The ability of HIV to establish long-lived latent infection is mainly due to transcriptional silencing of viral genome in resting memory T lymphocytes. Here, we show that new semi-synthetic ingenol esters reactivate latent HIV reservoirs. Amongst the tested compounds, 3-caproyl-ingenol (ING B) was more potent in reactivating latent HIV than known activators such as SAHA, ingenol 3,20-dibenzoate, TNF-α, PMA and HMBA. ING B activated PKC isoforms followed by NF-κB nuclear translocation. As virus reactivation is dependent on intact NF-κB binding sites in the LTR promoter region ING B, we have shown that. ING B was able to reactivate virus transcription in primary HIV-infected resting cells up to 12 fold and up to 25 fold in combination with SAHA. Additionally, ING B promoted up-regulation of P-TEFb subunits CDK9/Cyclin T1. The role of ING B on promoting both transcription initiation and elongation makes this compound a strong candidate for an anti-HIV latency drug combined with suppressive HAART.

The s.yn + anti equilibrium conformation about the glycosidic bond of purine nucleosides and 5'-nucleotides in different solvent systems has been investigated by means of 'H NMR spectroscopy. Quantitative values for the conformer populations were improved, relative to previous results, by a detailed study of, and a resultant derived correction for, the influence of the sugar exocyclic group carbons were employed to evaluate approximately the glycosidic angles x of the nucleosides in the conformations s,yn and anti. A critical examination is made of the applicability of relaxation methods, involving analysis of spin-lattice relaxation time of protons ( T I ) and the Overhauser effect, to determine the conformation of the base about the glycosidic bond; interpretations are provided for the lack of agreement between these methods and those based on chemical shifts in the present study. The foregoing results are also applied to an examination of the effect of the conformation of the base about the glycosidic bond on the enzymatic reactions catalyzed by 5'-nucleotidase and adenosine deaminase.We have previously described a procedure [l] for evaluation of the syn-anti conformer populations in nucleosides from the chemical shifts of the sugar protons, in particular of H-2'. Under conditions where there is a rapid exchange, on the N M R time-scale, between these two conformations, the observed chemical shift of H-2' is the mean of those for the fixed conformations syn and anti: dsVn and d o n t i , fiobs = Psyn d s y n + Punri d u n t i

BuPdGTP , the 2'-deoxyribonucleoside 5'-triphosphate of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n- butylphenyl )guanine, was examined with respect to its mechanism and its capacity to inhibit the mammalian DNA polymerases, pol alpha, pol beta, and pol gamma. BuP dGTP was specifically inhibitory for pol alpha, with no discernible activity on pol beta and pol gamma. The potency of BuP dGTP is unprecedented, with an apparent Ki less than 10 nanomolar. The unusual potency of the BuP dGTP is derived primarily from the 5' alpha and beta phosphoryl moieties, whose binding to enzyme complements that of the base-linked butylphenyl substituent. BuP dGTP is competitive with dGTP and apparently not subject to polymerization. Experiments employing BuP dGTP in the presence of a non-complementary template suggest that the core polymerase or an associated coprotein contains dNTP binding sites which recognize specific nucleic acid bases. The partial sensitivity of selected, non-mammalian DNA polymerases suggests that modification of the N2 substituent of dGTP will be a useful route to the design of novel, polymerase-specific affinity-probes.

N2-(p-n-Butylphenyl)-2'-deoxyguanosine (BuPdG) and its 5'-triphosphate (BuPdGTP), expected to be inhibitors of eukaryotic DNA polymerase alpha, have been synthesized. BuPdG was synthesized by two methods and characterized by 1H NMR and by chemical relation to guanosine. Direct synthesis involving silylated N2-(p-n-butylphenyl)guanine (BuPG) and 1-chloro-3,5-di-p-toluoyl-2-deoxyribofuranose in the presence of trimethylsilyl trifluoromethanesulfonate gave one alpha and two beta isomers of deoxyribonucleoside as determined by 1H NMR. However, NMR and UV spectra were equivocal in distinguishing between 7 and 9 isomers. The identity of the desired 9-beta-BuPdG was ultimately proved by its independent synthesis from the corresponding ribonucleoside. 1H NMR spectra of the O'-acetylated ribonucleosides of BuPG showed characteristic patterns of O'-acetylated guanosines, and their identity was proved by relating the products of the reaction of isomeric O'-acetylated 2-bromoinosines with p-n-butylaniline and with ammonia: the 2-bromoinosine which gave guanosine also gave the suspected 9-beta-ribonucleoside, BuPGr, and that which gave N7-beta-ribofuranosylguanine also gave the 7-beta isomer of BuPGr. BuPGr was transformed in a multistep procedure to give BuPdG, identical with the major beta isomer obtained by direct deoxynucleoside synthesis. The 5'-monophosphate of BuPdG was obtained by treatment of the nucleoside with phosphoryl chloride in trimethyl phosphate; the monophosphate reacted as the phosphoimidazolyl derivative with pyrophosphate to yield the 5'-triphosphate, BuPdGTP.(ABSTRACT TRUNCATED AT 250 WORDS)