Among the most promising chemopreventive agents, certain natural polyphenols have recently received a great deal of attention because of their demonstrated inhibitory activity against tumorigenesis. In view of their anticancer properties, these compounds also hold great promise as potential chemotherapeutic agents. However, to translate these chemopreventive agents into chemotherapeutic compounds, their exact mechanisms of action must be delineated. By using a multidisciplinary approach guided by modern nuclear magnetic resonance spectroscopy techniques, fluorescence polarization displacement assays, and cell-based assays, we have begun to unravel the mechanisms of actions of certain polyphenols such as Gossypol (a compound from cotton seed extracts) and Purpurogallin (a natural compound extracted from Quercus sp. nutgall) and their derivatives. Our findings suggest that these natural products bind and antagonize the antiapoptotic effects of B-cell lymphocyte/leukemia-2 (Bcl-2) family proteins such as Bcl-x(L). Our in vitro and in vivo data not only open a window of opportunities for the development of novel cancer treatments with these compounds but also provide structural information that can be used for the design and development of novel and more effective analogues.

Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small molecule furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addition of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochemical cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Molecular modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-LysArg-CH 2Cl. The compounds presented here are bona fide synthetic small molecule furin inhibitors that exhibit potency in the nanomolar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furindependent diseases.serine endoprotease ͉ prohormone ͉ proprotein convertase F urin is a membrane-anchored, calcium-dependent serine endoprotease and is expressed in all tissues and cell lines examined (1-3). It is the first and, so far, the best-characterized member of the mammalian subtilisin-like family of prohormone/proprotein convertases (PCs), which convert precursors of many secreted proteins and peptide hormones into their biologically active forms (1-3). Furin is predominantly localized in the trans-Golgi network and cycles between this compartment, the cell surface, and the endosomes (2, 4). Hence, furin is able to access and efficiently process a diverse spectrum of substrates including growth factors, receptors, serum proteins, matrix metalloproteinases, viral envelope glycoproteins, and bacterial toxins, typically at sites with the consensus sequence Arg-Xaa-Lys/Arg-Arg 2 (2).Although furin plays an essential role in embryogenesis and homeostasis, this endoprotease has also been implicated in the neurodegeneration associated with Alzheimer's disease, and tumor metastasis and the activation and virulence of many bacterial and viral pathogens (2). It has been demonstrated that furin inhibitors modulate tumor growth (5) and attenuate the toxicity of anthrax (6) and Pseudomonas aeruginosa (7) toxins and cytomegalovirus (8) in cell culture and animal models. Therefore, furin inhibitors hold great promise as potential therapeutic agents for treating furinmediated diseases and viral and bacterial infections, particularly for short-term therapy.To date, most furin inhibitors reported in the literature have been proteins (9-19) or peptides (20-24), which show excellent potency against furin, and largely mimic the substrate in bindi...

To avoid detection and targeting by the immune system, the plague-causing bacterium Yersinia pestis uses a type III secretion system to deliver a set of inhibitory proteins into the cytoplasm of immune cells. One of these proteins is an exceptionally active tyrosine phosphatase termed YopH, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. Because Y. pestis strains lacking YopH are avirulent, we set out to develop small molecule inhibitors for YopH. We used a novel and cost-effective approach, in which leads from a chemical library screening were analyzed and computationally docked into the crystal structure of YopH. This resulted in the identification of a series of novel YopH inhibitors with nanomolar K i values, as well as the structural basis for inhibition. Our inhibitors lack the polar phosphate-mimicking moiety of rationally designed tyrosine phosphatase inhibitors, and they readily entered live cells and rescued them from YopHinduced tyrosine dephosphorylation, signaling paralysis, and cell death. These inhibitors may become useful for treating the lethal infection by Y. pestis.

A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar. Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first. Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar. These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function.