Four novel bis-(naphtho-gamma-pyrones) isolated from Fusarium species as inhibitors of HIV-1 integrase

Integration of viral DNA into host cell DNA is an essential step in retroviral (HIV-1) replication and is catalyzed by HIV-1 integrase. HIV-1 integrase is a novel therapeutic target and is the focus of efforts to identify effective inhibitors that will prevent/or cure HIV infections. Four novel naphtho-gamma-pyrones, belonging to the chaetochromin and ustilaginoidin family, were discovered as inhibitors of HIV-1 integrase from the screening of fungal extracts using a recombinant in vitro assay. These compounds inhibit both the coupled and strand transfer activity of HIV-1 integrase with IC(50) values of 1-3 and 4-12 microM, respectively. The discovery, structure elucidation, chemical modification and the structure-activity relationship of these compounds are described.     

Stability-activity relationships of a family of G-tetrad forming oligonucleotides as potent HIV inhibitors. A basis for anti-HIV drug design

Recently, we have demonstrated that T30695, a G-tetrad-forming oligonucleotide, is a potent inhibitor of human immunodeficiency virus, type I (HIV-1) integrase and the K(+)-induced loop folding of T30695 plays a key role in the inhibition of HIV-1 integrase (Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). Here we have modified T30695 by introducing a hydrophobic bulky group, propynyl dU, or a positively charged group, 5-amino dU, into the bases of T residues of the loops, and by substitution of the T-G loops by T-T loops. Physical measurements have demonstrated that the substitution of propynyl dU or 5-amino dU for T in the T residues of the loops did not alter the structure of T30695, and these derivatives also formed an intramolecular G-quartet structure, which is an essential requirement for anti-HIV activity. Measured IC(50) and EC(50) values show that these substitutions did not induce an apparent decrease in the ability to inhibit HIV-1 integrase activity and in the inhibition of HIV-1 replication in cell culture. However, the substitution of T-T loops for T-G loops induced a substantial decrease in both thermal stability and anti-HIV activity. The data analysis of T30695 and the 21 derivatives shows a significant, functional correlation between thermal stability of the G-tetrad structure and the capacity to inhibit HIV-1 integrase activity and between thermal stability of the G-tetrad structure and the capacity to inhibit HIV-1 replication, as assessed with the virus strains HIV-1 RF, IIIB, and MN in cell culture. This relationship between thermostability and activity provides a basis for improving the efficacy of these compounds to inhibit HIV-1 integrase activity and HIV-1 replication in cell culture.     

HIV-1整合酶四聚体结构模拟及其活性位点分析

HIV-1复制需要HIV-1整合酶将其环状DNA整合进宿主DNA中,这其中包括2个重要反应,即“3′-加工”和“链转移”,两者均由HIV-1整合酶催化完成.阻断其中的任一反应,都能达到抑制HIV-1复制的目的.因此,了解HIV-1整合酶的完整结构和聚合状态,对深入探讨其作用机理及设计新型抑制剂具有重要的指导作用.然而,迄今为止仅有HIV-1整合酶单独结构域的晶体结构可供参考,而其全酶晶体结构尚未获得解析.本研究利用分子模拟技术,通过蛋白质 蛋白质/DNA分子对接、动力学模拟等方法,构建了全长整合酶四聚体的结构模型、HIV-1 DNA与整合酶复合物的结构模型,进一步从理论上证实HIV-1整合酶是以四聚体形态发挥催化作用,明确“3′-加工”和“链转移”在HIV-1整合酶上的催化位点.同时,通过与作用机理相似的细菌转座子Tn5转座酶等的结构比对,推测HIV-1整合酶的核心结构域中应有第2个Mg^２＋存在,其位置螯合于Asp64与Glu152之间.在HIV-1整合酶结构研究的基础上,有望进一步设计出新的抗艾滋病药物.     

Integrase of Mason-Pfizer monkey virus

The gene encoding an integrase of Mason-Pfizer monkey virus (M-PMV) is located at the 3'-end of the pol open reading frame. The M-PMV integrase has not been previously isolated and characterized. We have now cloned, expressed, isolated, and characterized M-PMV integrase and compared its activities and primary structure with those of HIV-1 and other retroviral integrases. M-PMV integrase prefers untranslated 3'-region-derived long-terminal repeat sequences in both the 3'-processing and the strand transfer activity assays. While the 3'-processing reaction catalyzed by M-PMV integrase was significantly increased in the presence of Mn(2+) and Co(2+) and was readily detectable in the presence of Mg(2+) and Ni(2+) cations, the strand transfer activity was strictly dependent only on Mn(2+). M-PMV integrase displays more relaxed substrate specificity than HIV-1 integrase, catalyzing the cleavage and the strand transfer of M-PMV and HIV-1 long-terminal repeat-derived substrates with similar efficiency. The structure-based sequence alignment of M-PMV, HIV-1, SIV, and ASV integrases predicted critical amino acids and motifs of M-PMV integrase for metal binding, interaction with nucleic acids, dimerization, protein structure maintenance and function, as well as for binding of human immunodeficiency virus type 1 and Rous avian sarcoma virus integrase inhibitors 5-CI-TEP, DHPTPB and Y-3.     

Design and synthesis of novel nitrogen-containing polyhydroxylated aromatics as HIV-1 integrase inhibitors from caffeic acid phenethyl ester

A series of nitrogen-containing polyhydroxylated aromatics from caffeic acid phenethyl ester were designed and synthesized as HIV-1 integrase inhibitors. Most of these compounds exhibited potent inhibitory activities at micromolar concentrations against HIV-1 integrase in the 3′-end processing and the strand transfer. Their key structure–activity relationship was also discussed.     

Amide-containing diketoacids as HIV-1 integrase inhibitors: Synthesis,structure–activity relationship analysis,and biological activity

HIV-1 integrase, which catalyzes the integration of the viral genome into the cellular chromosome, is an essential enzyme for retroviral replication, and represents an attractive and validated target in the development of therapeutics against AIDS. In this paper, 17 amide-containing novel diketoacids were designed and synthesized, and their ability to inhibit HIV-1 integrase was tested. The structure–activity relationships were also analyzed.     

Chemical and enzymatic modifications of integric acid and HIV-1 integrase inhibitory activity

Integric acid (1), an acyl eremophilane sesquiterpenoid, was identified as an inhibitor of HIV-1 integrase, the enzyme responsible for provirus entry into the host cell nucleus and integration in to the host genome. Chemical and enzymatic modification of integric acid led to the preparation of several selective chemical derivatives of integric acid. Preparation, HIV-1 inhibitory activity, and the structure-activity relationship against coupled and strand transfer assays are described. It appears that most of the groups present in the natural product are required for inhibition of HIV-1 integrase strand transfer activity. In contrast, inhibition of 3' processing activity is less stringent suggesting distinct SAR for the two integrase reactions.     

Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates.

The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is required for integration of viral DNA into the host cell chromosome. In vitro, integrase mediates a concerted cleavage-ligation reaction (strand transfer) that results in covalent attachment of viral DNA to target DNA. With a substrate that mimics the strand transfer product, integrase carries out disintegration, the reverse of the strand transfer reaction, resolving this integration intermediate into its viral and target DNA parts. We used a set of disintegration substrates to study the catalytic mechanism of HIV-1 integrase and the interaction between the protein and the viral and target DNA sequence. One substrate termed dumbbell consists of a single oligonucleotide that can fold to form a structure that mimics the integration intermediate. Kinetic analysis using the dumbbell substrate showed that integrase turned over, establishing that HIV-1 integrase is an enzyme. Analysis of the disintegration activity on the dumbbell substrate and its derivatives showed that both the viral and target DNA parts of the molecule were required for integrase recognition. Integrase recognized target DNA asymmetrically: the target DNA upstream of the viral DNA joining site played a much more important role than the downstream target DNA in protein-DNA interaction. The site of transesterification was determined by both the DNA sequence of the viral DNA end and the structure of the branched substrate. Using a series of disintegration substrates with various base modifications, we found that integrase had relaxed structural specificity for the hydroxyl group used in transesterification and could tolerate distortion of the double-helical structure of these DNA substrates.     

LEDGF dominant interference proteins demonstrate prenuclear exposure of HIV-1 integrase and synergize with LEDGF depletion to destroy viral infectivity

Target cell overexpression of the integrase binding domain (IBD) of LEDGF/p75 (LEDGF) inhibits HIV-1 replication. The mechanism and protein structure requirements for this dominant interference are unclear. More generally, how and when HIV-1 uncoating occurs postentry is poorly defined, and it is unknown whether integrase within the evolving viral core becomes accessible to cellular proteins prior to nuclear entry. We used LEDGF dominant interference to address the latter question while characterizing determinants of IBD antiviral activity. Fusions of green fluorescent protein (GFP) with multiple C-terminal segments of LEDGF inhibited HIV-1 replication substantially, but minimal chimeras of either polarity (GFP-IBD or IBD-GFP) were most effective. Combining GFP-IBD expression with LEDGF depletion was profoundly antiviral. CD4(+) T cell lines were rendered virtually uninfectable, with single-cycle HIV-1 infectivity reduced 4 logs and high-input (multiplicity of infection = 5.0) replication completely blocked. We restricted GFP-IBD to specific intracellular locations and found that antiviral activity was preserved when the protein was confined to the cytoplasm or directed to the nuclear envelope. The life cycle block triggered by the cytoplasm-restricted protein manifested after nuclear entry, at the level of integration. We conclude that integrase within the viral core becomes accessible to host cell protein interaction in the cytoplasm. LEDGF dominant interference and depletion impair HIV-1 integration at distinct postentry stages. GFP-IBD may trigger premature or improper integrase oligomerization.     

Cellular co-factors of HIV-1 integration

To achieve productive infection, the reverse transcribed cDNA of human immunodeficiency virus type 1 (HIV-1) is inserted in the host cell genome. The main protein responsible for this reaction is the viral integrase. However, studies indicate that the virus is assisted by cellular proteins, or co-factors, to achieve integration into the infected cell. The barrier-to-autointegration factor (BAF) might prevent autointegration. Its ability to bridge DNA and the finding that the nuclear lamina-associated polypeptide-2alpha interacts with BAF suggest a role in nuclear structure organization. Integrase interactor 1 was found to directly interact with HIV-1 integrase and to activate its DNA-joining activity, and the high mobility group chromosomal protein A1 might approximate both long terminal repeat (LTR) ends and facilitate integrase binding by unwinding the LTR termini. Furthermore, the lens-epithelium-derived growth factor (LEDGF; also known as p75) seems to tether HIV-1 integrase to the chromosomes. Although a direct role in integration has only been demonstrated for LEDGF/p75, to date, each validated cellular co-factor for HIV-1 integration could constitute a promising new target for antiviral therapy.     

Inhibition of HIV-1 integrase dimerization and activity with crosslinked interfacial peptides

Alternative modes of inhibition for the design of anti-HIV therapies are sought due to the resistance of HIV to a number of the currently approved drugs. A non-active site strategy for generating potent inhibitors of HIV-1 integrase is described based on blocking protein association. Peptides α5 and α6 derived from the HIV-1 integrase dimeric interface have previously demonstrated efficacious dimerization inhibition of HIV-1 integrase. Due to the proximity of the termini of these peptides within the integrase structure, a focused library of tethered agents was designed based on crosslinking the peptides α5 and α6 to mimic a larger interfacial region. The best crosslinked inhibitors are approximately five-fold more potent against HIV-1 integrase than the individual peptides alone or in combination. The most active agents have an inhibitory constant in the mid-nM range and function via a dissociative mechanism of inhibition.     

Regiocontrolled synthesis and HIV inhibitory activity of unsymmetrical binaphthoquinone and trimeric naphthoquinone derivatives of conocurvone

Unsymmetrical biquinone and trimeric quinone derivatives were synthesized using halotriflate-biselectrophilic naphthoquinones through stepwise regioselective quinone substitution chemistry and evaluated for their ability to inhibit the cytopathogenic effects of HIV-1 using an MTT colorimetric assay. Compounds were also screened for their ability to inhibit the activity of HIV-1 integrase in vitro. Pyranylated trimeric quinones and biquinones exhibited both antiviral activity and integrase inhibitory activity. Conocurvone 1 and trimeric quinone 21 were the most potent HIV integrase inhibitors in the series. All of the biquinones showed HIV inhibitory activity. Simple methoxy substituted biquinones did not inhibit HIV-1 integrase.     

De novo design and synthesis of HIV-1 integrase inhibitors

Existing AIDS therapies are out of reach for most HIV-infected people in developing countries and, where available, they are limited by their toxicity and their cost. New anti-HIV agents are needed urgently to combat emerging viral resistance and reduce the side effects associated with currently available drugs. Toward this end, LeapFrog, a de novo drug design program was used to design novel, potent, and selective inhibitors of HIV-1 integrase. The designed compounds were synthesized and tested for in vitro inhibition of HIV-1 integrase. Out of the 25 compounds that were designed, and synthesized, four molecules (compounds 23, 26, 43, and 59) showed moderate to low inhibition of HIV-1 integrase for 3'-processing and 3'-strand transfer activities. Nonetheless, these compounds possess structural features not seen in known HIV-1 integrase inhibitors and thus can serve as excellent leads for further optimization of anti-HIV-1 integrase activity.     

HIV-1整合酶链转移反应抑制剂的荧光筛选方法

整合酶被认为是抗HIV-1药物研究的理想靶点之一。为了建立便捷高效的整合酶链转移反应抑制剂筛选方法,首先将HIV-1整合酶原核表达载体pNL-IN转化入大肠杆菌感受态细胞BL21(DE3)进行原核表达,并用镍琼脂糖凝胶进行亲和纯化,获得了纯度和活性均较高的整合酶重组蛋白;然后设计了生物素标记的供体DNA和FITC标记的靶DNA,用链霉亲和素磁珠捕获反应体系中的DNA产物;最后用荧光分析仪检测DNA产物的荧光信号,并计算待测样品的抑制率。用已知整合酶抑制剂S-1360和MK-0518对筛选方法进行了验证,测定结果与已有实验数据相当,表明本筛选方法能够有效应用于HIV-1整合酶链转移反应抑制剂的筛选。与现有的整合酶链转移反应抑制剂筛选方法相比,本筛选方法步骤更为简化、耗时更短、成本更低。     

Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein.

Retroviral integrase, an essential enzyme for replication of human immunodeficiency virus type-1 (HIV-1) and other retroviruses, contains three structurally distinct domains, an N-terminal domain, the catalytic core and a C-terminal domain. To elucidate their spatial arrangement, we have solved the structure of a fragment of HIV-1 integrase comprising the N-terminal and catalytic core domains. This structure reveals a dimer interface between the N-terminal domains different from that observed for the isolated domain. It also complements the previously determined structure of the C-terminal two domains of HIV-1 integrase; superposition of the conserved catalytic core of the two structures results in a plausible full-length integrase dimer. Furthermore, an integrase tetramer formed by crystal lattice contacts bears structural resemblance to a related bacterial transposase, Tn5, and exhibits positively charged channels suitable for DNA binding.     

HIV-1 integrase complexes with DNA dissociate in the presence of short oligonucleotides conjugated to acridine

The human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus and is therefore an attractive target for the development of new antiviral drugs. Among them, inhibitors which are capable of targeting the preassembled integrase/DNA complex are of particular interest, because they could suppress integrase activity in the context of the HIV-1 preintegration complex. Here, we study the mechanism of action of 11-mer oligonucleotides, which are efficient inhibitors of the catalytic activity of integrase, provided that they are conjugated to a hydrophobic compound, acridine. To understand the mechanism of the conjugate inhibitory action, we used a steady-state fluorescence anisotropy assay, which allowed us to study the stability of the integrase/DNA complex in various conditions. We found that oligonucleotide-acridine conjugates induced the efficient dissociation of preassembled integrase/DNA complexes. The simultaneous presence of both acridine and an oligonucleotidic moiety is required for the inhibitory activity of conjugates. However, the dissociation effect is not dependent on the oligonucleotide sequence. Finally, our results suggest that the conjugates bind directly to integrase within its complex with DNA at a site different from the viral DNA binding site.     

Interaction of HIV-1 integrase with DNA repair protein hRad18

We have previously shown that human immunodeficiency virus-1 (HIV-1) integrase is an unstable protein and a substrate for the N-end rule degradation pathway. This degradation pathway shares its ubiquitin-conjugating enzyme, Rad6, with the post-replication/translesion DNA repair pathway. Because DNA repair is thought to play an essential role in HIV-1 integration, we investigated whether other molecules of this DNA repair pathway could interact with integrase. We observed that co-expression of human Rad18 induced the accumulation of an otherwise unstable form of HIV-1 integrase. This accumulation occurred even though hRAD18 possesses a RING finger domain, a structure that is generally associated with E3 ubiquitin ligase function and protein degradation. Evidence for an interaction between integrase and hRad18 was obtained through reciprocal co-immunoprecipitation. Moreover we found that a 162-residue region of hRad18 (amino acids 65-226) was sufficient for both integrase stabilization and interaction. Finally, we observed that HIV-1 integrase co-localized with hRad18 in nuclear structures in a subpopulation of co-transfected cells. Taken together, these findings identify hRad18 as a novel interacting partner of HIV-1 integrase and suggest a role for post-replication/translesion DNA repair in the retroviral integration process.