New in silico and conventional in vitro approaches to advance HIV drug discovery and design.

INTRODUCTION: Recently, the new concept of the long-range intermolecular interactions in biological systems has been proposed. Combined use of molecular modeling techniques and the screening techniques based on the long-range interaction concept could significantly improve and accelerate discovery of new HIV drugs. However, any hit identified in silico needs to be characterized with respect to its biological target by enzymatic studies. Combined use of the in silico screening and the enzymatic studies allows an efficient selection of new anti-HIV drugs. AREAS COVERED: The focus of this article is on the in silico screening of molecular libraries for candidate new HIV drugs, which is based on the molecular descriptors determining the long-range interaction between the drugs and their therapeutic target. This article also reviews the techniques for enzyme kinetic studies which are required for optimization of in silico selected candidate anti-HIV drugs. EXPERT OPINION: The novel approach of combining in silico screening techniques with enzymatic studies enables the accurate measurement of the quantitative descriptors of ligand-enzyme interactions. This novel method is a powerful tool for new anti-HIV drug discovery which can also reduce the drug development costs.

N2-Phenyl-9-(hydroxyalkyl)guanines and related compounds are substrates for Herpes simplex virus thymidine kinases.

Herpes simplex virus (HSV) types 1 and 2 thymidine kinases (TK) are responsible for phosphorylation of antiherpes acyclonucleosides such as acyclovir (ACV) and 9-(4-hydroxybutyl)guanine (HBG). Related compounds, the N2-phenyl-9-(hydroxyalkyl)guanines, are devoid of direct antiviral activity, but potently inhibit the viral TKs and block viral reactivation from latency in vivo. The similarity in structure between the acyclonucleosides and TK inhibitors raised the question of the relevance of phosphorylation of certain of the latter analogs in their mechanisms of action. Using recombinant TKs and HPLC analysis of reaction mixtures, we report that the lead TK inhibitor N2-phenyl-9 -(4-hydroxybutyl)guanine (HBPG) and its pentyl homolog (HPnPG) are excellent substrates for the enzymes, approaching the efficiency with which the natural substrate thymidine is phosphorylated, and significantly better than ACV or HBG. Other 9-hydroxyalkyl congeners are substrates for the enzymes, but with much poorer efficiency. HBPG triphosphate was a poor inhibitor of HSV DNA polymerase, the target of acyclonucleoside triphosphates, suggesting that phosphorylation of HBPG is not important in its mechanism of blocking viral reactivation in vivo. The fact that HBPG is an efficient substrate is consistent, however, with its binding mode based both on molecular modeling studies and x-ray structure of the HBPG:TK complex.

Selective targeting of the HIV-1 reverse transcriptase catalytic complex through interaction with the "primer grip" region by pyrrolobenzoxazepinone non-nucleoside inhibitors correlates with increased activity towards drug-resistant mutants.

PBO (pyrrolobenzoxazepinone) derivatives are non-nucleoside reverse transcriptase inhibitors (NNRTIs), which display a selective interaction with the catalytic ternary complex of HIV-1 reverse transcriptase (RT) and its substrates. In order to develop novel PBOs with improved resistance profiles, we synthesised additional PBO derivatives, specifically designed to target highly conserved residues in the beta12-beta13 hairpin, the so-called "primer grip" region of HIV-1 RT. Here, we investigated the biochemical and enzymological mechanism of inhibition of HIV-1 RT wild type and carrying NNRTIs-resistance mutations, by these derivatives. Our kinetic analysis indicates that the ability of PBOs to selectively target the catalytic ternary complex of RT with its substrates directly correlates with greatly reduced sensitivity to NNRTIs-resistance mutations, particularly the K103N substitution. Molecular modeling and docking studies provided an explanation for this correlation at the structural level.

Sensitivity of monkey B virus (Cercopithecine herpesvirus 1) to antiviral drugs: role of thymidine kinase in antiviral activities of substrate analogs and acyclonucleosides.

Herpes B virus (B virus [BV]) is a macaque herpesvirus that is occasionally transmitted to humans where it can cause rapidly ascending encephalitis that is often fatal. To understand the low susceptibility of BV to the acyclonucleosides, we have cloned, expressed, and characterized the BV thymidine kinase (TK), an enzyme that is expected to "activate" nucleoside analogs. This enzyme is similar in sequence and properties to the TK of herpes simplex virus (HSV), i.e., it has a broad substrate range and low enantioselectivity and is sensitive to inhibitors of HSV TKs. The BV enzyme phosphorylates some modified nucleosides and acyclonucleosides and l enantiomers of thymidine and related antiherpetic analogs. However, the potent anti-HSV drugs acyclovir (ACV), ganciclovir (GCV), and 5-bromovinyldeoxyuridine were poorly or not phosphorylated by the BV enzyme under the experimental conditions. The antiviral activities of a number of marketed antiherpes drugs and experimental compounds were compared against BV strains and, for comparison, HSV type 1 (HSV-1) in Vero cell cultures. For most compounds tested, BV was found to be about as sensitive as HSV-1 was. However, BV was less sensitive to ACV and GCV than HSV-1 was. The abilities of thymidine analogs and acyclonucleosides to inhibit replication of BV in Vero cell culture were not always proportional to their substrate properties for BV TK. Our studies characterize BV TK for the first time and suggest new lead compounds, e.g., 5-ethyldeoxyuridine and pencyclovir, which may be superior to ACV or GCV as treatment for this emerging infectious disease.

Human DEAD-box ATPase DDX3 shows a relaxed nucleoside substrate specificity.

Human DDX3 (hDDX3) is a DEAD-box protein shown to possess RNA-unwinding and adenosine triphosphatase (ATPase) activities. The hDDX3 protein has been implicated in nuclear mRNA export, cell growth control, and cancer progression. In addition, a role of this protein in the replication of human immunodeficiency virus Type 1 and in the pathogenesis of hepatitis C virus has been recently proposed. Its enzymological properties, however, are largely unknown. In this work, we characterized its ATPase activity. We show that hDDX3 ATPase activity is stimulated by various ribo- and deoxynucleic acids. Comparative analysis with different nucleoside triphosphate analogs showed that the hDDX3 ATPase couples high catalytic efficiency to a rather relaxed substrate specificity, both in terms of base selection and sugar selection. In addition, its ability to recognize the L-stereoisomers of both 3' deoxy- and 2',3' dideoxy-ribose, points to a relaxed stereoselectivity. On the basis of these results, we hypothesize the presence of structural determinants on both the base and the sugar moieties, critical for nucleoside binding to the enzyme. Our results expand the knowledge about the DEAD-box RNA helicases in general and can be used for rational design of selective inhibitors of hDDX3, to be tested as potential antitumor and antiviral agents.

Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases beta and lambda.

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol lambda; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol lambda wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol beta in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.

APOBEC deaminases as cellular antiviral factors: a novel natural host defense mechanism.

The APOBEC (acronym for apolipoprotein B editing catalytic polypeptide) family of cytidine deaminases are widely distributed in the biological world and play a central role in diverse enzymatic pathways. Members of this family (APOBEC3G and APOBEC3F) have been recently shown to be able to restrict HIV-1 replication in physiologically relevant target cells (macrophages, lymphocytes), presumably by triggering extensive deamination of the viral RNA/DNA replication intermediates. This natural antiretroviral host defense mechanism is counteracted by the HIV-1 protein Vif, which is able to target APOBECs to degrade. The so-called "Vif/APOBEC3G paradigm" has been confirmed by a growing literature. However, evidence arising from recent studies has expanded this view, showing that the replication of other viruses is also restricted by APOBEC family members and suggesting antiviral mechanism(s) of action unrelated to the catalytic activity of these proteins. Furthermore, evolutionary investigations on primates have shown that APOBEC3 gene expansion might be related to an ancient adaptive selection to prevent endogenous genetic instability, indicating an additional ancient protective role of APOBECs. This article is aimed at broadening the current knowledge about the antiviral activity of the APOBEC members and to highlight the notion that their role(s) might be more general than previously anticipated.

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda.

DNA polymerase lambda (pol lambda) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol lambda either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol lambda had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn++ and Mg++ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol lambda; (iii) pol lambda preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol lambda, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol lambda and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol lambda are discussed.

Specific targeting highly conserved residues in the HIV-1 reverse transcriptase primer grip region. Design, synthesis, and biological evaluation of novel, potent, and broad spectrum NNRTIs with antiviral activity.

Pyrrolobenzoxazepinones (PBOs) represent a new class of human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 5. Molecular modeling studies based on the X-ray structures of HIV-1 RT prompted the synthesis of novel analogues which were tested as anti-HIV agents. The PBO derivatives specifically designed to target the highly conserved amino acid residues within the beta12-beta13 hairpin, namely primer grip, proved to be very potent against the most common mutant enzymes, including the highly resistant K103N mutant strain. Structure-activity relationships (SARs) are discussed in terms of a possible interaction with the RT binding site, depending on the nature of the substituents at C-6. Among the pyrrolobenzoxazepines investigated, 15c appeared to be the most promising NNRTI of the series characterized by potent antiviral activity, broad spectrum, and low cytotoxicity. 15c showed synergistic antiviral activity with AZT.

High potency of indolyl aryl sulfone nonnucleoside inhibitors towards drug-resistant human immunodeficiency virus type 1 reverse transcriptase mutants is due to selective targeting of different mechanistic forms of the enzyme.

Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation.

Incorporation of non-nucleoside triphosphate analogues opposite to an abasic site by human DNA polymerases beta and lambda.

A novel class of non-nucleoside triphosphate analogues, bearing hydrophobic groups sterically similar to nucleosides linked to the alpha-phosphate but lacking the chemical functional groups of nucleic acids, were tested against six different DNA polymerases (polymerases). Human polymerases alpha, beta and lambda, and Saccharomyces cerevisiae polymerase IV, were inhibited with different potencies by these analogues. On the contrary, Escherichia coli polymerase I and HIV-1 reverse transcriptase were not. Polymerase beta incorporated these derivatives in a strictly Mn++-dependent manner. On the other hand, polymerase lambda could incorporate some alkyltriphosphate derivatives with both Mg++ and Mn++, but only opposite to an abasic site on the template strand. The active site mutant polymerase lambda Y505A showed an increased ability to incorporate the analogues. These results show for the first time that neither the base nor the sugar moieties of nucleotides are required for incorporation by family X DNA polymerases.

Specific targeting of hepatitis C virus NS3 RNA helicase. Discovery of the potent and selective competitive nucleotide-mimicking inhibitor QU663.

Hepatitis C virus (HCV) infection is an emerging global epidemic, and no effective cure is yet available. Interferon-alpha (INFalpha) and pegylated INFs, in combination or otherwise with ribavirin, have proven to be effective in no more than 50% of chronically infected patients. New and better therapeutic strategies are therefore needed. HCV nonstructural protein 3 (NS3) RNA helicase (h) is a promising target for developing new therapeutics. QU663 was discovered as a potent new selective inhibitor of the helicase reaction of HCV NS3 (K(i) = 0.75 microM), competing with the nucleic acid substrate without affecting ATPase function, even at high concentrations. QU663 is one of a new generation of small-molecule nucleotide-mimicking inhibitors which are potential anti-HCV agents. A thorough molecular modeling study was carried out to explain the molecular basis of NS3h inhibition by QU663. The resulting three-dimensional interaction model is discussed.

Inhibition of herpes simplex virus thymidine kinases by 2-phenylamino-6-oxopurines and related compounds: structure-activity relationships and antiherpetic activity in vivo.

Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogues with bulky tertiary amino substituents were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and 0.005 microM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.

Diketo hexenoic acid derivatives are novel selective non-nucleoside inhibitors of mammalian terminal deoxynucleotidyl transferases, with potent cytotoxic effect against leukemic cells.

Mammalian terminal deoxyribonucleotidyl transferase (TDT) catalyzes the non-template-directed polymerization of deoxyribonucleoside triphosphates and has a key role in V(D)J recombination during lymphocyte and repertoire development. More than 90% of leukemic cells in acute lymphocytic leukemia and approximately 30% of leukemic cells in the chronic myelogenous leukemia crisis show elevated TDT activity. This finding is connected to a poor prognosis and response to chemotherapy and reduced survival time. On the other hand, recent data indicated that TDT is not the only terminal deoxyribonucleotidyl transferase in mammalian cells. Its close relative, DNA polymerase lambda, can synthesize DNA both in a template-dependent (polymerase) and template-independent (terminal deoxyribonucleotidyl transferase) fashion. DNA polymerase lambda might be involved in the nonhomologous end-joining recombinational repair pathway of DNA double-strand breaks. In this work, we report the characterization of the mechanism of action of three diketo hexenoic acid (DKHA) derivatives, which proved to be extremely selective for the terminal deoxyribonucleotidyl transferase activity of DNA polymerase lambda and TDT. They seem to be the first non-nucleoside-specific inhibitors of mammalian terminal transferases reported. Moreover, the DKHA analog 6-(1-phenylmethyl-1H-indol-3-yl)-2,4-dioxo-5-hexenoic acid (RDS2119) was not toxic toward HeLa cells (CC(50) > 100 muM), whereas it showed significant cytotoxicity against the TDT(+) leukemia cell line MOLT-4 (CC(50) = 14.9 muM), thus having the potential to be further developed as a novel antitumor agent.

Inhibition of mammalian DNA polymerases by resveratrol: mechanism and structural determinants.

Resveratrol, a natural compound found in many dietary plants and in red wine, plays an important role in the prevention of many human pathological processes, including inflammation, atherosclerosis and carcinogenesis. We have shown that the antiproliferative activity of resveratrol correlated with its ability to inhibit the replicative pols (DNA polymerases) alpha and delta in vitro [Stivala, Savio, Carafoli, Perucca, Bianchi, Maga, Forti, Pagnoni, Albini, Prosperi and Vannini (2001) J. Biol. Chem. 276, 22586-22594]. In this paper, we present the first detailed biochemical investigation on the mechanism of action of resveratrol towards mammalian pols. Our results suggest that specific structural determinants of the resveratrol molecule are responsible for selective inhibition of different mammalian pols, such as the family B pol alpha and the family X pol lambda. Moreover, the resveratrol derivative trans-3,5-dimethoxy-4-hydroxystilbene, which is endowed with a strong antiproliferative activity (Stivala et al., 2001), can inhibit pols alpha and lambda and also suppress the in vitro SV40 DNA replication. The potency of inhibition is similar to that of aphidicolin, an inhibitor of the three replicative pols alpha, delta and epsilon. Our findings establish the necessary background for the synthesis of resveratrol derivatives having more selective and potent antiproliferative activity.

DNA elongation by the human DNA polymerase lambda polymerase and terminal transferase activities are differentially coordinated by proliferating cell nuclear antigen and replication protein A.

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.

Drug resistance mutations in the nucleotide binding pocket of human immunodeficiency virus type 1 reverse transcriptase differentially affect the phosphorolysis-dependent primer unblocking activity in the presence of stavudine and zidovudine and its inhibition by efavirenz.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) derivatives with D113E, Y115F, F116Y, Q151E/N, and M184V mutations were studied for their phosphorolysis-mediated resistance to the nucleoside RT inhibitors (NRTIs) zidovudine and stavudine and for their inhibition by the nonnucleoside analogs (NNRTIs) efavirenz and nevirapine. The results presented here indicate that these single amino acid substitutions within the nucleotide binding pocket of the viral RT can independently affect different enzymatic properties, such as catalytic efficiency, drug binding, and phosphorolytic activity. Moreover, small local alterations of the physicochemical properties of the microenvironment around the active site can have profound effects on some NRTIs while hardly affecting other ones. In conclusion, even though different mutations within the nucleotide binding pocket of HIV-1 RT can result in a common phenotype (i.e., drug resistance), the molecular mechanisms underlying this phenotype can be very different. Moreover, the same mutation can give rise to different phenotypes depending on the nature of the substrates and/or inhibitors.

Microwave-assisted synthesis and biological evaluation of novel uracil derivatives inhibiting human thymidine phosphorylase.

New 5-chloro-6-substituted-uracil derivatives have been prepared by microwave assisted-synthesis and tested in vitro as thymidine phosphorylase inhibitors. One of these compounds showed potent inhibitory activity, with an IC50 value in the submicromolar range. The biological activity of the new compounds is discussed in terms of structure-activity relationship.