Effect of N-tritylated amino-adenosine-3 ’ , 5 ’-cyclic monophosphate The effecT of cerTain N-TriTylaTed phenylalanine conjugaTes of amino-adenosine-3 ’ , 5 ’-cyclic monophosphaTe on moloney murine leukaemia virus reverse TranscripTase acTiviTy

Vol. 106 No. 7/8 Page 1 of 5 ABSTRACT Moloney murine leukaemia virus (M-MuLV) is a member of the retrovirus family. Its cloned reverse transcriptase (RT), similarly to HIV type 1 reverse transcriptase (HIV-1 RT), exhibits DNA-polymerase and ribonuclease H (RNase H) activities capable of converting the single-stranded retroviral RNA genome into double-stranded DNA. The latter is then integrated into the host chromosome during viral infection. M-MuLV RT is, therefore, an attractive enzyme to help understand mutations in HIV1 RT and its use in inhibition studies can help facilitate new drug designs. In this study, conjugates consisting of N-trityl derivatives of p-fluoro, p-nitro and p-iodo-DL-phenylalanine were coupled to 8-(6-aminohexyl) amino-adenosine-3’,5’-cyclic monophosphate and examined for their effect on DNA synthesis by M-MuLV RT. Synthesis was studied in a system containing poly (rA).oligo d(pT)15 as a template-primer with [3H] dTTP. The iodo-derivative, N-trityl-p-iodo-DL-phenylalanine-8-(6aminohexyl) amino-adenosine-3’,5’-cyclic monophosphate was found to be a very active inhibitor of the RT enzyme (IC50 = 1 μM), while the p-nitro (IC50 = 45 μM) and p-fluoro (IC50 = 65 μM) were weak inhibitors. Further work will be aimed at determining the mode of binding of the N-tritylated conjugates and also of various substituted amino acids and short peptides to M-MuLV RT to elucidate the mechanisms of inhibition.


INTRODUCTION
In a recent article' we described the synthesis of a number of A r acylated derivatives of 8(6aminohexyl) aminoadenosine5'monophosphate.These various acylated derivatives, as well as the free \trityl substituted DLphenylalanyl amino acids, were tested for their effect on tlie DNA polymerase activity of the Moloney murine leukaemia virus reverse transcriptase (MMuLV RT).A number of these compounds inhibited the enzyme with IC W values (the concentration at which 50% inhibition of activity occurs) of 60 uM 100 uM.If one particular compound, ( Vtrityl/»ifluoroDLphenylalanine, was attached to the nucleotide 8<6aminohexyl) aminoadenosine5'monophosphate (Figure 1), the inhibitor)' activity increased, giving an IC W value of 5 uM.
MMuLV RT is a cloned enzyme expressed in Esclwrkhia toh as a single polypeptide (71 kDa> : and, similarly to HIV type 1 revcrsetranscriptase(HlVlRT),exhibitsboth DNApolymerase and ribonuclease H (RNase H) activities'; the common properties of MMuLV RT and HIV 1RT suggest that the two enzymes are able to carry out identical biological functions as far as the mechanism of DNA synthesis is concerned.Furthermore, the amino acid sequences of several drugresistant mutations of HIV1 RT have revealed similarities to the native sequences found in the MMul.V RT catalytic fragment,* which makes MMuLV RT an attractive enzyme to help understand mutations in HIV1 RT and its use in inhibition studies can help facilitate new drug designs.
In our previous paper, 1 we made the assumption that the nucleotide component (5'AMP) of Un conjugate A'tritylmfluoroDLphenyIalanine8(6aminohexyl) aminoadenosine5'monophosphate could possibly bind to the polymerase site (area) of MMuLV RT, while the A'tritylmfluoroDL phenylalanine grouping was likely to interact with a hydrophobic pocket or area situated 10 A 15 A away.The binding of the conjugate therefore resembled the interaction of certain chimeras with HIV1 RT.We now realise that the suggested binding of (he A'-lrityl conjugates mentioned above may have been entirely wrong and that other areas or sites on the RT enzyme may possibly have been involved (see Discussion).
It would have been logical to use m-derivatives (wfluoro, m-nitro and m-iodo) of Dl.-phenylalanine in these new conjugates with 3',5'-cyclic-AMP.Our reason for using the jV-tritylatedp-substituted DL-phenylalanine derivatives was that we had adequate amounts of crystalline A'-hydroxysuccinim (deactivated esters of these A'-tritylated-/>-substituted amino acids, while the m-substituted derivatives were not available.
The strongly inhibitory activity of the /»-iodo derivative is interesting; the iodine atom has a large radius (covalent radius = 133 A) and is lipophilic, whilst fluorine and bromine have covalent radii of 0.72 A and 1.14 A, respectively.The large covalent radius of the iodine atom may have had some bearing on the activity of the nucleotide conjugate.
In this study, the compounds synthesised and evaluated as inhibitors of the M-MuLV RT were originally visualised as chimeras.Several other researchers*' 10 "' 11 originally suggested the idea of cross-linking a non-nucleoside compound, specifically a non-nucleoside reverse transcriptase inhibitor (NNRTI), to substrates (i.e. the formation of a chimera).As reported in our previous paper, 1 the design features of the nucleotide-A'-trityl-m-fluoro-DL-phenylalanine appear to be consistent with these ideas.The concept of chimera structures binding to M-MuLV RT was prominent in our thinking when we began to carry out the work reported in the present paper.We now believe thai the evidence for a chimera-type inhibitor and the mechanism we proposed, that is, of conjugates binding to M-MuLV RT, is incorrect, for the reasons outlined below.Binding of A'-trityl-p-fluoro-DL-phenylalanine-8-(6-aminohexyl) amino-adenosine-3',5'-cyclic monophosphate and the p-nitro and p-iodo substituted DL-phenylalanine conjugates to M-MuLV RT 11IV-1 RT is possibly the most extensively studied enzyme and a multitude of studies on its crystal structure have been reported. 011lthough there are distinct structural differences between the monomeric M-MuLV RT and the heterodimeric HIV-1 RT, it is important to keep in mind that the respective polymerase domains of M-MuLV RT and HIV-1 RT are approximately 85% structurally similar.""This similarity allows the use of M-MuLV-RT as a tool in order better to understand the reverse transcription process and the design of novel inhibitors for HIV-1 RT.We do not know how the M-MuLV RT binds NNRTIs, whether there is a hydrophobic pocket present, or whether the inhibitor can induce allosteric changes in the enzyme structure.It is also unlikely that the nucleotide (5'-AMP or 3',5'-cyclic-AMP) component of the conjugate is binding near, or at, the polymerase site, as these are not deoxyribose nucleoside triphosphates but ribosc-containing nucleotides.Also, inhibition, when it occurs, should be non-competitive.When using A-tritylm-fluoro-DL-phenylalanine-8-<6-aminohexyl) amino-adenosine-5'-monophosphate in our original paper, 1 we performed competition experiments using varying concentrations of poly rA, d(pT) ls at fixed concentrations of jV-trityl-m-fluoro-DLphenylalanine-nucleotide, which showed the compound acted as a non-competitive inhibitor (results not published).
It is thus unlikely that the DL-phenyl conjugates would fit into both the polymerase binding area and the nearby allosteric pocket site.Based on our results, we propose that other areas of the M-MuLV RT enzyme are being used for the attachment and functioning of the (V-tritylated conjugates. A number of research groups have reported on the synthesis and biological evaluation of dimers, some of which behave as true chimeras, l *-l *• ,7 • ll,, Velazquez et ■L* M have prepared a number of dimers consisting of 3'-azido-2',3'-dideoxy thymidine (AZT) linked to the non-nucleoside (NNRTI) TSAO or HE PT.These can be written as lAZTMTSAO-T) and [AZT]-|HE PT].They can also be placed in a general formula: lddN]-(CH ; ) p -lNNRTIJ, where n -3-9.
The TSAO-T molecule does not bind in the hydrophobic pocket of the HIV-1 RT, but rather interacts with certain amino acids at the interface of the p5l and p66 subunits.Velazquez et al." believe that the (AZT)-[TSAO-T) dimer works only through the NNRTI portion of the molecule and not through the AZT (ddN) segment.It should be noted that TSAO is also a ribose derivative.The |AZT|-(CII : ) n -lTSAO-T) structure is shown in Figure 3.
A recent paper by Tomassi et al.* deals with the synthesis of new TSAO-T derivatives, particularly TSAO-BocT.The introduction of a Boc grouping to the \-3 position of the thymidine ring creates a good inhibitor of HIV-1 RT activity.Besides details of the synthesis and biological evaluation of this inhibitor, they report on computational analysis regarding the binding site of the enzyme.*Our further work will be aimed at determining the mode of binding of the /V-tritylated conjugates and also of various substituted amino acids and short peptides to M-MuLV RT, in order to resolve the abovementioned uncertainties.