Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide

Ruud Oerlemans, Celia R. Berkers, Yehuda G. Assaraf, George L. Scheffer, Godefridus J. Peters, Sue Ellen Verbrugge, Jacqueline Cloos, Jerry Slootstra, Rob H. Meloen, Robert H. Shoemaker, Ben A.C. Dijkmans, Rik J. Scheper, Huib Ovaa, Gerrit Jansen

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Background The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of β5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional β5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the β5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.

Original languageEnglish
Pages (from-to)797-809
Number of pages13
JournalInvestigational New Drugs
Volume36
Issue number5
DOIs
Publication statusPublished - 1 Oct 2018

Cite this

Oerlemans, Ruud ; Berkers, Celia R. ; Assaraf, Yehuda G. ; Scheffer, George L. ; Peters, Godefridus J. ; Verbrugge, Sue Ellen ; Cloos, Jacqueline ; Slootstra, Jerry ; Meloen, Rob H. ; Shoemaker, Robert H. ; Dijkmans, Ben A.C. ; Scheper, Rik J. ; Ovaa, Huib ; Jansen, Gerrit. / Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide. In: Investigational New Drugs. 2018 ; Vol. 36, No. 5. pp. 797-809.
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title = "Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide",
abstract = "Background The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of β5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional β5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the β5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.",
keywords = "ABC drug efflux transporters, Bortezomib, Cytotoxic peptides, Drug resistance, Proteasome, Proteasome inhibitors",
author = "Ruud Oerlemans and Berkers, {Celia R.} and Assaraf, {Yehuda G.} and Scheffer, {George L.} and Peters, {Godefridus J.} and Verbrugge, {Sue Ellen} and Jacqueline Cloos and Jerry Slootstra and Meloen, {Rob H.} and Shoemaker, {Robert H.} and Dijkmans, {Ben A.C.} and Scheper, {Rik J.} and Huib Ovaa and Gerrit Jansen",
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Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide. / Oerlemans, Ruud; Berkers, Celia R.; Assaraf, Yehuda G.; Scheffer, George L.; Peters, Godefridus J.; Verbrugge, Sue Ellen; Cloos, Jacqueline; Slootstra, Jerry; Meloen, Rob H.; Shoemaker, Robert H.; Dijkmans, Ben A.C.; Scheper, Rik J.; Ovaa, Huib; Jansen, Gerrit.

In: Investigational New Drugs, Vol. 36, No. 5, 01.10.2018, p. 797-809.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide

AU - Oerlemans, Ruud

AU - Berkers, Celia R.

AU - Assaraf, Yehuda G.

AU - Scheffer, George L.

AU - Peters, Godefridus J.

AU - Verbrugge, Sue Ellen

AU - Cloos, Jacqueline

AU - Slootstra, Jerry

AU - Meloen, Rob H.

AU - Shoemaker, Robert H.

AU - Dijkmans, Ben A.C.

AU - Scheper, Rik J.

AU - Ovaa, Huib

AU - Jansen, Gerrit

PY - 2018/10/1

Y1 - 2018/10/1

N2 - Background The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of β5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional β5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the β5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.

AB - Background The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of β5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional β5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the β5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.

KW - ABC drug efflux transporters

KW - Bortezomib

KW - Cytotoxic peptides

KW - Drug resistance

KW - Proteasome

KW - Proteasome inhibitors

UR - http://www.scopus.com/inward/record.url?scp=85041905364&partnerID=8YFLogxK

U2 - 10.1007/s10637-018-0569-x

DO - 10.1007/s10637-018-0569-x

M3 - Article

VL - 36

SP - 797

EP - 809

JO - Investigational New Drugs

JF - Investigational New Drugs

SN - 0167-6997

IS - 5

ER -