Synthesis of an Epoxide Derivative of Thymidine as a Potential Enzyme Inhibitor

Abstract

Attempts to prepare I -[7,8-anhydro-2,5,6-trideoxy-α-L-lyxo-(and β-n-ribo)-octofuranosyl]thymine (10) by treatment of halohydrins 6–9 with sodium hydride in DMF or sodium methoxide in methanol gave mixtures of the epoxides 10 or 11 and the 3′,8′-anhydronucleoside 12. The structure of 12 was confirmed by oxidation to the cyclic ketone 14. The successful synthesis of 10 involved a Wittig reaction on the thymidine-5′-aldehyde 16 to give the unsaturated ketoacetate 18 which was reduced in two steps to the diacetate 20. The 7′-O-tosyl derivative 21 upon treatment with sodium methoxide in chloroform gave the pure epoxide 10 which was marginally toxic to L1210 cells in culture (I₅₀=25 μM) and demonstrated borderline in vivo activity (24% ILS) against P388 murine leukemia.     

Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg²⁺ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.Selective pressure exerted by antiretroviral drugs, in conjunction with high viral mutation rates, promotes the inevitable emergence of drug-resistant HIV-1 variants. This necessitates an ongoing search for novel antiretroviral compounds that either have novel mechanisms and inhibit different stages of viral replication or inhibit targets that have acquired resistance to existing drugs. In the latter case, such newer next-generation agents should ideally display resistance profiles which are distinct and nonoverlapping with those of the first-generation drugs.Integration of viral cDNA into the host cell genome is a distinct feature of retroviral replication, and inhibitors of HIV-1 integrase have recently been added to the arsenal of clinically approved antiretroviral drugs. Raltegravir (RAL) was the first integrase strand transfer inhibitor (INSTI) to be approved by the U.S. Food and Drug Administration (FDA) after clinical trials showed that this drug promoted a rapid and sustained antiviral effect (13). Elvitegravir (EVG), another integrase inhibitor, is currently in phase III clinical trials (27). Resistance mutations common to both of these first-generation integrase inhibitors have been reported and can result in high levels of drug resistance (26). Mutations which engender cross-resistance between RAL and EVG have been reported in clinical trials, cell culture studies, and biochemical assays (9, 26). This has prompted the search for second-generation integrase inhibitors that might display novel patterns of resistance, allowing their use in patients who have failed therapy with RAL or EVG. MK-2048 (28) is a prototype second-generation INSTI that retains potency against viruses containing common single and double mutations observed in the clinic with first-generation agents with a 95% inhibitory concentration (IC₉₅) in the nM range. MK-2048 has been previously reported to be active against viruses resistant to RAL and EVG (28, 29). Given common mechanisms of action among INSTIs and a lack of structural information on integrase inhibitor complexes with resistance mutations, an understanding of resistance to second-generation agents such as MK-2048 is important.This article describes the selection of resistance to MK-2048 in tissue culture and the characterization of mutations associated with such resistance, G118R and E138K. The identification of distinct mutations which appear to confer resistance to MK-2048 and not to either RAL or EVG has potential implications for understanding the structural basis for the second-generation profile of this compound as well as future drug discovery and development efforts focused on this mechanism.     

Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC₅₀ as low as 8.4 μm and a TC₅₀ of >100 μm when tested against HIV-1_Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC₅₀ as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.     

Synthesis of Novel Mannoside Glycolipid Conjugates for Inhibition of HIV-1 Trans-Infection

Mannose-binding lectins, such as dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), are expressed at the surface of human dendritic cells (DCs) that capture and transmit human immunodeficiency virus type-1 (HIV-1) to CD4(+) cells. With the goal of reducing viral trans-infection by targeting DC-SIGN, we have designed a new class of mannoside glycolipid conjugates. We report the synthesis of amphiphiles composed of a mannose head, a hydrophilic linker essential for solubility in aqueous media, and a lipid chain of variable length. These conjugates presented unusual properties based on a cooperation between the mannoside head and the lipid chain, which enhanced the affinity and decreased the need for multivalency. With an optimal lipid length, they exhibited strong binding affinity for DC-SIGN (K(d) in the micromolar range) as assessed by surface plasmon resonance. The most active molecules were branched trimannoside conjugates, able to inhibit the interaction of the HIV-1 envelope with DCs, and to drastically reduce trans-infection of HIV-1 mediated by DCs (IC(50s) in the low micromolar range). This new class of compounds may be of potential use for prevention of HIV-1 dissemination, and also of infection by other DC-SIGN-binding human pathogens.     

Virtual Screening of Indonesian Herbal Database as HIV-1 Protease Inhibitor

HIV-1 (Human immunodeficiency virus type 1)׳s infection is considered as one of most harmful disease known by human, the survivability rate of the host reduced significantly when it developed into AIDS. HIV drug resistance is one of the main problems of its treatment and several drug designs have been done to find new leads compound as the cure. In this study, in silico virtual screening approach was used to find lead molecules from the library or database of natural compounds as HIV-1 protease inhibitor. Virtual screening against Indonesian Herbal Database with AutoDock was performed on HIV-1 protease. From the virtual screening, top ten compounds obtained were 8-Hydroxyapigenin 8-(2",4"-disulfatoglucuronide), Isoscutellarein 4''-methyl ether, Amaranthin, Torvanol A, Ursonic acid, 5-Carboxypyranocyanidin 3-O-(6"-O-malonyl-beta-glucopyranoside), Oleoside, Jacoumaric acid, Platanic acid and 5-Carboxypyranocyanidin 3-O-beta-glucopyranoside.     

Novel furocoumarins as potential HIV-1 integrase inhibitors

A series of seven novel, rationally designed N-substituted 3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propanamides have been prepared as potential HIV-1 integrase (IN) inhibitors via a five-step pathway commencing with resorcinol and diethyl 2-acetylglutarate, and the HIV-1 IN inhibition potential of these compounds has been examined relative to raltegravir, a known HIV-1 IN inhibitor.     

Novel piperidinylpyrimidine derivatives as inhibitors of HIV-1 LTR activation

Piperidinylpyrimidine derivatives, previously prepared as inhibitors of TNF-alpha production, were evaluated for their inhibitory activity against HIV-1 LTR activation. Some of these derivatives inhibited activation of HIV-1 LTR-directed CAT gene expression induced by PMA in Jurkat cells. In this report, we describe SAR in this series of compounds and show that the 3,4-methylenedioxybenzoyl (piperonyloyl) group on the nitrogen of piperidine and lipophilic substitution at the C(6)-position of pyrimidine are important for this inhibitory activity. Some of the synthesized compounds also inhibited HIV-1 LTR transactivation induced by viral protein Tat. These results suggest that piperidinylpyrimidines are useful as potent AIDS therapeutics that directly inhibit HIV-1 LTR activation and indirectly suppress TNF-alpha production.     

RANTES衍生物与HIV-1进入抑制剂

HIV-1-1进入抑制剂的研究是近年来艾滋病药物研发领域的新热点,其中最受关注的是以CCR5为靶点的新药研发。CCR5是病毒进入细胞的主要辅助受体,在HIV-1进入宿主细胞过程中起着非常重要的作用。作为CCR5的天然配体,CC类的趋化因子RANTES、MIP-1α和MIP-1β都是极具潜力的HIV-1抑制剂,特别是有关对RANTES的定向设计的研究尤为引人关注,其目的是设计出一种既有很强的抗病毒能力而又不引发炎症反应的HIV-1拮抗剂。就RANTES衍生物应用于抑制HIV进入细胞方面的研究进行了综述。     

Solid Phase Synthesis of a Hydroxypyrrolidine Derivative and its Use in Solid Phase Peptide Synthesis as Constrained Statine Mimic

As part of our efforts to design constrained peptide mimics and introduce them in peptide sequences, we set up the synthesis of racemic N-Fmoc protected hydroxypyrrolidine by reduction of the corresponding oxopyrroline. Hydroxypyrrolidines are synthesized using amino acid building block and β-ketoester via a 4-steps solid supported route on Wang resin beads. The hydroxypyrrolidine template can be seen as a constrained mimic of statine. As proof of concept, the pseudopeptide JMV 2776, incorporating this new statine mimic has been synthesized. We replaced the phenyl statine building block in the sequence of known BACE 1/2 inhibitors by 5-benzyl 2-methyl 4-hydroxypyrrolidine, using conventional Fmoc SPPS on Rink amide PS resin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Use of Sodium Diethyldithiocarbamate as a Respiratory Inhibitor

A technique for the application of dieca (diethyldithiocarbamate)to plant tissues is described. It is shown to differentiatealmost quantitatively between extracted ascorbic oxidase andpolyphenolase on the one hand and cytochrome oxidase on theother at a concentration of M/5,000. It produces strong, butnot total, inhibitions when applied to barley root-tips.     

RNAi as a treatment for HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) was the first primate virus shown to be inhibited by RNA interference (RNAi). Early studies used both synthetic and promoter expressed small interfering RNAs (siRNAs) or expressed short hairpin RNAs (shRNAs) to demonstrate that this virus was susceptible to RNAi. In addition to targeting the virus itself RNAi-mediated down-regulation of cellular targets that encode receptors required for viral entry also proved to be effective. The power of RNAi as an anti-HIV agent has propelled development of RNAi-based gene therapy approaches for the treatment of HIV infection in humans. Nevertheless, extensive in vitro experimentation has revealed potential problems of viral escape mutants and other toxicities caused by the si/shRNAs. This review covers the progress and problems in the development of RNAi for the treatment of HIV infection. Potential modalities for clinical application of RNAi in the treatment of HIV-1 infection are also described.     

Novel Bent Conformation of CD4 Induced by HIV-1 Inhibitor Indirectly Prevents Productive Viral Attachment

GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to determine the binding site of the adnectin on CD4 and to understand the mechanism of inhibition. Using hydrogen–deuterium exchange with mass spectrometry (HDX), CD4 peptides showed differential rates of deuteration (either enhanced or slowed) in the presence of the adnectin that mapped predominantly to the interface of domains 2 and 3 (D2-D3). In addition, an X-ray crystal structure of an ibalizumab Fab/CD4(D1-D4)/adnectin complex revealed an extensive interface between the adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/GSK3732394 complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the adnectin. In addition, amino acid L151 was shown to be a critical indirect determinant of the specificity for binding to the human CD4 protein over related primate CD4 molecules, as it appears to modulate CD4′s flexibility to adopt the adnectin-bound conformation. The significant conformational change of CD4 upon adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface, thereby re-orienting the gp120 binding site in a direction that is inaccessible to incoming virus due to a steric clash between gp160 trimers on the virus surface and the target cell membrane.     

Novel 1,2,3-thiadiazole derivatives as HIV-1 NNRTIs with improved potency: Synthesis and preliminary SAR studies

A novel synthetic route and anti-HIV activity evaluation of a new series of 2-(4-(2,4-dibromophenyl)-1,2,3-thiadiazol-5-ylthio)acetamide (TTA) derivatives are described. Bioactivity assay indicated that most of the title compounds showed good activities against HIV-1. In particular, compound 7c displayed the most potent anti-HIV-1 activity (EC₅₀ = 36.4 nM), inhibiting HIV-1 replication in MT-4 cells more effectively than NVP (by sevenfold) and DLV (by eightfold). The preliminary structure–activity relationships (SAR) of the newly synthesized congeners are discussed, and molecular modeling of compound 7c in complex with HIV-1 RT is described, allowing rationalization of some SAR conclusions.     

Synthesis of betulinic acid derivatives as entry inhibitors against HIV-1 and bevirimat-resistant HIV-1 variants

Betulinic acid derivatives modified at the C28 position are HIV-1entry inhibitors such as compound A43D; however, modified at the C3 position instead of C28 give HIV-1 maturation inhibitor such as bevirimat. Bevirimat exhibited promising pharmacokinetic profiles in clinical trials, but its effectiveness was compromised by the high baseline drug resistance of HIV-1 variants with polymorphism in the putative drug binding site. In an effort to determine whether the viruses with bevirimat resistant polymorphism also altered their sensitivities to the betulinic acid derivatives that inhibit HIV-1 entry, a series of new betulinic acid entry inhibitors were synthesized and tested for their activities against HIV-1 NL4-3 and NL4-3 variants resistant to bevirimat. The results show that the bevirimat resistant viruses were approximately 5- to10-fold more sensitive to three new glutamine ester derivatives (13, 15 and 38) and A43D in an HIV-1 multi-cycle replication assay. In contrast, the wild type NL4-3 and the bevirimat resistant variants were equally sensitive to the HIV-1 RT inhibitor AZT. In addition, these three new compounds markedly improved microsomal stability compared to A43D.     

Potential Elucidation of a Novel CTL Epitope in HIV-1 Protease by the Protease Inhibitor Resistance Mutation L90M

The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89–97 and PR 90–99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.     

2-Nitro-5-Thiosulfobenzoic Acid as a Novel Inhibitor Specific for Deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (bpDNase I) contains four cysteine residues forming two disulfide bonds. Though there are no free sulfhydryl groups, incubation of bpDNase I with 2-nitro-5-thiosulfobenzoic acid (NTSB) in the presence of Ca(2+) or Mg(2+) at pH 7.5 results in inactivation of the enzyme. Amino acid analysis shows that NTSB-treated bpDNase I still contains all 4 half-cystine residues. The only amino acid residues having reduced values are threonine and serine, indicating that these may be the reaction sites for NTSB. Plasmid scission assay and circular dichroism analysis reveal the structural integrity of the inactivated enzyme. Treatment of bpDNase I with NTSB does not result in fragmentation, as demonstrated by SDS-PAGE analysis. NTSB binds bpDNase I through covalent modification, since dialysis and gel filtration can not reverse the inactivation reaction. However, after dilution into an acid buffer of pH 4.7, the inactivated enzyme regains about 40% of its initial activity, suggesting a reversible inactivation by acid treatment. NTSB does not inactivate DNase II, ribonuclease, chymotrypsin and lysozyme, while it effectively inactivates rat parotid DNase I. These results strongly suggest that NTSB can be considered as a novel inhibitor specific for DNase I.