Random mutagenesis analysis of the Influenza A M2 proton channel reveals novel resistance mutants

TY - JOUR

T1 - Random mutagenesis analysis of the Influenza A M2 proton channel reveals novel resistance mutants

AU - Santner, Paul

AU - Martins, Joao Miguel da Silva

AU - Kampmeyer, Caroline

AU - Hartmann-Petersen, Rasmus

AU - Laursen, Jonas S.

AU - Stein, Amelie

AU - Olsen, Christian A

AU - Arkin, Isaiah T

AU - Winther, Jakob R.

AU - Willemoës, Martin

AU - Lindorff-Larsen, Kresten

PY - 2018/10/16

Y1 - 2018/10/16

N2 - The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M-089 (N13I, I35L, and F47L) and M-272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

AB - The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M-089 (N13I, I35L, and F47L) and M-272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

U2 - 10.1021/acs.biochem.8b00722

DO - 10.1021/acs.biochem.8b00722

M3 - Journal article

C2 - 30230310

SN - 0006-2960

VL - 57

SP - 5957

EP - 5968

JO - Biochemistry

JF - Biochemistry

IS - 41

ER -