Research Articles

Identification and Characterization of Small Molecule Inhibitors of the Calcium-Dependent S100B−p53 Tumor Suppressor Interaction

The binding of S100B to p53 down-regulates wild-type p53 tumor suppressor activity in cancer cells such as malignant melanoma, so a search for small molecules that bind S100B and prevent S100B-p53 complex formation was undertaken. Chemical databases were computationally searched for potential inhibitors of S100B, and 60 compounds were selected for testing on the basis of energy scoring, commercial availability, and chemical similarity clustering. Seven of these compounds bound to S100B as determined by steady state fluorescence spectroscopy (1.0 microM < or = K(D) < or = 120 microM) and five inhibited the growth of primary malignant melanoma cells (C8146A) at comparable concentrations (1.0 microM < or = IC(50) < or = 50 microM). Additionally, saturation transfer difference (STD) NMR experiments confirmed binding and qualitatively identified protons from the small molecule at the small molecule-S100B interface. Heteronuclear single quantum coherence (HSQC) NMR titrations indicate that these compounds interact with the p53 binding site on S100B. An NMR-docked model of one such inhibitor, pentamidine, bound to Ca(2+)-loaded S100B was calculated using intermolecular NOE data between S100B and the drug, and indicates that pentamidine binds into the p53 binding site on S100B defined by helices 3 and 4 and loop 2 (termed the hinge region).

Development of a microplate bioassay for monocyte chemoattractant protein-1 based on activation of p44/42 mitogen-activated protein kinase

Monocyte chemoattractant protein-1 (MCP-1) is a potential therapeutic target for the treatment of several inflammatory conditions, including rheumatoid arthritis and chronic obstructive pulmonary disease. Current cell-based assays for MCP-1 use monocyte chemotaxis or calcium flux as a readout. Here, we describe an alternative bioassay based on MCP-1-induced phosphorylation of the mitogen-activated protein kinases (MAPK) p44 (ERK1) and p42 (ERK2). Adherent cells expressing the MCP-1 receptor CCR2B are treated with MCP-1 in 96-well plates in the presence or absence of inhibitors, fixed and permeabilized with methanol, and then probed with a monoclonal antibody that selectively recognizes the doubly phosphorylated form of p44/42 MAPK. Bound antibody is detected with a secondary antibody-peroxidase conjugate and a chromogenic substrate. The phosphorylation of p44/42 MAPK as detected in this assay peaks after 3–5 min of MCP-1 treatment, and the concentration of MCP-1 required for half-maximal p44/42 MAPK phosphorylation is 1–3 nM. MCP-1-induced phosphorylation of p44/42 MAPK is dependent upon the expression of CCR2B. The assay can be used for screening and characterization of small molecule inhibitors and antibodies blocking the binding of MCP-1 to its receptor. Since the assay is rapid and simple, it may represent a useful alternative to chemotaxis or calcium mobilization assays for the analysis of MCP-1 inhibitors.

Production and Use of HIV‐1 Luciferase Reporter Viruses

This unit describes the production and use of HIV-1 luciferase reporter viruses. These viruses are used to rapidly and accurately quantify HIV-1 in cell culture. Protocols are also presented for screening and characterization of small molecule inhibitors of HIV-1 entry into cells.

Beta-amyloid production, aggregation, and clearance as targets for therapy in Alzheimer's disease.

1. Despite major efforts aimed at elucidating the molecular basis and physiopathology of Alzheimer's disease (AD), there is still no effective treatment available for this devastating disorder. The biological mechanisms underlying the development of AD are complex, as multiple factors appear to modulate (either positively or negatively) the progression of neurodegeneration in the brains of AD patients. Not surprisingly, a number of different therapeutic approaches aimed at distinct aspects of the disease are currently being pursued. Given its central role in the neuropathology of AD, the beta-amyloid peptide (Abeta) is the focus of many such approaches. 2. In this review, we discuss recent developments along three major lines of investigation: (i) identification and characterization of inhibitors of the enzymes involved in proteolytic processing of the amyloid precursor protein and production of Abeta; (ii) identification of the pathways involved in cerebral degradation and clearance of Abeta; and (iii) characterization of small-molecule inhibitors of amyloid aggregation that prevent cerebral amyloid deposition and neurotoxicity. 3. Significant progress has been achieved in these directions, opening up new perspectives toward the development of effective approaches for the treatment or prevention of AD.