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.

目前正在评估与HIV-1核素蛋白（NCP7）结合并失活的分子。特别是，已经证明，基于2-甲基苯二苯甲酰胺硫酯模板（SAMT）的衍生物已被证明通过酰基从硫酯转移到锌协调的固醇固醇固醇的硫酯，从NCP7的C末端锌结合结构域（ZD2）特别弹出锌。这些锌结合结构域是开发新抗逆转录病毒和微生物剂的绝佳目标，因为它们的结构保护以及NCP7在病毒生命周期中的广泛功能范围。我们的努力集中在评估SAMT化合物的治疗潜力以及具有增加活性的新类似物的鉴定上。我们以前的工作确定了三种SAMT化合物，这些化合物被证明是病毒性的，并抑制了共培养系统中HIV-1的细胞对细胞与细胞相关的传播。此外，基于对NCP7氨基酸序列的突变分析，我们能够将SAMT化合物的先前报道的作用机理扩展到将二酰基分子内酰基转移的二次S酰基转移后从硫酯转移后从硫酯转移到蛋白质中的半胱氨酸侧链。 基于SAMT化合物的这种详细的作用机理，我们合成了SAMT化学型的新型衍生物，这些新型衍生物比SAMT化合物更稳定。这种改进可能使新化合物可以系统地使用以治疗艾滋病毒。 我们研究了一系列化合物的体外金属射血活性，发现有些具有与铅SAMT化合物相似的活性。此外，初步质谱实验表明，新化合物也遵循与SAMT相似的反应机制。当前，新化合物正在测试共培养系统中的抗病毒活性。我们已经开始探索SAMT化合物作为潜在的菌心候选物的使用。我们发现，三种铅SAMT化合物可防止HIV的细胞到细胞传播，并抑制病毒从宫颈外植体中传播。我们进一步确定SAMT将其活性保留在凝胶制剂和合成粘液中。 此外，它们没有引起兔子的阴道刺激。一项初步研究发现，SAMT铅可阻止在阴道双感染试验中挑战的六个恒河猕猴中的五个感染。这些实验令人鼓舞，我们继续追求将SAMTs用作局部杀菌剂以防止HIV传播。最近的研究发现，基于凝胶的杀菌剂受到患者依从性的阻碍，这表明这种递送方法对良好的菌心不会是最佳的。因此，我们与爱尔兰皇后大学贝尔法斯特大学的Karl Malcolm博士合作研究了从硅环中释放SAMT化合物的释放，例如目前被批准用于节育的硅环。我们已经确定某些铅SAM可以在体外从环中充分释放。我们还开始与疾病控制与预防中心的詹姆斯·史密斯（James Smith）博士进行合作，以研究使用阴道环的非人类灵长类动物中病毒传播的抑制作用。初步结果已经确定，环中的SAMT在体内释放与体外测量的SAMT相似。目前正在进行研究以评估环中SAMT化合物在环上阻断病毒传播的功效。最后，我们正在使用质谱实验来阐明SAMT化合物的代谢和药代动力学。与Waters Corporation合作，我们确定了SAMT化合物的潜在代谢产物小组。我们正在努力验证代谢物的化学结构，并评估一些可能具有抗病毒活性的可能性。此外，我们准备从活检组织中鉴定SAMT化合物的代谢产物。我们正在继续研究细胞模型系统和非人类灵长类动物中铅SAMT化合物的活性和代谢。鉴定非人类灵长类动物中潜在的安全有效的单一或组合候选杀菌剂以及阐明其药代动力学的阐明，将使我们进行更正式的测试，以准备临床评估。