Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity

Allan H. Pang; Keith D. Green; Nishad Thamban Chandrika; Atefeh Garzan; Ankita Punetha; Selina Y.L. Holbrook; Melisa J. Willby; James E. Posey; Oleg V. Tsodikov; Sylvie Garneau-Tsodikova

doi:10.1016/j.ejmech.2022.114698

Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity

Allan H. Pang, Keith D. Green, Nishad Thamban Chandrika, Atefeh Garzan, Ankita Punetha, Selina Y.L. Holbrook, Melisa J. Willby, James E. Posey, Oleg V. Tsodikov, Sylvie Garneau-Tsodikova

Research output: Contribution to journal › Article › peer-review

5 Scopus citations

Abstract

A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC₅₀ ∼ 1 μM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc² 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.

Original language	English
Article number	114698
Journal	European Journal of Medicinal Chemistry
Volume	242
DOIs	https://doi.org/10.1016/j.ejmech.2022.114698
State	Published - Nov 15 2022

Bibliographical note

Funding Information:
This study was funded by grants from the National Institutes of Health (NIH) AI090048 (to S.G.-T.), the Firland Foundation (to S.G.-T.), and the Center for Chemical Genomics (CCG) at the University of Michigan (to S.G.-T.), as well as by startup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T. and O.V.T). We acknowledge S. Vander Roest, M. Larsen, and P. Kirchhoff from the CCG at the University of Michigan for their help with HTS. We thank the staff of sector SER-CAT of the Advanced Photon Source at the Argonne National Laboratories for assistance with remote X-ray diffraction data collection. The synchrotron access was supported, in part, by the Center for Structural Biology at the University of Kentucky . We thank the College of Pharmacy NMR Center ( University of Kentucky ) for NMR support. Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the U.S. Public Health Service, or the CDC. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies.

Funding Information:
This study was funded by grants from the National Institutes of Health (NIH) AI090048 (to S.G.-T.), the Firland Foundation (to S.G.-T.), and the Center for Chemical Genomics (CCG) at the University of Michigan (to S.G.-T.), as well as by startup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T. and O.V.T). We acknowledge S. Vander Roest, M. Larsen, and P. Kirchhoff from the CCG at the University of Michigan for their help with HTS. We thank the staff of sector SER-CAT of the Advanced Photon Source at the Argonne National Laboratories for assistance with remote X-ray diffraction data collection. The synchrotron access was supported, in part, by the Center for Structural Biology at the University of Kentucky. We thank the College of Pharmacy NMR Center (University of Kentucky) for NMR support. Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the U.S. Public Health Service, or the CDC. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies.

Publisher Copyright:
© 2022 Elsevier Masson SAS

Keywords

Crystal structure
Drug resistance
Enhanced intracellular survival
Enzyme kinetics
Mycobacterium tuberculosis
Structure-activity relationship
Tuberculosis

ASJC Scopus subject areas

Pharmacology
Drug Discovery
Organic Chemistry

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.ejmech.2022.114698

Cite this

Pang, A. H., Green, K. D., Chandrika, N. T., Garzan, A., Punetha, A., Holbrook, S. Y. L., Willby, M. J., Posey, J. E., Tsodikov, O. V., & Garneau-Tsodikova, S. (2022). Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity. European Journal of Medicinal Chemistry, 242, Article 114698. https://doi.org/10.1016/j.ejmech.2022.114698

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title = "Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity",

abstract = "A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC50 ∼ 1 μM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc2 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.",

keywords = "Crystal structure, Drug resistance, Enhanced intracellular survival, Enzyme kinetics, Mycobacterium tuberculosis, Structure-activity relationship, Tuberculosis",

author = "Pang, {Allan H.} and Green, {Keith D.} and Chandrika, {Nishad Thamban} and Atefeh Garzan and Ankita Punetha and Holbrook, {Selina Y.L.} and Willby, {Melisa J.} and Posey, {James E.} and Tsodikov, {Oleg V.} and Sylvie Garneau-Tsodikova",

note = "Funding Information: This study was funded by grants from the National Institutes of Health (NIH) AI090048 (to S.G.-T.), the Firland Foundation (to S.G.-T.), and the Center for Chemical Genomics (CCG) at the University of Michigan (to S.G.-T.), as well as by startup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T. and O.V.T). We acknowledge S. Vander Roest, M. Larsen, and P. Kirchhoff from the CCG at the University of Michigan for their help with HTS. We thank the staff of sector SER-CAT of the Advanced Photon Source at the Argonne National Laboratories for assistance with remote X-ray diffraction data collection. The synchrotron access was supported, in part, by the Center for Structural Biology at the University of Kentucky . We thank the College of Pharmacy NMR Center ( University of Kentucky ) for NMR support. Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the U.S. Public Health Service, or the CDC. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies. Funding Information: This study was funded by grants from the National Institutes of Health (NIH) AI090048 (to S.G.-T.), the Firland Foundation (to S.G.-T.), and the Center for Chemical Genomics (CCG) at the University of Michigan (to S.G.-T.), as well as by startup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T. and O.V.T). We acknowledge S. Vander Roest, M. Larsen, and P. Kirchhoff from the CCG at the University of Michigan for their help with HTS. We thank the staff of sector SER-CAT of the Advanced Photon Source at the Argonne National Laboratories for assistance with remote X-ray diffraction data collection. The synchrotron access was supported, in part, by the Center for Structural Biology at the University of Kentucky. We thank the College of Pharmacy NMR Center (University of Kentucky) for NMR support. Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the U.S. Public Health Service, or the CDC. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies. Publisher Copyright: {\textcopyright} 2022 Elsevier Masson SAS",

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doi = "10.1016/j.ejmech.2022.114698",

language = "English",

volume = "242",

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T1 - Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity

AU - Pang, Allan H.

AU - Green, Keith D.

AU - Chandrika, Nishad Thamban

AU - Garzan, Atefeh

AU - Punetha, Ankita

AU - Holbrook, Selina Y.L.

AU - Willby, Melisa J.

AU - Posey, James E.

AU - Tsodikov, Oleg V.

AU - Garneau-Tsodikova, Sylvie

N1 - Funding Information: This study was funded by grants from the National Institutes of Health (NIH) AI090048 (to S.G.-T.), the Firland Foundation (to S.G.-T.), and the Center for Chemical Genomics (CCG) at the University of Michigan (to S.G.-T.), as well as by startup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T. and O.V.T). We acknowledge S. Vander Roest, M. Larsen, and P. Kirchhoff from the CCG at the University of Michigan for their help with HTS. We thank the staff of sector SER-CAT of the Advanced Photon Source at the Argonne National Laboratories for assistance with remote X-ray diffraction data collection. The synchrotron access was supported, in part, by the Center for Structural Biology at the University of Kentucky . We thank the College of Pharmacy NMR Center ( University of Kentucky ) for NMR support. Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the U.S. Public Health Service, or the CDC. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies. Funding Information: This study was funded by grants from the National Institutes of Health (NIH) AI090048 (to S.G.-T.), the Firland Foundation (to S.G.-T.), and the Center for Chemical Genomics (CCG) at the University of Michigan (to S.G.-T.), as well as by startup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T. and O.V.T). We acknowledge S. Vander Roest, M. Larsen, and P. Kirchhoff from the CCG at the University of Michigan for their help with HTS. We thank the staff of sector SER-CAT of the Advanced Photon Source at the Argonne National Laboratories for assistance with remote X-ray diffraction data collection. The synchrotron access was supported, in part, by the Center for Structural Biology at the University of Kentucky. We thank the College of Pharmacy NMR Center (University of Kentucky) for NMR support. Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the U.S. Public Health Service, or the CDC. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies. Publisher Copyright: © 2022 Elsevier Masson SAS

PY - 2022/11/15

Y1 - 2022/11/15

N2 - A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC50 ∼ 1 μM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc2 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.

AB - A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC50 ∼ 1 μM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc2 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.

KW - Crystal structure

KW - Drug resistance

KW - Enhanced intracellular survival

KW - Enzyme kinetics

KW - Mycobacterium tuberculosis

KW - Structure-activity relationship

KW - Tuberculosis

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VL - 242

JO - European Journal of Medicinal Chemistry

JF - European Journal of Medicinal Chemistry

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ER -

Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

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