Bifunctional Anti-Human Immunodeficiency Virus Type 1 Small Molecules with Two Novel Mechanisms of Action

A class of betulinic acid derivatives was synthesized to targettwo critical steps in the human immunodeficiency virus type1 (HIV-1) replication cycle, entry and maturation. Each mechanismof HIV-1 inhibition is distinct from clinically available anti-HIVtherapeutics. The viral determinants of the antientry and antimaturationactivities are the bridging sheet of HIV-1 gp120 and the P24/p2cleavage site, respectively.

INTRODUCTION

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Introduction

Betulinic acid derivatives are a class of small molecules thatexhibit anti-human immunodeficiency virus type 1 (anti-HIV-1)activity (6, 7, 11). The targets of betulinic acid derivativesare varied, depending primarily on the side chain structuresof the compounds (6) (Fig. 1). For example, betuliniyl aminooctanoylamino-3R-hydroxy-6-methylheptanoicacid (IC9564), which has aside chain at position 28 of betulinicacid, is a potent HIV-1 entry inhibitor (5). In contrast, dimethyl-succinyl-betulinicacid (DSB), which has a side chain at position 3, exhibits potentanti-HIV activity by inhibiting the maturation of the viralprogeny (4, 7). IC9564 and DSB share a five-membered betulinicacid ring structure. Based on the molecular targets and chemicalstructures of IC9564 and DSB, we synthesized a class of compoundspossessing both of these structural features, which resultedin an anti-HIV-1 profile superior to those of IC9564 and DSB.N-[3-O-(3',3'-dimethylsuccinyl)-lup-20(29)-en-28-oyl]leucine(LH15) and N-[3-O-(3',3'-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-11-aminoundecanoicacid (LH55) are two of these compounds with potent anti-HIVactivity.

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FIG. 1. Chemical structures of anti-HIV-1 betulinic acid derivatives.

Synthesis and anti-HIV-1 activity of bifunctional small molecules LH15 and LH55.

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Synthesis and Anti-hiv-1...

The protocol used to synthesize LH15 and LH55 was modified frompreviously described procedures (4, 12). The antientry functionalgroups, leucine and aminoundecanoic acid methyl esters, wereintroduced into the backbone of betulinic acid at position 28.The resulting intermediates were refluxed with 2,2-dimethylsuccinicanhydride in the presence of pyridine and dimethylaminopyridineto introduce the antimaturation side chains at position 3. Thefinal products were purified with high-performance liquid chromatographyto yield LH15 and LH55. ¹H nuclear magnetic resonance with signalassignment and mass spectrometry were performed to verify thestructures.

The anti-HIV activity of these compounds was evaluated withan HIV-1 infectivity assay described previously (5). A dilutedHIV-1 stock at a multiplicity of infection of 0.001 50% tissueculture infective dose per cell was used to infect MT4 cellsin the presence of various concentrations of the compounds.The compounds IC9564 and DSB (4, 5) inhibit various HIV-1 isolatesat submicromolar concentrations but not the protease inhibitor-resistantstrain PI-R, which is less sensitive to DSB (Table 1). LH15and LH55, in general, are at least a log more potent than eitherIC9564 or DSB. The results shown in Table 1 also demonstratethat LH15 and LH55 are potent inhibitors of the multiple-protease-inhibitor-resistantstrain PI-R (2) and the multiple-reverse transcriptase (RT)-inhibitor-resistantstrain RTI-R (10).

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TABLE 1. Anti-HIV activity of betulinic acid derivatives

Inhibition of HIV-1 entry.

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Inhibition of hiv-1 entry.

The side chain at position 28 is critical for the antifusionactivity of the betulinic acid derivatives. Betulinic acid derivativeswith the same side chains as LH15 and LH55 at position 28 butwithout a functional side chain at position 3 exhibit antifusionactivity (12). Figure 2a shows that the potency of LH55 againstNL4-3 envelope-induced membrane fusion is similar to that ofIC9564 and T20. DSB, lacking a side chain at position 28, doesnot significantly affect the envelope-mediated membrane fusion.It has previously been demonstrated that the key determinantfor IC9564 sensitivity is gp120 (5). However, the detailed mechanismof fusion inhibition remains unclear. IC9564 does not affectCD4-gp120 interaction. The binding of gp120 to CD4 and the subsequentinteraction with chemokine receptors are two critical fusionevents for HIV-1 entry. Thus, it is likely that the chemokinereceptor interactive site on gp120 is a key determinant forthe antifusion activity of IC9564.

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FIG. 2. (a) LH15 and LH55 inhibit HIV-1 entry. A fusion assay modified from a previously reported protocol (5) was used in this study. The fusions between COS cells expressing NL4-3 envelope glycoproteins and HeLa cells expressing CD4 and CXCR4 (1) were performed in the presence of the anti-HIV-1 betulinic acid derivatives and T20, as indicated in the figure. T20 is a peptide derived from gp41 of HIV-1. The fused syncytia were stained blue and enumerated as previously described (5, 15). Each data point in the figure represents the mean of three independent experiments. (b) An amino acid in the bridging sheet of gp120 determines the sensitivity of HIV-1 to IC9564. The same fusion assay described for Fig. 2a was used for the experiments. Each data point in the figure represents the mean of three independent experiments.

To test this hypothesis, we chose a pair of HIV-1 strains, NLDH120and M2-NLDH, that exhibit significant differences in the accessibilityof their chemokine receptor interactive sites (15). The twoviruses differ in one amino acid at position 198 in the bridgingsheet of gp120 (15). The envelope sequences of these two viruseswere cloned into an expression vector, pSRHS, and used in anenvelope-mediated membrane fusion assay (5). The fusion mediatedby the M2-NLDH envelope, M2-pSW120, is approximately 10-foldmore sensitive to IC9564 than that of the NLDH120 envelope,pSW120 (Fig. 2b). Since the bridging sheet is a critical structuralmotif involved in HIV-1 entry (9), the results strongly supportthe notion that the bridging sheet is involved in IC9564 sensitivity.

Interference with the processing of p25 resulted in an inhibition of HIV-1 maturation.

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Interference with the processing...

Based on our previous observation that DSB does not affect theproduction of HIV-1 viral particles (4), we speculated thatDSB treatment might lead to the production of immature viralparticles that have lost infectivity. In order to test thishypothesis, HIV-1 particles produced in the presence of LH55were lysed and analyzed using Western blots. There was an accumulationof p25 in the virus produced in the presence of LH55 (Fig. 3).Accumulation of p25 was also observed in the virus particlesproduced in the presence of DSB (data not shown). The processingof p25 to p24 and p2 is the last step in sequential proteasecleavage of Gag precursor into mature Gag proteins, which iscritical for viral infectivity (3, 8, 13). The unique mode ofaction of DSB or LH55 is that these compounds affect the processingof only p25. The processing of other Gag proteins, such as p17,is not affected by these compounds (Fig. 3).

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FIG. 3. LH55 inhibits HIV-1 maturation. HIV-1 viral particles were produced from ACH-2 cells in the absence (A) or presence (B) of LH55. The virus particles were purified by ultracentrifugation using a sucrose gradient (14). The collected viral samples were lysed and analyzed using a sodium dodecyl sulfate-12.5% polyacrylamide gel followed by a Western blot using an HIV-1-positive human serum. kd, molecular mass standards in kilodaltons.

The key structural feature that enables LH55 and its analogsto possess the dual mode of action is the presence of both sidechains at position 3 and position 28. The unique biologicalactivity of these compounds is that they not only inhibit theviruses resistant to HIV-1 RT and protease inhibitors but alsoinhibit viruses that are resistant to compounds which bear theside chains only at position 3 or position 28 (data not shown).The bridging sheet of gp120 and the maturation of p24 are thecritical determinants for drug sensitivity to this class ofcompounds. The antifusion activity of LH55 or LH15 allows thecompound to block HIV-1 before it enters the cell. The virusthat survives this blockage will have to face the antimaturationactivity of these compounds. Since the molecular targets ofthese bifunctional anti-HIV betulinic acid derivatives are differentfrom those of clinically available anti-HIV-1 drugs, it is possiblethat LH15, LH55, or their derivatives might have the potentialto become useful additions to current anti-HIV therapy.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grantR01AI49096.

FOOTNOTES

* Corresponding author. Mailing address: Duke University Medical Center, Surgical Oncology Research Facility, LaSalle Street Extension, Durham, NC 27710. Phone: (919) 684-3819. Fax: (919) 684-3878. E-mail: chc{at}acpub.duke.edu.

REFERENCES

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