Structural Requirements for Enzymatic Activation of Acyclonucleotide

Chapter 3

Structural Requirements for Enzymatic Activation of Acyclonucleotide Analogues

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Relationship to Their Mode of Antiherpetic

Action

Richard L. Tolman Merck Sharp & Dohme Research Laboratories, Box 2000, Rahway, NJ 07065 The structure-substrate relationships of the viral specified thymidine kinase (TK) as well as host phosphorylating enzymes ( G M P kinase) have been studiea and relevant acyclonucleoside conformations amenable to phosphorylation have been proposed based o n molecular modelling. The modes of action of other acyclonucleoside antiviral agents, which are not viral DNA polymerase inhibitors, and therefore TK-independent, e.g. 2'nor-cGMP, have also been discussed. The most safe and effective agents of the present generation of antivirals for members of the group of herpes viruses are guanine acyclonucleosides. These agents have been shownQL,2) to ultimately inhibit the viral replication process as their triphosphate derivatives.

Ganciclovir

Thymidine

It is a viral-specified enzyme, thymidine kinase (TK), which accomplishes the first phosphorylation® of guanine acyclonucleosides, such as acyclovir (ACV), to monophosphate tnereby accounting for the great selectivity of their antiviral action. Our initial interest i n this area was i n understanding the manner of this remarkable enzymatic event i n accepting a guanine derivative as a surrogate for the natural substrate thymidine. The structuresubstrate relationships for acyclonucleoside derivatives were examined for herpes (HSV-1) thymidine kinase as well as for the host enzyme G M P kinase, which converts monophosphate to diphosphate(4). There are many 0097-6156/89/0401-0035$06.00/0 © 1989 A m e r i c a n Chemical Society

Martin; Nucleotide Analogues as Antiviral Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE

36

ANALOGUES


enzymes® which convert acyclonucleoside diphosphate to triphosphate, the final agent which inhibits viral D N A polymerase and thereby viral replication (Fig. 1). Molecular modelling was found to be a source of stimulation toward the synthesis of new chemical entities and to provide plausible scenarios for enzymatic phosphorylation. Molecular modelling studies are arguably less relevant in their application to substrate studies as there are two components in substrate activity, binding and efficacy. It is the binding component which is more easily dealt with in the absence of an X-ray structure ot the enzyme. H S V Thymidine Kinase Keller et Λ/.(6) have examined in a cursory fashion the substrate requirements of herpes thymidine kinase (HSV-TK) particularly with regard to guanine derivatives. The structural requirements of pyrimidine nucleoside analogs have been examined(7-10) for substrate activity. It has been demonstrated that 5-substitution of the pyrimidine ring is important for substrate activity. Thymidine is a much better substrate than uricfine which has hydrogen as a 5-substituent. The ability of other bulkier and more hydrophobic 5substituents to function even more effectively than thymidine as substrates has led to the thesis that the binding site of T K which accommodates the 5methyl of thymidine is in fact a long cylindrical hydrophobic pocket©. 2'Deoxy-cytidme is also a quite a good substrate(one-twentieth as effectively phosphorylated as thymidine, Π) despite the fact that it has hydrogen at C5. The phosphorylation of ganciclovir (GCV) by H S V - T K has been shown to be stereospecific for the pro-S hydroxyl(12) as evidenced by the fact that enzymaticallv-prepared G C V - M P is rapidly converted by G M P kinase to G C V - D P , wnereas the chemically-prepared M P (racemic) is only 50% converted to D P under the same conditions. Molecular Models. A statement of Garland Marshall's(13) describes the promise of molecular modelling for studies of the type of the thymidine kinase structure-substrate study: "Without detailed information about the three-dimensional nature of the receptor, conventional physicochemically based approaches are not possible. One can only attempt to deduce an operational model of the receptor that gives a consistent explanation of the known data and, ideally, provides predictive value when considering new compounds for synthesis and biological testing." U s i n g a modified M M 2 force field(14), tnree dimensional energy maps were generated for thymidine and ganciclovir (Figs 2 & 3) using two sidechain dihedral angles(15). The conformationally-constrained thymidine was seen to have a number of low energy wells corresponding to anti-conformers with gauche- or gauche+ 5'-hydroxyls, whereas permutations of G C V ' s side chain position produced little variation i n energy (total energy range = 10 kcal). The C 8 - N 9 - C l ' - 0 torsion angles from the X-ray structures of A C V Q 6 18) and GCV(19,15) (-91 and -110° respectively) fell i n a low energy portion of the contour plot, but were not the lowest energy structures i n that energy well. The key to understanding how H S V - T K could accept a guanine acyclonucleoside as a substrate seemed to be i n the positioning of the purine on the active site of the enzyme. The effect of some purine substituent changes upon T K substrate ability have been examined by others(20,6). Table I shows the relative substrate ability of various substituted guanine derivatives within any of three side-chain series. A n y change of the 6-oxo function abolished substrate activity [the substrate activity of 2'β


Structural Requirements for Enzymatic Activation

3. T O L M A N

Ο


Ganciclovir (GCV)

OH

OH HSV Thymidine Kinase

GMP kinase (host)

/

HN

II

Ν

Μ

[Ι

cr

o-

2

OH

host enzymes Ν

HN 2

}

if

ο

?

Î

o'NHS^Nr o- o- oOH

Figure 1. Viral Activation of Acyclonucleosides


37

38

N U C L E O T I D E ANALOGUES

Thymidine

Energy


-30.00

-0

90

180

270

Figure 2. Energy Contour Plot for Thymidine (X-ray Structure) for Two Torsions, θΐ (C8-N9-Cl'-0) and Θ2 (0-C4'-C5'-05')


360

3.

TOLMAN

39


Ganciclovir


Energy ι

-0

90

180 θ

270

36

2

Figure 3. Energy Contour Plot for Gandclovir (X-ray Structure) for T w o Torsions, θΐ (C8-N9-Cl'-0) and Θ2 ( Ο Ζ ^ ' ^ ' - Ο δ ' )


NUCLEOTIDE ANALOGUES


40

deoxycytidine might have predicted the 6-amino derivative to be a substrate]. Similarly, changes were not tolerated at the purine-2-position of guanine, except for the 2-methylamino(6) which retained weak substrate activity. However, hydrophobic substituents at the 8-position (particularly halogen or alkyl) enhanced substrate activity, while hydrophilic substituents were not substrates. A likely explanation for the substrate activity of 8-substituteduanines is that the hydrophobic 8-bromo or -methyl fits into the postulated ydrophobic pocket which exists i n our model to account for the enhanced substrate activity of 5-substituted thymidines. Table I. Structure-Substrate Relationship for Modified Guanines

R = C H 2 O C H 2 C H 2 O H (+) R = C H 2 0 C H ( C H 2 0 H ) 2 (+++) R = C H 2 C H 2 C H 2 C H 2 O H (++)

N O T E :

A = OH(-), H(-), NHMe(+) NHCH2Ph(-), N H N H 2 ( - ) , Cl(-) Β = H , SH, NH2, N H M e , OMe, N H n P r , N H C H 2 P h , all(-) C = OH(-),NH2(-),SH(-) 8-aza(+), Br(++), Me(+++)

- , not a substrate; +, poor substrate; + + ,

good substrate; and + + + ,

excellent

substrate. Assay protocol, ref. 21.

In order to test the hypothesis by superposition of molecular models, randomly generated ganciclovir conformational structures were optimized and grouped into families when atoms differed i n position by 100

2'Nor-cGMP Still another type of antiherpetic mode of action employing phosphorylated acyclonucleosides is typified by 2'nor-cGMP(31-33). This antiviral agent has potent activity against members of the herpes group and other D N A viruses, including cytomegalovirus(34,35), adenovirus(36), papilloma virus(31), varicella-zoster(31), and herpes keratitis(37). Cell culture studies and animal studies have shown that 2'nor-cGMP is equally effective against TK-deficient (or minus) H S V strains, indicating that this activity i n independent of viral thymidine kinase. Although some hydrolysis of the cyclic pnosphate occurs intracellularly in vivo to produce levels of G C V - M P (and therefore GCV-TP),

American Chemical Society Library

1155 16th as S tAntiviral . N.W.Agents Martin; Nucleotide Analogues Washington, C 20036 ACS Symposium Series; American Chemical D Society: Washington, DC, 1989.


48


the amount of T P produced is not enough to account for the antiviral activity observed(38).

Unlike G C V - M P , 2'nor-cGMP is taken up intact from the gut [10% of a labelled dose (10mg/kg p.o., rats) is recovered i n the urineQS)] and is relatively stable i n plasma (80% of drug recovered from urine is intact cyclic phosphate). The mechanism of antiherpetic action of 2'nor-cGMP remains unknown, since the cyclic phosphate directly inhibits neither HSV-1 D N A polymerase nor C M V polymerase. Summary Molecular modelling has proven to be a positive factor i n the development of structure-activity relationships as a source of stimulation and predictive models. Although the means is available to design acyclonucleoside substrates which w i l l be phosphorylated i n herpes-infected cals, there is no assurance that the resultant monophosphates w i l l be converted to higher phosphates by G M P and other kinases. It is also not possible to predict a rion whether these triphosphates w i l l inhibit viral polymerases and/or ave antiherpetic activity. Other acyclonucleoside or acyclonucleotides have antiviral activity whicn is independent of D N A polymerase or viral activation by viral thymidine kinase or both.

K

Acknowledgments The author is very grateful to the many contributors to this work, especially to Dr. Malcolm MacCoss, Dr. Wallace T. Ashton, and Dr. John D. Karkas who helped to shape and guide this work and to Dr. Dennis Underwood who helped to solve the molecular modelling problems. Literature Cited Elion, G. B. Am. J. Med. 1982, 73, 7 (Acyclovir Symposium). Elion, G. B.; Furman, P. Α.; Fyfe, J. Α.; de Miranda, P.; Beauchamp, L.; Schaeffer, H. J. Proc. Natl. Acad. Sci. USA 1977, 74, 5716. 3. Fyfe, J. Α.; Keller, P. M.; Furman, P. Α.; Miller, R. L.; Elion, G. B.J.Biol. Chem. 1978, 253, 8721. 4. Miller, W. H.; Miller, R. L.J.Biol. Chem. 1980, 255, 7204. 5. Miller, W. H.; Miller, R. L. Biochem. Pharmacol. 1982, 31, 3879. 6. Keller, P. M.; Fyfe, J. Α.; Beauchamp, L.; Lubbers, C. M.; Furman, P. Α.; Schaeffer, H. J.; Elion, G. B. Biochem. Pharmacol. 1981, 30, 3071. 7. Schildkraut, I.; Cooper, G. M.; Greer, S. Mol Pharmacol. 1975 11, 153. 8. Cheng, Y.-C.; Dutschman, G.; De Clercq, E.; Jones, A. S.; Rahim, S. G.; Verhelst, G.; Walker, R. T. Mol. Pharmacol. 1981, 20, 230. 9. Sim, I. S.; Goodchild, J.; Meredith, D. M.; Porter, R. Α.; Raper, R. H.; Viney, J.; Wadsworth, H. J. Antimicrob. Ag. Chemother. 1983, 23, 416. 10. Cheng, Y.-C. Ann. Ν. Y. Acad. Sci. 1977, 284, 594. 1. 2.


3.

11. 12.


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TOLMAN


Cheng, Y.-C.; Ostrander, M. J. Biol. Chem. 1976, 251, 2605. Karkas, J. D.; Germershausen, J. G.; Tolman, R. L.; M . MacCoss; Wagner, A . F.; Liou, R.; Bostedor, R. Biochem. Biophys. Acta 1987, 911, 127. Marshall, G.; Trends Biol. Sci. 1988, 236. The MM2-extended force field (MM2-X), developed internally at Merck, shares many parameters with M M 2 ; it differs principally i n that lone pairs on neteroatoms are not used (compensating changes have been made in torsional parameters and charge distributions), and that electrostatic interactions take place between atom-centered charges, thus allowing proper treatment of charged systems: M M 2 - X has been parameterized for a wide ranee of systems. Birnbaum, G . I.; Shugar, D. i n Nucleic A c i d Structure, Part 3, Topics i n Molecular and Structural Biology, ed. S. Neidle, M a c M i l l a n (London, 1987) p. 1. Birnbaum, G . I.; Cygler, M . ; Kusmierek, J. T.; Shugar, D. Biochem. Biophys. Res. Commun. 1981, 103, 968. Birnbaum, G. I.; Cygler, M . ; Shugar, D. Can. J. Chem. 1984, 62, 2646. Birnbaum, G . I.; Johansson, N. G.; Shugar, D. Nucleosides & Nucleotides 1987, 6(4), 775. Stolarski, R.; Lassota, P.; Kazimierczuk, Z.; Shugar, D. Z. Naturforsch 1988, 43c, 231. Beauchamp, L. M . ; Dolmatch, B. L.; Schaeffer, H . J.; Collins, P.; Bauer, D. J.; Keller, P. M . ; Fyfe, J. A . J. Med. Chem. 1985, 28, 982. Ashton, W. T.; Canning, L. F.; Reynolds, G. F.; Tolman, R. L.; Karkas, J. D.; L i o u , R.; Davies, M.-E. M . ; DeWitt, C. M . ; Perry, H . C.; Field, A. K. J. Med. Chem. 1985, 28, 926. Stein, J. M . ; Stoeckler, J. D.; Li, S.-Y.; Tolman, R. L.; MacCoss, M . ; Chen, Α.; Karkas, J. D.; Ashton, W. T.; and Parks, R. E., Jr. Biochem. Pharmacol. 1987, 36, 1237. Tolman, R. L.; Ashton, W. T.; MacCoss, M . ; Karkas, J. D.; Underwood, D.; Meurer, L. C.; Cantone, C. C.; Johnston, D. B. R.; Hannah, J.; Liou, R. J. Med. Chem. 1989, submitted. Martin, J. C.; McGee, D. P.C.; Jeffrey, G. Α.; Hobbs, D. W.; Smee, D. F.; Matthews, T. R.; Verheyden, J. P. H. J. Med. Chem. 1986, 29, 1384. McGee, D. P. C.; Martin, J. C.; Smee, D. F.; Matthews, T. R.; Verheyden, J. P. H . J. Med. Chem. 1985, 28, 1242. Karkas, J. D.; Ashton, W. T.; Canning, L. F.; Liou, R.; Germershausen, J.; Bostedor, R.; Arison, B.; Field, A . K.; Tolman, R. L. J. Med. Chem. 1986, 29, 842. Larsson, Α.; Tao, Pei-zhen Antimicrob. Ag. Chemother. 1984, 25, 524. Ashton, W. T.; Meurer, L. C.; Cantone, C. L.; Field, A . K.; Hannah, J.; Karkas, J. D.; L i o u , R.; Patel, G. F.; Perry, H . C.; Wagner, A . F.; Walton, E.; Tolman, R. L. J. Med. Chem. 1988, 31, 2304. Field, A . K.; Perry, H . C. personal communication. Bradley, M. O.; Sharkey, N. A . Nature 1978, 274, 608. Tolman, R. L.; Field, A. K.; Karkas, J. D.; Wagner, A . F.; Germershausen, J.; Crumpacker, C.; Scolnick, Ε. M. Biochem. Biophys. Res. Comm. 1985, 128, 1329. Field, A. K.; Davies, M.-E.; DeWitt, C. M . ; Perry, H . C.; Schofield, T. L.; Karkas, J. D.; Germershausen, J . ; Wagner, A . F.; Cantone, C. L.; MacCoss, M . ; Tolman, R. L. Antiviral Res. 1986, 6, 329. Prisbe, E. J . ; Martin, J. C.; Baker, M . F.; Smee, D. F.; Duke, A . E.; Matthews, T. R.; Verheyden, J. P. H. J. Med. Chem. 1986, 29, 671. Duke, A . E.; Smee, D. F.; Chernow, M . ; Boehme, R.; Matthews, T. R. Antiviral Res. 1986, 6, 299. Martin; Nucleotide Analogues as Antiviral Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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50 35. 36. 37.


38.

39.


Yang, Ζ. H . ; Lucia, H. L.; Hsiung, G. D.; Tolman, R. L.; Colonno, R. J. Antimicrob. Ag. Chemother. 1989 submitted. Baba, M . ; M o r i , S.; Shigeta, S.; DeClercq, Ε. Antimicrob. Ag. Chemo. 1987, 31, 337. Gordon, Y. J.; Capone, Α.; Sheppard, J.; Gordon, Α.; Romanowski, E.; Araullo-Cruz, Τ Current Eye Res. 1987, 6, 247. Germershausen, J. G.; L i o u , R.; Field, A . K.; Wagner, A . F.; MacCoss, M . ; Tolman, R. L.; Karkas, J. D. Antimiaob. Ag. Chemother. 1986, 29, 1025. Hucker, H . ; Germershausen, J. personal communication.

R E C E I V E D June 9,

1989


Structural Requirements for Enzymatic Activation of Acyclonucleotide

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