Discovery, identification, and characterization of candidate pharmacodynamic markers of methionine aminopeptidase-2 inhibition.
The catalytic activity of methionine aminopeptidase-2 (MetAP2) has been pharmacologically linked to cell growth, angiogenesis, and tumor progression, making this an attractive target for cancer therapy. An assay for monitoring specific protein changes in response to MetAP2 inhibition, allowing pharmacokinetic (PK)/pharmacodynamic (PD) models to be established, could dramatically improve clinical decision-making. Candidate MetAP2-specific protein substrates were discovered from undigested cell culture-derived proteomes by MALDI-/SELDI-MS profiling and a biochemical method using (35)S-Met labeled protein lysates. Substrates were identified either as intact proteins by FT-ICR-MS or applying in-gel protease digestions followed by LC-MS/MS. The combination of these approaches led to the discovery of novel MetAP2-specific substrates including thioredoxin-1 (Trx-1), SH3 binding glutamic acid rich-like protein (SH3BGRL), and eukaryotic elongation factor-2 (eEF2). These studies also confirmed glyceraldehye 3-phosphate dehydrogenase (GAPDH) and cyclophillin A (CypA) as MetAP2 substrates. Additional data in support of these proteins as MetAP2-specific substrates were provided by in vitro MetAP1/MetAP2 enzyme assays with the corresponding N-terminal derived peptides and 1D/2D Western analyses of cellular and tissue lysates. FT-ICR-MS characterization of all intact species of the 18 kDa substrate, CypA, enabled a SELDI-MS cell-based assay to be developed for correlating N-terminal processing and inhibition of proliferation. The MetAP2-specific protein substrates discovered in this study have diverse properties that should facilitate the development of reagents for testing in preclinical and clinical environments.
Warder
Scott E
SE
Advanced Technology and Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-6202, USA. scott.warder@abbott.com
Tucker
Lora A
LA
McLoughlin
Shaun M
SM
Strelitzer
Tamara J
TJ
Meuth
Joseph L
JL
Zhang
Qian
Q
Sheppard
George S
GS
Richardson
Paul L
PL
Lesniewski
Rick
R
Davidsen
Steven K
SK
Bell
Randy L
RL
Rogers
John C
JC
Wang
Jieyi
J
D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000818:Animals; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006801:Humans; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D051379:Mice; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000145:classification / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods; D013997:Time Factors
D014408:Biomarkers, Tumor; D011480:Protease Inhibitors; D000626:Aminopeptidases; C106541:methionine aminopeptidase 2; D008666:Metalloendopeptidases