Bcl-2 and related proteins are key regulators of apoptosis or programmed cell death implicated in human disease including cancer. We recently showed that cell-permeable Bcl-2 binding peptides could induce apoptosis of human myeloid leukemia in vitro and suppress its growth in severe combined immunodeficient mice. Here we report the discovery of HA14-1, a small molecule (molecular weight ‫؍‬ 409) and nonpeptidic ligand of a Bcl-2 surface pocket, by using a computer screening strategy based on the predicted structure of Bcl-2 protein. In vitro binding studies demonstrated the interaction of HA14-1 with this Bcl-2 surface pocket that is essential for Bcl-2 biological function. HA14-1 effectively induced apoptosis of human acute myeloid leukemia (HL-60) cells overexpressing Bcl-2 protein that was associated with the decrease in mitochondrial membrane potential and activation of caspase-9 followed by caspase-3. Cytokine response modifier A, a potent inhibitor of Fas-mediated apoptosis, did not block apoptosis induced by HA14-1. Whereas HA14-1 strongly induced the death of NIH 3T3 (Apaf-1 ؉͞؉ ) cells, it had little apoptotic effect on Apaf-1-deficient (Apaf-1 ؊͞؊ ) mouse embryonic fibroblast cells. These data are consistent with a mechanism by which HA14-1 induces the activation of Apaf-1 and caspases, possibly by binding to Bcl-2 protein and inhibiting its function. The discovery of this cell-permeable molecule provides a chemical probe to study Bcl-2-regulated apoptotic pathways in vivo and could lead to the development of new therapeutic agents.

Sirtuins with an extended N-terminal domain (NTD), represented by yeast Sir2 and human SIRT1, harbor intrinsic mechanisms for regulation of their NAD-dependent deacetylase activities. Elucidation of the regulatory mechanisms is crucial for understanding the biological functions of sirtuins and development of potential therapeutics. In particular, SIRT1 has emerged as an attractive therapeutic target, and the search for SIRT1-activating compounds (STACs) has been actively pursued. However, the effectiveness of a class of reported STACs (represented by resveratrol) as direct SIRT1 activators is under debate due to the complication involving the use of fluorogenic substrates in in vitro assays. Future efforts of SIRT1-based therapeutics necessitate the dissection of the molecular mechanism of SIRT1 stimulation. We solved the structure of SIRT1 in complex with resveratrol and a 7-amino-4-methylcoumarin (AMC)-containing peptide. The structure reveals the presence of three resveratrol molecules, two of which mediate the interaction between the AMC peptide and the NTD of SIRT1. The two NTD-bound resveratrol molecules are principally responsible for promoting tighter binding between SIRT1 and the peptide and the stimulation of SIRT1 activity. The structural information provides valuable insights into regulation of SIRT1 activity and should benefit the development of authentic SIRT1 activators.

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1␣) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1␣ or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1␣ and vMIP-II and HIV-1 envelope protein gp120. Hypothetical threedimensional structures were proposed by molecular modeling studies of the CXCR4⅐SDF-1␣ complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1␣. As an HIV-1 co-receptor, we found that the Nterminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.Chemokines are a family of 8 -10-kDa small proteins that act as chemoattractants of various types of leukocytes to sites of inflammation and to secondary lymphoid organs. Based on the positions of two conserved cysteine residues in their N termini, chemokines can be divided into four subfamilies: CC, CXC, CX3C, and C (1, 2). The stromal cell-derived factor-1 (SDF-1␣) 1 is one of the CXC chemokines, which plays critical roles in the migration, proliferation, and differentiation of leukocytes. SDF-1␣ is the only known natural ligand of CXCR4 receptor (3, 4). CXCR4 can also be recognized by an antagonistic ligand, the viral macrophage inflammatory protein-II (vMIP-II) encoded by the Kaposi's sarcoma-associated herpesvirus (5). vMIP-II acts as a selective chemoattractant for T helper 2 cells and monocytes and is an agonist for CCR8 (6). vMIP-II displays a broader spectrum of receptor activities than any mammalian chemokine, as it binds with high affinity to a number of both CC and CXC chemokine receptors including CXCR4 and CCR5 and inhibits cell entry of human immunodeficiency virus type I (HIV-1) mediated by these receptors (7,8).CXCR4 belongs to the family of seven transmembrane Gprotein-coupled receptors that transduce signals via heterotrimeric G-proteins (9). Recent studies with knockout mice of CXCR4 have demonstrated that this molecule plays an important role in immunomodulation, organogenesis, hematopoiesis, and derailed cerebellar neuron migration (10 -12). CXCR4 has...

The chemokine receptor CXCR4 is critical for many biological functions, such as B-cell lymphopoiesis, regulation of neuronal cell migration, and vascular development (1-3). In addition, CXCR4 together with another chemokine receptor CCR5 are two principal co-receptors for the cellular entry of the human immunodeficiency virus type 1 (HIV-1) 1 (4 -7). The stromal cell-derived factor-1 (SDF-1␣) is the only known natural ligand of CXCR4 and plays important roles in migration, proliferation, and differentiation of leukocytes (8, 9). The viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus 8 (10) is an antagonistic chemokine ligand of CXCR4 (11, 12). vMIP-II also interacts with other chemokine receptors such as CCR5 and CCR3 and inhibits HIV-1 entry mediated by these co-receptors.CXCR4 and other chemokine receptors belong to the superfamily of seven transmembrane G-protein-coupled receptors (GPCRs) (13). These membrane proteins transmit signals from extracellular ligands to intracellular biological pathways via heterotrimeric G-proteins and have been a major class of therapeutic targets for a wide variety of human diseases (14). As such, characterizing the mechanism of biological recognition between these receptors and their ligands is essential for understanding the physiological or pathological processes that they mediate and devising novel strategies for clinical intervention. For CXCR4, studies have been carried out by a number of laboratories using chimeric chemokine receptors and site-specific mutants to study multiple domains of CXCR4 that are important for interacting with chemokine ligands and HIV-1 (15-23). However, because there is no high resolution crystal structure available for CXCR4 (or any other chemokine receptor) alone or complexed with ligands, the structural and biochemical basis of ligand binding and signaling through these important membrane receptors remains poorly understood.To further define the structure-function relationship of the chemokine receptor-ligand interaction, theoretical computer modeling and site-directed mutagenesis were combined to predict plausible structural models for chemokine receptors and their complexes with ligands, such as interleukin-8 receptor ␤ (24) and CCR5 (25,26). Structural models of CXCR4 and its complex with ligands were also proposed (27, 28). Complementary to modeling and mutational analyses of the receptors,

The entry of human immunodeficiency virus type 1 (HIV-1) into the cell is initiated by the interaction of the viral surface envelope protein with two cell surface components of the target cell, CD4 and a chemokine coreceptor, usually CXCR4 or CCR5. The natural ligand of CXCR4 is stromal cell-derived factor 1␣ (SDF-1␣). Whereas the overlap between HIV-1 and SDF-1␣ functional sites on the extracellular domains of CXCR4 has been well documented, it has yet to be determined whether there are sites in the transmembrane (TM) helices of CXCR4 important for HIV-1 and/or SDF-1␣ functions, and if such sites do exist, whether they are overlapping or distinctive for the separate functions of CXCR4.

The direct fusion of viral and target cell membranes required for human immunodeficiency virus type 1 (HIV-1) entry is initiated by the primary receptor, CD4, and a chemokine receptor, usually CXCR4 or CCR5. Chemokine receptors are members of the G-protein-coupled receptor (GPCR) superfamily that possess seven transmembrane (TM) domains. Because of its importance in the development of AIDS, CXCR4 has been explored as a new target for drug discovery to combat the AIDS epidemic (3,8,10). As the natural ligands of chemokine receptors, chemokines are small soluble proteins of about 70 amino acid residues that play prominent roles in leukocyte activation and inflammation (5, 11). Most of the known human chemokines are broadly categorized into the CXC and CC chemokines based on the position of two conserved cysteine residues in their amino (N)-terminal domains (3, 11). The natural chemokines of CXCR4 or CCR5 can inhibit HIV-1 infection (4, 13) by blocking HIV-1 gp120 binding sites (2, 14) and/or inducing receptor internalization (1, 9).Despite their important roles in the pathogenesis of AIDS and other human diseases, the lack of receptor selectivity of natural chemokines has made their direct clinical applications problematic. It is common knowledge that a chemokine receptor can often be recognized by multiple ligands, while a chemokine ligand binds to several different receptors (15), illustrating the apparent redundancy and the lack of selectivity in the chemokine ligand-receptor interaction network. As such, we have been working toward the development of a systematic chemical biology approach based on chemokine protein structures and chemistry to generate synthetically and modularly modified (SMM) chemokines that have higher receptor binding selectivity and improved pharmacological profiles compared with natural chemokines. This SMM chemokine approach was recently applied to generate novel ligands selective for CXCR4 or CCR5 by modifying the N-terminal (1-10) sequence module of viral macrophage inflammatory protein II (vMIP-II) or stromal cell-derived factor 1␣ (SDF-1␣) (unpublished data). Importantly, some of these SMM chemokines,

Abeta peptides cleaved from the amyloid precursor protein are the main components of senile plaques in Alzheimer's disease. Abeta peptides adopt a conformation mixture of random coil, beta-sheet, and alpha-helix in solution, which makes it difficult to design inhibitors based on the 3D structures of Abeta peptides. By targeting the C-terminal beta-sheet region of an Abeta intermediate structure extracted from molecular dynamics simulations of Abeta conformational transition, a new inhibitor that abolishes Abeta fibrillation was discovered using virtual screening in conjunction with thioflavin T fluorescence assay and atomic force microscopy determination. Circular dichroism spectroscopy demonstrated that the binding of the inhibitor increased the beta-sheet content of Abeta peptides either by stabilizing the C-terminal beta-sheet conformation or by inducing the intermolecular beta-sheet formation. It was proposed that the inhibitor prevented fibrillation by blocking interstrand hydrogen bond formation of the pleated beta-sheet structure commonly found in amyloid fibrils. The study not only provided a strategy for inhibitor design based on the flexible structures of amyloid peptides but also revealed some clues to understanding the molecular events involved in Abeta aggregation.

Chemokines and their receptors play important roles in numerous physiological and pathological processes. To develop natural chemokines into receptor probes and inhibitors of pathological processes, the lack of chemokine-receptor selectivity must be overcome. Here, we apply chemical synthesis and the concept of modular modifications to generate unnatural synthetically and modularly modified (SMM)-chemokines that have high receptor selectivity and affinity, and reduced toxicity. A proof of the concept was shown by transforming the nonselective viral macrophage inflammatory protein-II into new analogs with enhanced selectivity and potency for CXCR4 or CCR5, two principal coreceptors for human immunodeficiency virus (HIV)-1 entry. These new analogs provided insights into receptor binding and signaling mechanisms and acted as potent HIV-1 inhibitors. These results support the concept of SMM-chemokines for studying and controlling the function of other chemokine receptors.