Carbonyl J Derivatives: A New Class of HIV-1 Integrase Inhibitors

Integration of a DNA copy of the HIV-1 genome is required for viral replication and pathogenicity, and this highly specific molecular process is mediated by the virus-encoded integrase protein. The requirement for integration, combined with the lack of a known analogous process in mammalian cells, makes integrase an attractive target for therapeutic inhibitors of HIV-1 replication. While many reports of HIV-1 IN inhibitors exist, no such compounds have yet emerged to treat HIV-1 infection. As such, new classes of integrase inhibitors are needed. We have combined molecular modeling and combinatorial chemistry to identify and develop a new class of HIV-1 integrase inhibitors, the Carbonyl J [N,N'-bis(2-(5-hydroxy-7-naphthalenesulfonic acid)urea] derivatives. This new class includes a number of compounds with sub-micromolar IC(50) values for inhibiting purified HIV-1 integrase in vitro. Herein we describe the chemical characteristics that are important for integrase inhibition and cell toxicity within the Carbonyl J derivatives. Copyright 2000 Academic Press.     

Inhibition of HIV-1 virion production by a transdominant mutant of integrase interactor 1

Integase interactor 1 (INI1), also known as hSNF5, is a protein that interacts with HIV-1 integrase. We report here that a cytoplasmically localized fragment of INI1 (S6; aa183-294) containing the minimal integrase-interaction domain potently inhibits HIV-1 particle production and replication. Mutations in S6 or integrase that disrupt integrase-INI1 interaction abrogated the inhibitory effect. An integrase-deficient HIV-1 transcomplemented with integrase fused to Vpr was not affected by S6. INI1 was specifically incorporated into virions and was required for efficient HIV-1 particle production. These results indicate that INI1 is required for late events in the viral life cycle, and that ectopic expression of S6 inhibits HIV-1 replication in a transdominant manner via its specific interaction with integrase within the context of Gag-Pol, providing a novel strategy to control HIV-1 replication.     

Overexpression of the lens epithelium-derived growth factor/p75 integrase binding domain inhibits human immunodeficiency virus replication

We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.     

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.     

Simian virus 40-based replication of catalytically inactive human immunodeficiency virus type 1 integrase mutants in nonpermissive T cells and monocyte-derived macrophages

Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the nef position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.     

8-Hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors

A series of potent novel 8-hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 12 is active against replication of HIV-1 in cell culture with a CIC(95) of 0.31microM. Further SAR exploration led to the preparation of pseudosymmetrical tricyclic pyrrolopyrazine inhibitors 23 and 24 with further improvement in antiviral activity.     

HIV-1整合酶及其抑制剂的研究进展

HIV-1整合酶是由HIV病毒pol基因编码的分子量为32KD的蛋白质,是HIV病毒复制的必需酶之一,它催化病毒DNA整合入宿主染色体DNA。人类细胞中没有HIV 整合酶的类似物[1],理论上抑制整合酶对人体副作用很小。因此HIV-1整合酶成为继HIV-1蛋白酶,逆转录酶后治疗艾滋病的富有吸引力和合理的靶标。本文综述了HIV整合酶结构,抑制剂的研究以及以HIV-1 整和酶为靶点治疗AIDS方法的最新研究进展。     

Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.     

Genetic analysis of the human immunodeficiency virus type 1 integrase protein.

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.     

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.     

Design and synthesis of substituted 4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides, novel HIV-1 integrase inhibitors

A series of 4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-2-carboxamides was synthesized and tested for their inhibition of HIV-1 integrase catalytic activity and HIV-1 replication in cells. Structure-activity studies around lead compound 5 indicated that a coplanar relationship of metal-binding heteroatoms provides optimal binding to the integrase active site. Identification of potency-enhancing substituents and adjustments in lipophilicity provided 17b which inhibits integrase-catalyzed strand transfer with an IC(50) value of 74 nM and inhibits HIV-1 replication in cell culture in the presence of 50% normal human serum with an IC(95) value of 63 nM.     

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.     

Integracides: tetracyclic triterpenoid inhibitors of HIV-1 integrase produced by Fusarium sp

HIV-1 integrase is a critical enzyme in the replication of HIV-1. It is absent in the host cells and therefore is a good target for treatment of HIV-1 infections. Integracides are members of the tetracyclic triterpenoids family that were isolated from the fermentation broth of a Fusarium sp. Integracide A, a sulfated ester, exhibited significant inhibitory activity against strand transfer reaction of HIV-1 integrase. The discovery, structure elucidation including single crystal X-ray structure and HIV-1 inhibitory activity of these compounds are described.     

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.