Molecular Immunopharmacology of Kunming Primate Research Center

1 Principal Investigator: Yong-Tang Zheng PhD

2 Research Fields
In recent years, the HIV/AIDS epidemic has become more serious in China. By June 2003, the accumulated number of reported HIV cases in the whole country was 45, 092, of which 3, 532 were AIDS patients, with 1, 800 deaths. According to estimates by experts from the China CDC, the accumulated number of HIV infections and of AIDS patients has reached 840, 000 and 80, 000 respectively. Although the adult prevalence rate is less than 0.1%, the epidemic has spread to 31 provinces (autonomous regions and municipalities) and the number of reported HIV/AIDS cases has increased significantly.

The status of the HIV/AIDS epidemic is quite different in different areas. The HIV epidemic is more severe in certain areas and among specific populations. For example, Yunnan, which borders heroin-producing Myanmar (formerly Burma), has more HIV-infected people than any other Chinese province. By June 2003, Yunnan has cumulatively reported 13, 948 HIV infections. According to estimates by experts, the accumulated number of HIV infections has reached 80, 000.

Anti-HIV drugs are still the most important for AIDS treatment because there is no breakthrough in HIV vaccine research. There are, at present, 24 compounds that have been formally approved by US FDA for the treatment of HIV infection. All these compounds were obtained from chemical synthesis, none originated from natural sources.

Although China has locally produced four anti-HIV drugs for two combination therapy regimes, it is far from sufficient both in terms of quantity produced, and in the number of different anti-HIV drugs available, to maximize the options for treatment.

Now, It is a hotspot in new drugs development field to search for new anti-HIV drugs or lead compound from the Traditional Chinese Medicine and natural resources. A large variety of natural products have been described as anti-HIV agents, and for a portion thereof the target of interaction has been identified.

3 General Research Objectives
Research is aimed at making full use of the rich natural resources in the Southwest of China and studying the natural anti-HIV compounds extracted from animals, plants and halobios, determining the structure-activity relationship of lead compounds; Searching for anti-HIV agents based on a new concept of interfering the cell signal transduction.

4 Summary of Achievements
Screening and researching anti-HIV-1 natural products. More than 2000 purified compounds purified from animals, plants and halobios were screened on anti-HIV-1 activity. Many lead natural compounds such as Xanthohumol (from the hop Humulus lupulus.), Concentricolide (from Daldinia sp.), Scutellarin (from Erigeron breviscapus (Van.) Hand–Mazz.), Sulfated Polyhydroxysterol (from marine sponge Halichondria rugosa Ridley&Dendy), Rutin sulphate (from Sophora japonica), Trichobitacin (from the root tubers of Trichosanthes kirilowii), Zinc complex of Baicalin (baicalin extracted from the Chinese medicinal plant Scutellaria baicalensis), Lectins (from marine invertebrates), Maxinin (from the skin secretion of Chinese red belly toad Bombina maxima.), Rare-earth compounds (La (NO3)3.6H2O, Ce (NO3)3.6H2O) and venom L-amino acid oxidase (TSV-LAO) (from Trimeresurus stejnegeri) have been shown strong anti-HIV activity.

Xanthohumol. Xanthohumol, prenylchacone flavonoid, is a natural product with multi-biofunctions purified from the hop Humulus lupulus. Xanthohumol inhibited HIV-1 induced cytopathic effects (CPE), the production of viral p24 antigen and reverse transcriptase (RT) in C8166 lymphocytes at non-cytotoxic concentration. The EC50 values were 0.82, 1.28 and 0.50 μg/ml respectively. The Therapeutic index (TI) was about 10.8. Xanthohumol also inhibited HIV-1 replication in PBMC with EC50 value of 20.74 μg/ml. The activity of recombinant HIV-1 RT and the HIV-1 entry were not inhibited by xanthohumol. The results suggested that xanthohumol is effective against HIV-1 and might serve as an interesting leading compound.
Zinc complex of baicalin. Baicalin (BA) has been shown with anti-HIV-1 activity. We studied the anti-HIV-1 activity of zinc complex of baicalin (BA-Zn) in vitro and compared the anti-HIV-1 activities between BA and BA-Zn. BA-Zn was with lower cytotoxicity and higher anti-HIV-1 activity compared with BA in vitro. The EC50s of BA-Zn on inhibition of CPE, HIV-1 p24 antigen, HIV-1 RT production and HIV-1 RT activity were lower than those of BA. The antiviral activity of BA and BA-Zn may owe to inhibition of HIV-1 RT activity and HIV-1 entry into cells. Zinc coupling enhanced the anti-HIV-1 activity of baicalin.

L-amino acid oxidase. A novel L-amino acid oxidase, named TSV-LAO, has been purified and cloned from the snake Trimeresurus stejnegeri. 50% cytotoxic concentrations (CC50) of TSV-LAO on C8166 cells were 24 nM and 390 nM in the absence or presence of catalase (400 nM), respectively. However, at concentrations that showed little effect on cell viability, TSV-LAO displayed dose dependent inhibition on HIV-1 infection and replication. The antiviral selectivity (CC50/EC50) was 16 and 6, respectively, corresponding to the measurements of syncytium formation and HIV-1 p24 antigen expression. Interestingly, the presence of catalase resulted in an increase of its antiviral selectivity to 52 and 38. Under the same conditions, no anti-HIV-1 activity was observed by exogenous addition of H2O2. The complete amino acid sequence of TSV-LAO, as deduced from its cDNA, exhibits a high degree of sequence identity with other snake venom LAOs.
Maximin. Two groups of antimicrobial peptides have been isolated from the skin secretion of Chinese red belly toad Bombina maxima. Peptides in the first group, which are composed of 27 amino acid residues and termed maximins 1, 2, 3, 4 and 5 respectively, are structurally related to bombinin-like peptides (BLPs) from toads Bombina orientalis and Bombina variegata. However, unlike BLPs which share a constant C-terminal region and differ only by on or two substitutions in the N-terminal 13 residues, sequence identities among maximins are ranging from 40% to 78% and mutations occurred all through the molecules. Maximin peptides exhibited a potent antimicrobial activity with a wide overlap in specificity but also significant differences in potency among each other. In addition, maximins 1 and 3 manifested a remarkable cytotoxic activity against human tumor cells. Furthermore, maximin 3 possessed a significant anti-HIV activity. Exposure of human motile sperms to maximins 1 and 3 resulted in the loss of sperm motility. Additionally, maximins 1 and 3 were toxic to mice with an acute lethal toxicity LD50 values of 8.2, 4.3 mg/kg respectively.

The mechanism and structure/function relationship of trichosanthin on anti-HIV activity

Structure-function relationship

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing anti-tumor and antiviral activity, including HIV. The mechanism of these actions are not entirely clear, but is generally attributed to its ribosome inactivating property. In order to study the relationship between the anti-HIV-1 activity of TCS and its structure, 8 TCS mutants with different ribosome inactivating (RI) activities were constructed by using site-directed mutagenesis. and the anti-HIV-1 activities of the mutants were tested in vitro.

Results showed that 2 TCS mutants, namely TCS M (120-123), TCSE160A/E189A, with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCSR122G, which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The RI activity of mutants with C-terminal deletion mutants (TCSC2、TCSC4 and TCSC14) decrease by 1.2-3.3 folds with parallel down shifting of its anti-HIV-1 activity (1.4-4.8 folds). The results suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property. But an exception was demonstrated that two TCS mutants retaining almost all RI activity but devoid of anti-HIV-1 activity. TCSC19aa and TCSKDEL having 19 amino acids extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS.

Phase I/II clinical trial of TCS had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Through site-directed mutagenesis, these 3 antigenic amino acids (Ser-7, Lys-173 and Gln-219) were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kD (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG20k-S7C, PEG20k-K173C, and PEG20k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, Q219C was decreased by about 1.5-5.5 fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20-30 fold) in anti-HIV activity. Cytotoxicity was however weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life.

The antiviral mechanism
The antiviral mechanism of TCS is not clear. The present results suggested that antiviral action may be mediated through selective apoptosis on infected cells. TCS induced apoptosis in cells and this action was more potent in those infected with HIV-1. In flow cytometry study, TCS induced larger population of apoptotic H9 cells chronically infected with HIV-1 in a dose dependent manner. At TCS concentration of 25 μg/ml, 6% of normal H9 cells were found to be apoptotic whereas the same concentration induced 20% in HIV-1 infected cells. Such difference was not found in control experiments. Two other studies supported this action. Cytotoxicity study showed that cell viability was always lower in HIV-1 infected cells after TCS treatment, and DNA fragmentation study confirmed more laddering in infected cells. The mechanism of TCS induced apoptosis is not clear. It is known that both RIP and HSV-1 infection affected the MAPK pathways. In this study, HSV-1 infection induced an elevation of phosphorylated p38 and Bcl-2 which can be blocked by TCS. Both viral replication and host cells viability in infected cells were lowered after TCS. This suggested that TCS may interfere with MAPK signals generated by infection leading to selective apoptosis of infected cells. In summary, our result demonstrated that TCS is more effective in inducing apoptosis in HIV-1 and HSV-1 infected cells. The MAPK signal pathway is implied to participate in the mechanism.

Combination of TCS and other antiviral agents
The anti-HSV-1 activity, toxicity and combination of TCS on Vero cells were measured. Results showed that the ED50, TD50 and TI were 38.5 ?g/ml, 416.5 ?g/ml and 10.9 respectively. Antiviral activity of TCS was substantially potentiated when it was used in conjunction with other antiviral agent. The ED50 of TCS was reduced 125 folds by acyclovir at a concentration of 0.001 ?g/ml, which was practically devoid of significant antiviral activity. Similarly the ED50 of TCS was reduced 100 folds by interferon-?2a at a concentration of 100 IU/ml. In conclusion, TCS is effective against HSV-1 and other antiviral agents such as acyclovir or interferon can potentiate its action substantially.

Toxicity of trichosanthin

It is generally believed that TCS toxicity is mediated through intracellular ribosome inactivation. Therefore TCS toxicity should be determined by the amount inside cells rather than outside. Three different cell types IC21, JAR and Vero cell lines were chosen with high, medium and low sensitivity to TCS. Intracellular concentrations of FITC labeled TCS were determined by laser scanning confocal microscopy. A good relationship was demonstrated between intracellular TCS concentration and toxicity. Highest intracellular concentration was found in IC21, followed by JAR, and lowest in Vero cells. When the intracellular TCS concentrations in these cells were reduced by using a competitive inhibitor to block cell entry, cytotoxicity was not observed. In conclusion, there is strong evidence to indicate that cytotoxicity of TCS is dependent on its intracellular concentration. Variation of cytotoxicity in different cells may be related to the mechanisms affecting its internalization.

Structure-function relationship of 2-nitroethenylbenzene on anti-HIV-1 RT activity
2-nitroethenylbenzene was with some anti-HIV-1 activity and excellent inhibitory activity on recombinant HIV-1 RT. We took 2-nitroethenylbenzene as lead compound and synthesized 26 derivatives and tested the inhibitory activities of these 27 Nitrooelfins on recombinant HIV-1 RT activity. The results demonstrated that there’s an obvious structure-function relationship of nitroolefins between their structures and their inhibitory activities on HIV-1 RT. Ortho substitution of one of the hydrogens in the phenyl ring of 2-nitroethenylbenzene increased the inhibitory activities of nitroolefins whereas meta and para substitution did not. If the substituent was electron donor, it may increase the electronic density of the phenyl ring and increase the inhibitory activities of nitroolefins. Conjugating between the substituient and the phenyl ring and the group of nitroethenyl also increased the inhibitory activities of Nitrooelfins. When more than one of the hydrogens in the phenyl ring of 2-nitroethenylbenzene was substituted, there was steric hindrance between the substituents, leading to decreasing of the inhibitory activities of nitroolefins. Substitution of the whole phenyl ring of 2-nitroethenylbenzene with heteroautom aromatic ring increased the inhibitory activities of nitrooelfins. Nitroolefins showed severe cytotoxicities on C8166 cells. Substituting nitro (NO2) group with acetylamino (NHCOCH3) group or benzoylamino group (NHCOPh) was expected to reduce the cytotoxicities of nitroolefins. However, these substitutions decreased the inhibitory activities on HIV-1 RT dramatically. It may be helpful to find more potent and less cytotoxic nitroolefins if we study the quantitative structure-activity relationship of niroolefins using bioinformatics methods.

5 Scientific contributions
[1] Zheng YT, Ben KL, Jin SW, 2000, Anti-HIV-1 activity of trichobitacin, a novel ribosome-inactivating protein. Acta Pharmacol.Sin.21: 179-182

[2] Zheng YT, Chan WL, Chan P, Tam SC. 2001, Enhancement of the anti-herpetic effects of trichosanthin by acyclovir and interferon. FEBS lett. 496: 139-142

[3] Lai R, Zheng YT, Shen JH, Liu GJ, Liu H, Lee WH, Tang SZ, Zhang Y. 2002, ，Antimicrobial peptides from the skin secretion of Chinese red belly toad Bombina maxima. Peptides 23 (3): 427-435

[4] Chan WL, Zheng YT, Huang H, Tam SC. 2002, Relationship between trichosanthin cytotoxicity and its intracellular concentration. Toxicology 177: 245-251

[5] Wang YF, Chen JJ, Yang Y, Zheng YT, Tang SZ, Luo SD. 2002, New rotenoids from Roots of Mirabilis jalapa. Helvetica Chimica Acta 85 (8): 2342-2348

[6] Wang JH, Nie HL, Tam SC, Huang H, Zheng YT. 2002, Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity. FEBS Lett 531：295-298

[7] Wang JH, Nie HL, Huang H, Tam SC, Zheng YT. 2003, Independency of anti-HIV-1 activity from ribosome inactivating activity of trichosanthin. Biochem. Biophys. Res. Commun. 302 (1): 89-94

[8] Zhang YJ, Wang JH, Li WH, Wang Q, Liu H, Zheng YT, Zhang Y. 2003, Molecular characterization of Trimeresurus stejnegeri venom L-amino acid oxidase with potential anti-HIV activity. Biochem. Biophys. Res. Commun. 309: 598-604.

[9] Wang JH, Tam SC, Huang H, Ouyang DY, Wang YY, Zheng YT. 2004, Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro. Biochem. Biophys. Res. Commun. 317 (4): 965-971

Books
[1] Zheng YT, Ben KL, 2000, Trichosanthin (2th ed). Wang Y, Sciences Press，Beijing. pp373-381

[2] Chen JJ, Luo SD, Zheng YT, Wang YF. 2002, The cellection of active compounds of anti-HIV/AIDS. Yunnan Sci-Tech Press.

Patents
Authorized: 7; in application: 7.

6 HONORS AND AWARDS
"The study of RIPs with anti-HIV activities and structure-function relationship" by Yong-Tang Zheng, Kun-Long Ben and Jian-Hua Wang won the Second Class of Yunnan Natural Science Awards in 2003.

7 RESEARCH FUNDING (2000-2004)
2000-2002 RMB 110, 000 (National Natural Science Foundation of China), Structure-function of trichosanthin on anti-HIV activities

2001-2003 RMB 200, 000 (Special grant of CAS), Research and development of HIV p24 antigen diagnosis assay.

2000-2003 RMB 100, 000 (Youth innovation grant of CAS), The roles of selectively apoptosis in treatment of AIDS with trichosanthin.

2003-2006 RMB 300, 000 (Natural Science Foundation of Yunnan), Cellular and molecular anti-SARS virus assays and screening of natural products.

2002-2005 RMB 60, 000 (Natural Science Foundation of Yunnan), The mechanism of selectively apoptosis in treatment of AIDS with trichosanthin.

2002-2005 RMB 500, 000 (CAS Knowledge Innovation Projects), The interaction of HIV and host cells.

2000-2002 RMB 60, 000 ( (National Natural Science Foundation of China), Discovery and structure modify of natural lead compounds with anti-HIV activity.

2002-2005 RMB 100, 000 (Hi-tech Research and Development Program of China), Research and development of a new therapeutic HIV vaccine.

2003-2005 RMB 60, 000 (Hi-tech Research and Development Program of China), Research and development of NOF with anti-HIV RT activity.

2003-2005 RMB 250, 000 (Hi-tech Research and Development Program of China), Research and development of anti-HIV drugs and vaccine.

2003-2004 RMB 70, 000 (Natural Science Foundation of Yunnan), Anti-HIV compounds based on MAPK signal transduction.

2004-2005 RMB 400, 000 (Hi-tech Research and Development Program of China), The preclinical study of “Qisilei” capsule, a Chinese Traditional Medicine.

8 INTERNATIONAL CO-OPERATION
“Trichosanthin as an antiviral agent – mechanism and action” is a cooperation with Prof. Michael SC Tam of the Chinese University of Hong Kong (CUHK).

9 Structure of the laboratory
Staffs: 4; PhD Students: 6; M.Sci. Students: 4; two M. Sci. students received degree; Undergraduate Students: 7.

10 RESEARCH INTERESTS
Studying the natural anti-HIV compounds from natural resource and the structure-activity relationship of lead compounds;

Searching for anti-HIV agents based on a new concept of interfering the cell signal transduction;

Developing novel HIV vaccines;

Establishing Nonhuman primate animal model for AIDS and exploring the pathogenic mechanism of AIDS.