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In clarifying the antiviral role of PAA it is helpful to compare the roles and properties of normal mammalian cellular DNA polymerases and the herpesvirus- induced enzyme. The cellular polymerases are necessary for cell division and DNA repair, while the virus-induced enzyme is required forvDNA replication.

Of the three biophysically and biochemically distinct cellular DNA polymerases, a, B and y (see Table 1.3.6.1), the herpesvirus induced enzyme is most similar to DNA polymerase a. Both enzymes are able to copy efficiently, double stranded DNase "activated" DNA as well as the synthetic primer-template (dG)^^^-

(dc)n (Wagner, et al., 1974; Weissbach, et al., 1973).

The enzymes can only be separated by phosphocellulose chromatography (Weissbach, et al., 1973) . The virally induced enzymes are most active at high ionic strengths, at which host cell polymerases are totally inactive

(Weissbach, et al. , 1973). DNA polymerase a is

inhibited by PAA but at concentrations up to 100 times higher than required to inhibit the herpesvirus-induced enzymes (Bolden, et al. , 1975; Miller and Rapp, 1976; Sabourin, et al. , 1978) , whereas DNA polymerase B and y are relatively unaffected by high concentrations of PAA.

PAA resistant mutants of herpesvirus strains have been isolated and characterised (Honess and

unequivocally that PAA inhibits herpesvirus replication by direct interaction with and inactivation of the viral DNA polymerase.

1.3.6.2 Mechanism of Action and Structure-Activity Relationships

Independent studies by Helgstrand, et al. (1978) and Reno, et a l . (1978) led to the evaluation of phosphonoformic acid (PFA) (2) (Fig. 1.3.6.2) as an antiviral compound, which was found to inhibit herpesvirus DNA polymerase and herpesvirus replication in a manner similar to PAA.

h o' o h (2) 0 * ? . O R , C P , RgO' OR2 (3)

Fig. 1.3.6.2 Phosphonoformic acid (2) and esters (3) .

The activity of PFA revealed that separation of the phosphono and carboxyl group negative charges is not mandatory for antiherpes activity, and

subsequently, despite the structure of PAA providing little opportunity for chemical modification to optimise activity, a number of analogues have been synthesised and tested on isolated viral enzymes in cell culture and animal models (Table 1.3.6.2).

A n a lo g A c tiv ity R efe re n ce I. C a r b o x y l e s te r s E th y l; p r o p y l; r-b u ty l Y es (eth y l) He k k in e t a l. (1 9 7 7 ) C y c lo h e x y l; o c ty l; b en z y l Y es (o cty l) He r r in et al. (1 9 7 7 ) II. M o n o p h o s p h a te e s te rs M e th y l; p r o p y l; h exyl N o He r r in e t al. (1 9 7 7 ) III. T r ie s te r s T r im e th y l N o Lee e t al. (1976) T r ie th y l N o Sh ie k o w it z e t a l. (1973) IV. M o n o p h o s p h i n ic a c id s P h e n y l; 4 - m e t h o x y p h e n y l; m e th y l N o He r r in e t al. (1977) V. P h o s p h o n o a n a lo g s S u lf o a c e ta te N o Le in b a c h e t a l. (1976) M a lo n a te N o Le in b a c h e t a l. (1976) A r s e n o a c e ta te Y es Ne w t o n (1979) V I. C a r b o x y l a n a lo g s P h o s p h o n o a c e ta ld e h y d e N o Bo e z i (1979) P h o s p h o n o a c e ta m id e N o Bo e z i (1979) /V - M e th y lp h o s p h o n o a c e ta m id e N o Bo e z i (1979) /V -P ro p y l-; N - b u ty l- ; A /-cy cIo h e x y l-; Y es v o n Es c h (1 9 7 8 ) • /V - a m a n ty lp h o s p h o n o a c e ta m id e A c e to n y l p h o s p h a te N o Bo e z i (1979) A m in o m e th y lp h o s p h o n a te N o Lee e t al. (1976)

cr-A m ino e th y l p h o s p h o n a te N o Lee e t al. (1976) N -{ p h o s p h o n o a c e ty i) - L - a s p a r ta te N o Bo e z i (1979) M e th y le n e d i p h o s p h o n a t e N o Lee et al. (1976) M e th y le n e d ia r s o n a te Y es Ne w t o n (1 9 7 9 ) A r s o n o m e th y l p h o s p h o n a te Y es Ne w t o n (1979) V II. M e th y le n e a n a lo g s P h o s p h o n o f o r m a te Y es Re n o e t al. (1 9 7 8 ) P h o s p h o n o p r o p i o n a te N o S m f K o w r r z e t al. (1973) P h o s p h o n o b u t y r a te N o Sh i i-k o w it z e t al. (1973) a - P h o s p h o n o p r o p io n a t e N o Le in b a c h e t a l. (1976) a - M e t h y l - 2 - p h o s p h o n o p r o p i o n a tc N o Le in b a c h e t a l. (1976) a - P h e n y l p h o s p h o n o a c e ta te N o Le in b a c h e t a l. (1976) c r - A m in o p h o s p h o n o a c e ta tc N o Le in b a c h e t a l. (1976) V III. O th e r r e la te d c o m p o u n d s P h o s p h o g ly c o la te N o Le in b a c h e t a l. (1976) I m i d o d ip h o s p h o n a tc N o Bo e z i (1979) C a r b a m y l p h o s p h a te N o Bo e z i (1979) 2 '- D e o x y r ib o th y m id in c - 5 - N o Bo e z i (1979) p h o s p h o r o p h o s p h o n o a c c ta tc P u r in e - 5 '- m o n o - c a r b o x y m e th y l- Y es Heim er a n d Nu s sb a u m (1977) p h o s p h o n a te P y r im id in e - 5 - m o n o - c a r b o x y m e th y l- Y es Heim er a n d Nu s sb a u m (1977) p h o s p h o n a te

Table 1.3.6.2 PAA analogues and their antiherpes activity. (From Overby, 1982)

Comoound HSV-1 DN A-pol H S V -2 DNA-pol HCMV DNA-pol Influenza R N A -pol Cellular DNA-pol<* HO OH > 5 0 0 > 5 0 0 >5 0 0 4 5 0 > 5 0 0 HO ? V ?OH HO H OH > 5 0 0 - > 5 0 0 > 5 0 0 > 5 0 0 HO OH 1 0 2 7 6 2 8 0 ₁₀₀ HO ? ^ °>0H 'P - C - P . HO OH > 5 0 0 > 5 0 0 _— 10 3 5 0 HO.? JJ _{> - c - c}J > HO ^ OH 0-5 0-7 0 -4 3 0 0 3 5 HO ? Ç hl O > C-C HO )!< OH 1 5 5 5 0 1 8 > 5 0 0 > 5 0 0 H O .? 5 > P -c -c EtO h "OH > 5 0 0 > 5 0 0 > 5 0 0 > 5 0 0 > 5 0 0 HO ? > ~ Ç ~ C. HO H OEt 5 0 - - - > 5 0 0 H O .? V /,° HO A 'N H , > 5 0 0 > 5 0 0 — > 5 0 0 > 5 0 0 HO ? ? * 0 > s - C - C HO ^ sOH — 2 70 - — - H O .? ¡J 0^ S S C'O H > 5 0 0 — — — > 5 0 0 H° . t c " ° MO' ' OH 0-3 0-5 0- 3 3 0 4 0 HOv? ?^.OH H O ' ^ O H 1 5 0 1 2 1 5 0 > 5 0 0 2 5 0 V c ' ° MO "OH 1 1 5 4 0 0 1 3 0 » 5 0 0 > 5 0 0

Structures of some pyrophosphate analogues and their reported inhibitory properties.

Substitution of the phosphono group by carboxyl and sulphono groups results in loss of activity against the DNA polymerases induced by the herpesvirus of turkeys (Leinbach, et at., 1976). Replacement of the phosphono and/or carboxyl group by arseno groups results in comparable activities for methylenebisarsonate and arsenoacetic acid in plaque reduction assays with HSV-1 (Newton, 1979) but no activity is observed in vivo (Mao, et at., 1985). Substitution of the carboxyl group by moieties other than arseno- or phosphono-generally abolishes antiviral activity, with the exception of some amide derivatives

(Mao, et at. , 1985). Replacement of the carboxyl group of PAA by a phosphono-group yields methylenebisphosphonate, which, like pyrophosphate, is devoid of significant

antiherpes activity (Eriksson, et at. , 1980) , carbonylbisphosphonate however, shows significant inhibition of HSV-1 DNA polymerase (Eriksson, et at., 1980).

Substitution of the methylene group of PAA with various alkyl or aryl groups resulted in a reduction in activity against the polymerase enzyme and reduced inhibition of HSV-1 plaque formation. In general esters have proved to be inactive relative to PAA and PFA, including aliphatic and aromatic

mono-, di- and triesters of PFA (Norén, et at. , 1983) . None showed inhibition of HSV-1 DNA polymerase in vitro. Effective antiherpes activity was, however, observed

for several compounds, probably due to hydrolysis by non-specific esterases. More recently, Mao, et al.

(1985) reported that small alkyl esters such as methyl, ethyl and propyl, only slightly decrease in activity against the HSV-induced DNA polymerase, suggesting that, contrary to earlier reports, an ionisable carboxylic acid is not an absolute

requirement for activity. Compounds of this type, and bridge substituted PAA, would be expected to traverse the cell membrane with more ease than the unesterified compound, none were more active than the parent compounds however.

1.3.6.3 Mechanism of Action of PAA

PAA inhibits herpesvirus DNA polymerase by interacting with the proposed pyrophosphate binding site at the enzyme active site (Leinbach, et al., 1976; Mao and Robishaw, 1975). Non-competitive inhibition is exhibited by both PAA and pyrophosphate against the virus-induced enzyme with respect to the four deoxyrubo- nucleoside triphosphates and non-competitive inhibition with respect to DNA at low deoxyribonucleoside

triphosphate concentrations but almost uncompetitive inhibition at high concentrations.

Inhibition studies with pyrophosphate gave apparent inhibition constants in the mM range, whereas for PAA values of 1-2 yM were obtained. PAA is a

competitive inhibitor of pyrophosphate in the pyrophosphate- deoxyribonucleoside 5'-triphosphate exchange reaction

and in multiple inhibition analyses pyrophosphate and PAA function as mutually exclusive inhibitors and hence bind at the same site. Additional evidence for the shared binding site of these

compounds comes from the observation that PAA resistant mutants of HSV are also more resistant to PFA and

pyrophosphate (Eriksson and Oberg, 1979; Hay and Subak-Sharpe, 1976; Reno, et al. , 1978).

An alternate product mechanism has been used to describe the inhibition patterns observed with PAA

(Leinbach, et al. , 1976). Pyrophosphate is predicted to act as a substrate for the reverse reaction of the polymerisation, i.e., pyrophosphate-deoxyribonucleoside 5'-triphosphate exchange. However, nucleotides

containing [2-^H]PAA have not been recovered from DNA polymerisation reactions containing [2-^H]PAA (Cload, 1983) and, furthermore, nucleotide analogues such as dAMP-PAA and dTMP-PAA are neither substrates nor inhibitors of HSV DNA polymerase (Cload, 1983; Boezi, 1979) .

Further insight into the mechanism of action of pyrophosphate analogues has come from a consideration of their metal-binding abilities. DNA and RNA

polymerases are zinc-requiring enzymes (Mildvan and Leob, 1979) as is reverse-transcriptase (Poesz, et a l . , 1974) which is inhibited by PFA (Sundquist and Oberg, 1979). The stability constants for some metal complexes of PAA and other pyrophosphate analogues have been

determined (Stünzi and Perrin, 1979; Cload and Hutchinson, 1983) and both PAA and PFA found to form strong complexes with zinc in particular. It has been suggested that these compounds are active by virtue of their ability to chelate with an essential metal ion in the herpes DNA polymerase (Perrin and Stünzi, 1981) .

As shown by the spectrum of activity of PAA and PFA (Table 1.3.6.3(i), as well as inhibiting

herpesvirus DNA polymerase and the reverse transcriptases, PFA in particular is also an effective inhibitor of

influenza virus RNA polymerase activity (Helgstrand, et al. , 1978; Stridh, et al. , 1979) .

I n h ib itio n o f p o ly m e r a s e s b y P F A a n d P A A E n z y m e C o n c e n tr a ti o n g iv in g 5 0 Vo in h ib itio n , p M P F A P A A R N A p o l y m e r a s e s I n f lu e n z a v ir u s (M g f+) 20 300 I n f lu e n z a v ir u s (M n ,+) 0.3 0.9 vsv 500 _ R eo v iru s > 5 0 0 _ C a lf th y m u s I > 5 0 0 > 5 0 0 C a lf th y m u s II > 5 0 0 > 5 0 0 E . c o l i > 5 0 0 > 5 0 0 R e v e r s e tr a n s c r i p t a s e s A M V 7 4,000 R M u L V 0.7 600 D N A p o l y m e r a s e s H SV -1 ( s tr a in C 42) 0.4 0.5 H SV -1 ( s tr a in 124) 3.5 7.0 H e p a titis B v iru s 20 > 5 0 0 C a lf th y m u s a (25 U /a s s a y ) 50 75 C a lf th y m u s a ( 2.5 U /a s s a y ) 3.5 6.5 C a lf liv e r y _{> 5 0 0 > 5 0 0} M i c r o c o c c u s l u t e u s > 5 0 0 > 5 0 0 E . c o l i > 5 0 0 > 5 0 0

Table 1.3.6.3(i) Inhibition of polymerases by PAA and PFA.

The RNA polymerase activity of influenza virus is known to be zinc requiring (Oxford and

Perrin, 1977) and subsequent studies on the inhibition of influenza RNA transcriptase systems has provided the best evidence supporting the hypothesis that

pyrophosphate analogues inhibit these enzyme activities by complexing with an essential zinc ion at the active site of the enzyme and thus inhibiting, either by preventing the binding of incoming nucleoside triphos­ phates or preventing the release of inorganic pyrophos­ phate once the internucleotide bond has been formed by the enzyme.

Recent studies on influenza virus have shown that PFA does not inhibit the initiation of mRNA

formation, and in the presence of PFA the primer of about thirteen bases can be elongated with about twelve bases, but further elongation is inhibited

(Stridh and Datema, 1984). It was suggested that after addition of the conserved nucleotides, changes in the viral core proteins may result in an increased sensitivity to pyrophosphate analogues by, for example, allowing zinc chelation, resulting in enzyme inhibition. Alternatively, a different protein such as PA may be

involved in the synthesis of the message-specific sequences and only this enzyme may be PFA sensitive.

A good correlation between the pK^i (-log^g dissociation constant of zinc ion-pyrophosphate analogue complex)) of pyrophosphate analogues, as

determined by gel filtration, and their effectiveness as inhibitors of the RNA transcriptase activity of influenza A has been obtained (Cload and Hutchinson, 1983). PFA and PAA have relatively high pKd , values and are inhibitors of the enzyme (Table 1.3.6.3(H) and Fig. 1.3.6.3(D) whereas compounds such as

phosphonopropionic acid (PPA) and esters of PAA have lower pK^i values and are ineffective inhibitors.

TABLE

INHIBITORY EFFECT OF PYROPHOSPHATE ANALOGUES ON RNA POLYMERASE FROM INFLUENZA VIRUS AND

CALF THYMUS DNA POLYMERASE a

Compound PKd .*

Concn. (uM) producing 50% inhibition 'flu RNA polymerase DNA polymer­ ase a (1) r c h2c o o h 5.5 275 45 (2) RCOOH 5.6 35 35 (3) r c h2c h2c o o h < 4 > 500 > 500 (4) RCHMeCOOH *v 5 > 500 > 500 (5) r c o n h2 < 4 > 500 > 500 (6) (EtO)2P(0)CH2COOH < 4 > 500 > 500 (7) ROR 5.7 125 > 500 (8) RNHR 5.7 50 > 500 (9) r c h2r 5.3 > 500 > 500 (10) RCHO.R > 6 85 > 500 (11) r c ç i2r > 6 75 > 500 (12) RCBr2R > 6 10 350 (13) RCOR 5.4 20 100 whore R - (H0)2P(0)

•Dissociâtion constant of complex formed with zinc ions, measured at pH 8.0 as described in text.

Table 1.3.6.3(ii) Inhibitory effect of pyrophosphate analogues on RNA transcriptase from influenza virus and calf thymus DNA polymerase a. (Cload and Hutchinson, 1983)

Fig. 1.3.6.3(i ) Relationship between pK^i (pH 8) and effectiveness as inhibitors of the RNA transcriptase activity of

influenza A. (Taken from Cload and Hutchinson, 1983.)

The steric factors determining the type of chelate ring formed are likely to be of importance, with PFA forming the most stable 5-membered ring. PAA and PPA would form 6- and 7-membered rings

respectively (Fig. 1.3.6.3(ii)) and show progressively less antiviral activity. Particularly active

inhibitors were the halogenated methylenebisphosphonates, dibromomethylenebisphosphonate being the most active

-1---c * ° ° ^ o ? V

1

A

J

Fig. 1.3.6.3(H) Metal chelates of (1) PFA, (2) PAA, and (3) PPA.

However, with the exception of carbonyl- bisphosphonate, these compounds showed no antiherpes activity despite forming very stable zinc ion complexes. It appears therefore that much more stringent structural requirements exist for inhibition of HSV DNA polymerase than for influenza RNA transcriptase, which is

presumably due to differences in the pyrophosphate binding sites of the two enzymes.

1.3.6.4 Clinical Evaluation