Design of Antiviral Drugs Effective Against the HIV-1 Virus

有效对抗 HIV-1 病毒的抗病毒药物的设计

• 批准号: 7732934
• 负责人: ETTORE APPELLA
• 金额: $ 18.12万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732934
• 关键词: Amino Acid Sequence; Anti-Retroviral Agents; Antiviral Agents; Binding; Biochemical; Biological Models; Biopsy; C-terminal; Cell model; Cells; Centers for Disease Control and Prevention (U.S.); Cervical; Chemical Structure; Coculture Techniques; Collaborations; Cysteine; Development; Drug Formulations; Drug Kinetics; Evaluation; Gel; Goals; HIV; HIV Infections; HIV-1; Human; Human immunodeficiency virus 1 NCP7 protein; In Vitro; Infection; Infection prevention; Ireland; Lead; Libraries; Life Cycle Stages; Local Microbicides; Macaca mulatta; Mass Spectrum Analysis; Measures; Metabolism; Metals; Methods; Modeling; Molecular; Mucous body substance; Nucleocapsid Proteins; Oryctolagus cuniculus; Preparation; Proteins; Range; Reaction; Reporting; Research; Secondary to; Series; Side; Silicones; Sulfur; System; Testing; Therapeutic; Tissues; Topical application; Universities; Vagina; Vaginal Ring; Viral; Virus; Virus Inhibitors; Water; Work; Zinc; analog; base; birth control; compliance behavior; design; in vivo; inhibitor/antagonist; irritation; microbicide; nonhuman primate; novel; prevent; rectal; research clinical testing; research study; thioester; transmission process; virucide

Molecules that bind to and inactivate the HIV-1 nucleocapsid protein (NCp7) are currently being evaluated as new antiviral drugs. In particular, derivatives based on a 2-mercaptobenzamide thioester template (SAMTs) have been shown to specifically eject zinc from the C-terminal zinc-binding domain (ZD2) of NCp7 via acyl transfer from the thioester to the sulfur of a zinc-coordinating cysteine residue. These zinc-binding domains are excellent targets for the development of new antiretroviral and microbicidal agents because of their structural conservation and the broad range of function of NCp7 in the viral life cycle. Our efforts have focused on the evaluation of the therapeutic potential of the SAMT compounds and the identification of new analogs with increased activity. Our previous work identified three SAMT compounds that were shown to be virucidal and to inhibit cell-to-cell associated transmission of HIV-1 in co-culture systems. In addition, based on mutational analysis of the NCp7 amino acid sequence, we were able to extend the previously reported mechanism of action of the SAMT compounds to include a secondary S to N intramolecular acyl transfer that occurs after the primary acyl transfer from the thioester to a cysteine side chain in the protein. Based upon this detailed mechanism of action of SAMT compounds, we have synthesized novel derivatives of the SAMT chemotype that are predicted to be more stable than the SAMT compounds. This improvement may enable the new compounds to be used systemically to treat HIV. We have studied the in vitro metal ejection activity of a series of these compounds and found that some have similar activity as the lead SAMT compounds. In addition, preliminary mass spectrometry experiments demonstrate that the new compounds also follow a similar reaction mechanism as the SAMTs. The new compounds are currently being tested for antiviral activity in co-culture systems. We have begun to explore the use of the SAMT compounds as potential microbicide candidates. We found that three lead SAMT compounds prevent cell-to-cell transmission of HIV and inhibit the dissemination of virus from cervical explants. We further determined that the SAMTs retain their activity in gel formulation and in synthetic mucus. Furthermore, they did not induce vaginal irritation in rabbits. A preliminary study found that a SAMT lead prevented infection in five of six rhesus macaques challenged in a vaginal dual-infection trial. These experiments were encouraging, and we have continued to pursue the use of the SAMTs as topical microbicides to prevent the transmission of HIV. Recent studies have found that gel-based microbicides are hampered by patient compliance, suggesting that this method of delivery would not be optimal for a good microbicide. Thus, we initiated a collaboration with Dr. Karl Malcolm at the Queens University Belfast in Ireland to study the release of the SAMT compounds from silicone rings, such as those currently approved for use as birth control. We have determined that certain lead SAMTs are able to be sufficiently released from the ring in vitro . We have also begun a collaboration with Dr. James Smith at the Centers for Disease Control and Prevention to study the inhibition of virus transmission in non-human primates using the vaginal rings. Preliminary results have established that the SAMT in the ring is released in vivo similarly to what was measured in vitro . Studies are currently underway to assess the efficacy of a SAMT compound in a ring to block viral transmission. Finally, we are using mass spectrometry experiments to elucidate the metabolism and pharmacokinetics of the SAMT compounds. In collaboration with the Waters Corporation, we have identified a panel of potential metabolites of the SAMT compounds. We are working to validate the chemical structure of the metabolites and evaluate the possibility that some may have antiviral activity. In addition, we are preparing to identify metabolites of the SAMT compounds from biopsy tissue. We are continuing to investigate the activity and metabolism of the lead SAMT compounds in cellular model systems and non-human primates. Identification of a potentially safe and efficacious single or combination candidate microbicide in non-human primates, and the elucidation of their pharmacokinetics, will lead us to more formal testing in preparation for clinical evaluation.

与HIV-1核衣壳蛋白(NCp7)结合并使其失活的分子目前正在被评估为新的抗病毒药物。特别是，基于2-硫代苯甲酰胺硫酯模板(SAMTS)的衍生物已被证明通过从硫酯到与锌配位的半胱氨酸残基的硫的酰基转移，从NCp7的C-末端锌结合结构域(ZD2)特异性地排出锌。这些锌结合结构域是开发新的抗逆转录病毒和杀微生物剂的良好靶点，因为它们的结构保守，而且NCp7在病毒生命周期中具有广泛的功能。我们的努力集中在对SAMT化合物的治疗潜力的评估和对活性增加的新类似物的鉴定上。我们之前的工作鉴定了三个SAMT化合物，它们被证明是杀毒的，并在共培养系统中抑制HIV-1的细胞间相关传播。此外，基于NCp7氨基酸序列的突变分析，我们能够扩展先前报道的SAMT化合物的作用机制，包括第二次S到N分子内的酰基转移，该转移发生在蛋白质中的硫酯到半胱氨酸侧链的初级酰基转移之后。基于SAMT化合物的这种详细的作用机制，我们合成了新的SAMT化学型衍生物，预计它们比SAMT化合物更稳定。这一改进可能使新化合物能够系统地用于治疗艾滋病毒。我们研究了一系列这类化合物的体外金属喷射活性，发现其中一些化合物具有与先导SAMT化合物相似的活性。此外，初步的质谱学实验表明，新化合物也遵循与SAMTS相似的反应机理。新化合物目前正在共培养系统中进行抗病毒活性测试。我们已经开始探索使用SAMT化合物作为潜在的杀菌剂候选者。我们发现，三种先导SAMT化合物可以防止HIV病毒在细胞间传播，并抑制宫颈组织中病毒的传播。我们进一步确定SAMT在凝胶制剂和合成粘液中保持其活性。此外，它们不会引起兔的阴道刺激。一项初步研究发现，在一项阴道双重感染试验中，六只恒河猴中有五只受到挑战，SAMT铅预防了其中五只的感染。这些试验令人鼓舞，我们继续使用SAMTS作为局部杀微生物剂，以防止艾滋病毒的传播。最近的研究发现，以凝胶为基础的杀微生物剂会受到患者依从性的阻碍，这表明这种给药方法对于良好的杀菌剂来说并不是最佳的。因此，我们开始与爱尔兰贝尔法斯特女王大学的卡尔·马尔科姆博士合作，研究SAMT化合物从硅胶环中的释放，例如那些目前被批准用于节育的化合物。我们已经确定某些铅SAMTs能够在体外从环上充分释放。我们还开始与疾病控制和预防中心的詹姆斯·史密斯博士合作，研究使用阴道环在非人类灵长类动物中抑制病毒传播。初步结果表明，环内SAMT在体内的释放与体外测定的结果相似。目前正在进行研究，以评估环状SAMT化合物阻断病毒传播的有效性。最后，我们正在使用质谱学实验来阐明SAMT化合物的代谢和药代动力学。在与Waters Corporation的合作下，我们已经确定了一组SAMT化合物的潜在代谢物。我们正在努力验证代谢物的化学结构，并评估其中一些可能具有抗病毒活性的可能性。此外，我们正在准备从活检组织中鉴定SAMT化合物的代谢物。我们正在继续研究先导SAMT化合物在细胞模型系统和非人类灵长类动物中的活性和代谢。在非人类灵长类动物中鉴定一种潜在安全有效的单一或联合候选杀微生物剂，并阐明其药代动力学，将引导我们进行更正式的测试，为临床评估做准备。