Musharrafieh Rami, Lagarias Panagiotis, Ma Chunlong, Hau Raymond, Romano Alex, Lambrinidis George, Kolocouris Antonios, Wang Jun
Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, United States.
Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721, United States.
ACS Pharmacol Transl Sci. 2020 Mar 31;3(4):666-675. doi: 10.1021/acsptsci.0c00018. eCollection 2020 Aug 14.
Recent efforts in drug development against influenza A virus (IAV) M2 proton channel S31N mutant resulted in conjugates of amantadine linked with aryl head heterocycles. To understand the mechanism of drug resistance, we chose a representative M2-S31N inhibitor, compound , as a chemical probe to identify resistant mutants. To increase the possibility of identifying novel resistant mutants, serial viral passage experiments were performed with multiple strains of H1N1 and H3N2 viruses in different cell lines. This approach not only identified M2 mutations around the drug-binding site, including the pore-lining residues (V27A, V27F, N31S, and G34E) and an interhelical residue (I32N), but also a new allosteric mutation (R45H), in addition to L46P previously identified, located at the C-terminus of M2 that is more than 10 Å away from the drug-binding site. The effects of each mutation were next investigated using electrophysiology, recombinant viruses, and molecular dynamics (MD) simulations. The reduced sensitivity in channel blockage correlated with increased drug resistance in antiviral assays using recombinant viruses. The MD simulations show that the V27A, V27F, G34E, and R45H mutations increase the diameter and hydration state of the pore in complex with compound . The Molecular Mechanics Generalized Born (MM-GBSA) calculations result in more positive binding free energies for the complexes of resistant M2 (V27A, V27F, G34E, R45H) with compound compared to the stable complexes (S31N and I32N). Overall, this is the first systematic study of the drug resistance mechanism of M2-S31N channel blockers using multiple viruses in different cell lines.
近期针对甲型流感病毒（IAV）M2质子通道S31N突变体进行的药物研发工作产生了金刚烷胺与芳基头部杂环连接的缀合物。为了解耐药机制，我们选择了一种具有代表性的M2 - S31N抑制剂化合物 作为化学探针来鉴定耐药突变体。为了增加鉴定新型耐药突变体的可能性，我们在不同细胞系中对多种H1N1和H3N2病毒株进行了连续病毒传代实验。这种方法不仅鉴定出了药物结合位点周围的M2突变，包括构成孔道内壁的残基（V27A、V27F、N31S和G34E）以及一个螺旋间残基（I32N），还鉴定出了一个新的变构突变（R45H），此外还有先前鉴定出的位于M2 C末端且距离药物结合位点超过10 Å的L46P。接下来，我们使用电生理学、重组病毒和分子动力学（MD）模拟研究了每个突变的影响。通道阻断敏感性的降低与使用重组病毒进行的抗病毒试验中耐药性的增加相关。MD模拟表明，V27A、V27F、G34E和R45H突变增加了与化合物 结合时孔道的直径和水合状态。与稳定复合物（S31N和I32N）相比，分子力学广义玻恩（MM - GBSA）计算得出耐药性M2（V27A、V27F、G34E、R45H）与化合物 的复合物具有更正的结合自由能。总体而言，这是首次在不同细胞系中使用多种病毒对M2 - S31N通道阻滞剂的耐药机制进行的系统研究。
