Philip Lin Huang

MAP 30: a new inhibitor of HIV-1 infection and replication.

A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordica charantia. This compound, MAP 30 (Momordica Anti‐HIV Protein), is a basic protein of about 30 kDa. It exhibits dose‐dependent inhibition of cell‐free HIV‐1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM‐ss monolayers; (II) viral core protein p24 expression; and (iii) viral‐associated reverse transcriptase (RT) activity in HIV‐1 infected H9 cells. The doses required for 50% inhibition (ID50) in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV‐1 infections. The sequence of the N‐terminal 44 amino acids of MAP 30 has been determined.

Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment

We investigated the antiviral activity of olive leaf extract (OLE) preparations standardized by liquid chromatography-coupled mass spectrometry (LC-MS) against HIV-1 infection and replication. We find that OLE inhibits acute infection and cell-to-cell transmission of HIV-1 as assayed by syncytia formation using uninfected MT2 cells co-cultured with HIV-1-infected H9 T lymphocytes. OLE also inhibits HIV-1 replication as assayed by p24 expression in infected H9 cells. These anti-HIV effects of OLE are dose dependent, with EC(50)s of around 0.2 microg/ml. In the effective dose range, no cytotoxicity on uninfected target cells was detected. The therapeutic index of OLE is above 5000. To identify viral and host targets for OLE, we characterized gene expression profiles associated with HIV-1 infection and OLE treatment using cDNA microarrays. HIV-1 infection modulates the expression patterns of cellular genes involved in apoptosis, stress, cytokine, protein kinase C, and hedgehog signaling. HIV-1 infection up-regulates the expression of the heat-shock proteins hsp27 and hsp90, the DNA damage inducible transcript 1 gadd45, the p53-binding protein mdm2, and the hedgehog signal protein patched 1, while it down-regulates the expression of the anti-apoptotic BCL2-associated X protein Bax. Treatment with OLE reverses many of these HIV-1 infection-associated changes. Treatment of HIV-1-infected cells with OLE also up-regulates the expression of the apoptosis inhibitor proteins IAP1 and 2, as well as the calcium and protein kinase C pathway signaling molecules IL-2, IL-2Ralpha, and ornithine decarboxylase ODC1.

Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon.

MAP30 is an anti-HIV plant protein that we have identified and purified to homogeneity from bitter melon (Momordica charantia). It is capable of acting against multiple stages of the viral life cycle, on acute infection as well as replication in chronically infected cells. In addition to antiviral action, MAP30 also possesses anti-tumor activity, topological inactivation of viral DNA, inhibition of viral integrase and cell-free ribosome-inactivation activities. We have cloned and expressed the MAP30 gene. The objective of this study is to characterize recombinant MAP30 (re-MAP30) and to determine its anti-HIV, anti-tumor and other activities. We report here that re-MAP30 inhibits HIV-1 and certain human tumors to the same extent as its native counterpart, natural MAP30 (nMAP30). The anti-HIV activity was measured by quantitative focal syncytium formation on CEM-ss cell monolayers, viral core protein p24 expression and viral-associated reverse transcriptase activity in HIV-1-infected H9 cells. The anti-tumor activity was measured by metabolic labeling of protein synthesis in tumor cells. In the dose range of the assay, re-MAP30 exhibits little toxicity to the uninfected viral target cells and other normal human cells. Identical to nMAP30, re-MAP30 is also active in topological inactivation of viral DNA, inhibition of viral DNA integration and cell-free ribosome inactivation. The cloning and expression of the gene encoding biologically active re-MAP30 provides an abundant source of homogeneous material for clinical investigations, as well as structure-function studies of this novel antiviral and anti-tumor agent.

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: Part I. Integrase inhibition

We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose-dependent inhibition on HIV-1 fusion core formation with EC(50)s of 66-58nM, with no detectable toxicity. Our findings on effects of HIV-1 integrase are reported in the subsequent article.

A new class of anti‐HIV agents: GAP31, DAPs 30 and 32

Three inhibitors of human immunodeficiency virus (HIV) have been isolated and purified to homogeneity from Euphorbiaceae himalaya seeds (Gelonium multiflorum) and carnation leaves (Dianthus caryophytlus). These proteins. GAP 31 (Gelonium Anti‐HIV Protein 31 kDa) and DAPs 30 and 32 (dianthus anti‐HIV proteins, 30 and 32 kDa), inhibit HIV‐1 infection and replication in a dosc‐dependent manner with little toxicity to target cells. The therapeutic indices of these compounds are in the order 104, suggesting that they may be clinically important agents in the treatment of AIDS. The N‐terminal amino acid sequences of these proteins show little homology to those of previously described anti‐HIV proteins. The structure‐function features of these HIV inhibitors, based on the 40–60 amino acid residues of N‐terminal sequences, are examined.

Solution Structure of Anti-HIV-1 and Anti-Tumor Protein MAP30: Structural Insights into Its Multiple Functions

We present the solution structure of MAP30, a plant protein with anti-HIV and anti-tumor activities. Structural analysis and subsequent biochemical assays lead to several novel discoveries. First, MAP30 acts like a DNA glycosylase/apurinic (ap) lyase, an additional activity distinct from its known RNA N-glycosidase activity toward the 28S rRNA. Glycosylase/ap lyase activity explains MAP30s apparent inhibition of the HIV-1 integrase, MAP30s ability to irreversibly relax supercoiled DNA, and may be an alternative cytotoxic pathway that contributes to MAP30s anti-HIV/anti-tumor activities. Second, two distinct, but contiguous, subsites are responsible for MAP30s glycosylase/ap lyase activity. Third, Mn2+ and Zn2+ interact with negatively charged surfaces next to the catalytic sites, facilitating DNA substrate binding instead of directly participating in catalysis.

ANTI‐ERYTHROPOIETIN ANTIBODIES IN HYPERVISCOSITY SYNDROME ASSOCIATED WITH GIANT LYMPH NODE HYPERPLASIA (GLNH; CASTLEMAN'S DISEASE)

The authors report a case of GLNH, plasma cell type, with demonstration of anti-Ep antibodies, hyperviscosity, anaemia, and subsequent response to steroid therapy

COMPUTATIONAL DESIGN OF NORBORNANE-BASED HIV-1 PROTEASE INHIBITORS

A series of norbornane-based HIV-1 protease (PR) inhibitors are designed theoretically to displace the tetrahedrally coordinated internal water molecule that bridges inhibitor to flaps via hydrogen bonds. These designed inhibitors use the norbornenone oxygen atom to mimic this structural water molecule and contain diols to interact with the carboxylate oxygens of catalytic aspartates. The binding free energies were estimated by modified linear interaction energy approach [Zoete H, Michielin O, Karplus M, J Comput Aided Mol Des17:861, 2003], in which the binding free energy is written as a linear combination of the electrostatic interaction energy between PR and the ligand, Eelec, the van der Waals interaction energy between PR and the ligand, EvdW, and the difference of the solvation free energies of the complex, the receptor, and the isolated ligand, ΔGsolv. The equation obtained in previous work [Da W. Zhang, Philip Lin Huang, Sylvia Lee-Huang, John Z. H. Zhang, J Theor Comput Chem7:485, 2008] is applied directly to calculate the binding free energy of designed norbornane-based HIV-1 PR inhibitors.

Live-cell real-time imaging reveals role of mitochondria in cell-to-cell transmission of HIV-1

We used live-cell, real-time fluorescence imaging of co-cultures of HIV-1 infected T cells and uninfected target cells to examine the action of mitochondria during cell-to-cell transmission of the virus. We find that mitochondria of HIV infected cells enter uninfected target cells and advance viral spread. We show that human mitochondria serve as viral reservoirs and carriers and that they can move between cells. This was confirmed by our results that purified mitochondria from HIV infected cells are infectious, and that mitochondrial inhibitors block HIV transmission. Viral infection and replication in the target cells were verified by syncytial formation and HIV-1 core protein p24 production. Our results offer new insights into the cellular mechanisms of viral transmission and identify mitochondria as new host targets for viral infection.

Oleuropein and Related Compounds Reduce Atherosclerosis

Diabetes, obesity, and the metabolic syndrome all increase the risk for cardiovascular disease. Epidemiologic studies have demonstrated that a Mediterranean diet rich in olive oil is associated with decrease in risk for cardiovascular disease, obesity, and diabetes. Although some of the protection may be from the unsaturated fatty acid components of such a diet, additional small molecules found in olive oil and olive plants may confer protection, including the polyphenol oleuropein and hydroxytyrosol. We report here studies in mice that document the bioavailability of these molecules when administered orally. Using the apoE knockout mouse model fed a high fat diet, we assessed the effects of orally administered oleuropein on diet-induced atherosclerosis. Oleuropein significantly reduced the extent of atherosclerosis found in the aorta of apoE knockout mice. Molecular modeling studies indicate that oleuropein binds to the peroxisome proliferator activating receptors (PPAR)  ,  , and  , but it does so with distinct conformations that differ from the way that PPAR  -,  -, and  -specific agonists bind to their active sites. The manner with which oleuropein binds uniquely to the various PPARs offers a possible explanation for its effects on metabolism and cardiovascular disease.