Nakane and Ono27 reported that epicatechin and epigallocatechin gallates (Figure 8.8) had
a marked capacity to inhibit the polymerase reaction catalyzed by the human immuno- deficiency virus reverse transcriptase (HIV-RT). Interestingly, inhibition was much greater for HIV-RT than for a representative group of other viral and cellular polymerases that were also tested. Unhappily, the agents were not active in cellular assays against the virus. The authors speculated that these gallates were probably not entering the cell, where any antiviral activity would have to have been expressed. At the same time they noted that hydrolysis of the gallate ester in either epicatechin gallate or in epigallocatechin gallate
FIGURE 8.8
Structures of catechin gallates with HIV-RT inhibitory activity.
Reactive Quinones: From Chemical Defense Mechanisms in Plants to Drug Design 117
would result in two molecular fragments that were, in both cases, inactive against the enzyme. This suggested that premature hydrolysis of both reagents by cellular esterases, or a combination of slow penetration of the membrane and enzyme-assisted hydrolysis of the gallate ester linkage, could have been major reasons for the observed lack of antiviral activity.
Considerable simplification of the structure of the catechins and modification (rather than removal) of the gallate ester linkage with a hydrolytically stable linkage might allow retention of HIV-RT inhibition and facilitate membrane transport. Then, because of better cell penetration behavior and reagent stability in vivo, one might expect to observe antiviral activity directly. The idea of simplifying a complex natural product and retaining signifi- cant, even improved inhibition, from which pharmaceutical agents might then be devel- oped, is not new. This approach represents one of several strategies in lead compound modification,28 and led not only to an understanding of the relatively smaller complex of
atoms on the surface of the morphine molecule required for receptor recognition (the phar- macophore), but eventually to the development of pain remedies (darvon and demerol) with less addictive properties and fewer severe side effects.28
Initially, we removed one and then two rings from the catechins, and then reduced as much as possible the number of oxidation-activating reactive phenolic groups. The nature of the molecular simplifications undertaken is illustrated in Figure 8.9. At the present stage of development, these compounds are only 10 to 100 times less active than the catechins on which they are based. However, we also discovered these agents to represent a fundamen- tally new class of inhibitors: they illustrated a nearly uncompetitive pattern of inhibition. This was a surprise, as there are no other such HIV-RT inhibitors described. Indeed, we already suspected that we had discovered a new class of inhibitors, because the activity
FIGURE 8.9
Strategy for the simplification of catechin gallate structures.
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against the A-17 mutant enzyme (K103N–Y181C) was nearly equivalent to the level of inhi- bition against wild-type enzyme, despite the fact that the A-17 mutant enzyme is resistant to all known noncompetitive non-nucleoside inhibitors. Thus, inhibitors based on these catechins are of special interest.
A second, unexpected benefit from this study derived from simultaneous measurement of DNA-strand-transfer-inhibiting properties of the simplified catechins. We discovered that the level of residual polymerase inhibition was different in some cases from that observed for strand-transfer inhibition. Some of the agents with simplified structure had IC50 values for the DNA strand-transfer inhibition at less than 10 µM, and were without any
inhibitory effect on the polymerase reaction at inhibitor concentrations as high as 100 µM. Two DNA strand transfers must occur during complete copying of the viral RNA into a double-stranded DNA form prior to integration of the DNA into the genome of the host. Many of the mutations associated with the hypermutability of the virus occur during DNA strand transfer. Thus, it could be important to develop DNA-strand-transfer inhibitors with little polymerase inhibitory capacity to study both (1) the effects associated with direct DNA-strand-transfer inhibition of the virus and (2) the possibility of inhibiting the DNA- strand-transfer process at a sublethal level in the absence of polymerase inhibition, which would allow the virus to reproduce while slowing considerably the formation of escape mutations. While complete inhibition of DNA-strand-transfer process would itself be expected to be antiviral, limited inhibition of the process could affect the rate of viral muta- tion while allowing, albeit slower, replication of the virus. A depressed rate of mutation thus achieved could allow the immune system of the host an opportunity to mount a defen- sive response against a less chameleon-like virus far easier to target.
8.4
Conclusions
In summary, the studies reviewed here use diverse strategies to take advantage of the redox properties of two classes of catechol–quinone compounds present in nature to design new compounds of pharmaceutical interest. In a third class of naturally occurring compounds of complex structure, simplification and removal of the redox-sensitive elements may be key to providing target structures with a novel antiviral character.
ACKNOWLEDGMENTS: This work was partially supported by Grants NS 14491, NS 22851, and NS 35305 from the National Institute of Childhood Disorders and Stroke (NIH) and in part by grants from the DeArce Foundation and from the Ohio Board of Regents Research Challenge Program. We also wish to thank Parke-Davis, Ann Arbor, MI for cloned enzymes and reagents and for generous preliminary financial support of the HIV-RT work.
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