Phytomedicines of Europe - American Chemical Society

Chapter 8

Downloaded by NORTH CAROLINA STATE UNIV on September 29, 2012 | http://pubs.acs.org Publication Date: April 15, 1998 | doi: 10.1021/bk-1998-0691.ch008

Secondary Metabolites from Plants as Antiretroviral Agents: Promising Lead Structures for Anti-HIV Drugs of the Future

Eckart Eich Institute für Pharmazie II, Pharmazeutische Biologie, Freie Universität Berlin, Königin-Luise-Str. 2+4, D-14195 Berlin, Germany Retroviral integrase, a virally encoded enzyme (like reverse transcriptase and protease) could be another interesting target for the therapy of HIV infections. Herbal constituents and their derivatives belonging to different structural classes, e.g. lignans, flavonoids, and curcuminoids, turned out to befirstinhibitors of this enzyme. Studies on structure/activity relationships showed that the most potent of these compounds may be described in general as consisting of two aryl units containing the 1,2-dihydroxy pattern each, thus forming bis-catechols separated by an appropriate linker segment. On the basis of these specific integrase inhibiting leads which, however, lack antiviral activity, the development of HIV-inhibiting compounds is in progress as recent results with a bis-coumarin derivative show.

Herbal extracts, as well as pure plant constituents, with elucidated chemical structures have been screened from species of numerous plant families with respect to antiviral properties. This has been so, particularly during the past two decades. Indeed a large number of compounds from almost all classes of secondary metabolites, show confirmed activity in special in vitro screens of many different pathogenic viruses. Nevertheless, the contribution of phytomedicines to the treatment of viral infections has been rather limited to date. A cream containing 1 % of a specially prepared dried extract from lemon balm leaves (Melissa officinalis L., Labiatae), has been introduced to the German market for local therapy of herpes simplex of the skin. The effect of this melissa cream in the topical treatment, is statistically significant as proven by clinical studies (1, 2 J. On the other hand, constituents of plants could serve as useful leads for developing the antiviral drugs of the future. This review focuses on such leads from well-known medicinal plants in the field of human immuno-deficiency viruses (HIV), with emphasis on a novel target, retroviral integrase.

©1998 American Chemical Society

In Phytomedicines of Europe; Lawson, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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84


Arctigenin, the First Integration Inhibiting Agent in H I V Infected Cells (-)-Arctigenin 1, a lignan isolated from the pantropical climbing shrub Ipomoea cairica (L.) SWEET belonging to the morning glory family (Convolvulaceae) (3), proved to be a potent inhibitor of HTV-1 replication in different human cell systems (4,5). I. cairica is a medicinal plant of various ethnomedicines in West Africa (6). On the other hand, 1 is also a constituent of certain species of the daisy family (Compositae), e.g. Arctium lappa L., the greater burdock (7), which is used in Chinese traditional medicine (Niu bang) (8). This lignanolide turned out to be an efficient inhibitor of the nuclear matrix associated D N A topoisomerase n activity.

R = C H : H-Arctigenin (1) R = H: 3-O-Demethylarctigenin (3) 3

Thus, initially it was assumed that it showed anti-HIV activity due to prevention of the increase of activity of this mammalian enzyme involved in virus replication. However, using the polymerase chain reaction (PCR) technique to determine the degree of integration of proviral D N A into the cellular D N A genome after 6 days of incubation, a strong suppression of the HTV-1 integration could be observed (9,10). From these results it was concluded that the anti-HIV effect of arctigenin 1 is primarily caused by inhibition of the HIV integration reaction rather than by inhibition of D N A topoisomerase II. Until this discovery, no compounds with HIV integration inhibiting properties had been found. HTV Integrase in vitro Assay In principal there is the possibility for the development of multiple classes of antiviral agents that could act at each respective stage of HIV replication, e.g. attachment, penetration, uncoating, and reverse transcription. These antiviral agents could act at an early stage of the HIV life cycle, or at different later stages, e.g. as protease inhibitors in the maturation stage. To date, only inhibitors of reverse transcriptase and protease have been found to be of therapeutic relevance. For many years, pharmacological antiretroviral research, with virally encoded enzymes as targets, has focused principally on agents that inhibit these two enzymes. The protein catalyzing the integration observed in the studies with arctigenin 1 is a target which had been vastly neglected until the early 1990s. This protein is called retroviral or HIV integrase and is contained within the virus particle. It integrates a double stranded D N A copy of the viral R N A genome, synthesized by reverse transcriptase, into a host chromosome (11, 12). This represents the last, and somehow decisive, step of the viral infection: the point of no return.


85 The development of an in vitro assay for HIV-1 integrase function, in the early 1990s, permitted testing of potential inhibitors. The recombinant integrase enzyme produced via an Escherichia coli expression vector uses a blunt-ended 21-mer duplex oligonucleotide as both the specific donor and nonspecific target substrates. The initial step in the enzymatic reaction involves nucleolytic cleavage (= 3'-processing). It liberates the 3'-terminal dinucleotide GT, producing a 19-mer oligonucleotide from the P-labeled 21-mer duplex substrate. The resultant 3'-termi-nal dA hydroxyl is the donor substrate for the second step, the subsequent joining to a 5'-phosphate of a second 21-mer duplex oligonucleotide. The nucleophilic attack on the phosphodiester bond of the target substrate leads to the D N A strand transfer or integration reaction. Thus, this reaction is a transesterification cutting the target D N A (in vivo: the host DNA) and binding its 5'-end to the viral DNA's just shortened 3'-end. It results in the insertion of one 3'-processed oligonucleotide into another nucleotide, yielding higher molecular weight species with slower migration in electrophoretic separation procedures than the 21-mer substrate (for further details see 13-16).


32

Plant Constituents as Leads for HIV Integrase Inhibitors Arctigenin and CAPE. The results in this enzyme assay for arctigenin 1, however, were not up to the expectations: the compound was almost inactive (13-16). A structural comparison of 1 with another recently discovered HIV-1 integrase inhibitor, caffeic acid phenethylester (CAPE) 2 (17), a secondary metabolite from the buds of poplar trees (Populus spp., Salicaceae) as well as a constituent of bee propolis, shows surprising similarities. On the other hand, an important difference is the presence (2) or lack (1) of a catechol partial structure (two adjacent hydroxyl groups). This led to the

Caffeic acid phenethylester (CAPE) (2)

assumption that the corresponding 3-O-demethylated derivative 3 ought to be active. Indeed, this was confirmed (13-16). Compound 3 exhibited remarkable activities in both integrase assays: the cleavage or 3'-processing step was inhibited by 57 % at a 100 p M concentration of the compound, and the integration or strand transfer step was inhibited by 52 % (Figure 1). Thus, the observed integration inhibiting effect of 1, in the HIV-infected human cell systems (9,10), could be due to metabolization of 1 to the 3-0-demethylated derivative 3 during the incubation. Therefore, arctigenin 1 probably acts as a "prodrug" which may easily enter the cell due to its more lipophilic character compared with 3. Such a metabolization was confirmed at least in V79


86

H C0. 3


Q

a) LiBH / THF 4

b) p-toluenesulfonic acid/CH CI 2

57%

2

52%

3-O-Demethylarctigenin (3)

benzylamine/ CH CI 2

3,4-0,0-Didemethylbrassilignan (4) LiBH /THF 4

2

H CO,

CHoOH

H,CO'

CH 0H

3

< 5%

< 5%

3 - 0 Demethyl-arctigenJc acid benzylamide (5)

Phytomedicines of Europe - American Chemical Society

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