ABSTRACT
Identifying major histocompatibility complex (MHC) class I immunopeptide antigens represents a key step in the development of immune-based targeted therapeutics and vaccines. However, the complete characterization of these antigens by tandem mass spectrometry remains challenging due to their short sequence length, high degree of hydrophobicity, and/or lack of sufficiently basic amino acids. This study seeks to address the potential for 193 nm ultraviolet photodissociation (UVPD) to improve the analysis of MHC class I immunopeptides by offering enhanced characterization of these sequences in lower charge states and differentiation of prominent isomeric leucine and isoleucine residues in the HLA-A*02:01 motif. Although electron transfer dissociation-higher energy collisional dissociation (EThcD) offered some success in the differentiation of leucine and isoleucine, 193 nm UVPD was able to confirm the identity of nearly 60% of leucine and isoleucine residues in a synthetic peptide mixture. Furthermore, 193 nm UVPD led to significantly more peptide identifications and higher scoring metrics than EThcD for peptides obtained from immunoprecipitation of MHC class I immunopeptides from in vitro cell culture. Additionally, 193 nm UVPD represents a promising complementary technique to higher-energy collisional dissociation (HCD), in which 424 of the 2593 peptides identified by 193 nm UVPD were not identified by HCD in HLA-A*02:01-specific immunoprecipitation and 804 of the 3300 peptides identified by 193 nm UVPD were not identified by HCD for pan HLA-A, -B, and -C immunoprecipitation. These results highlight that 193 nm UVPD offers an option for the characterization of immunopeptides, including differentiation of leucine and isoleucine residues.
Subject(s)
HLA-A Antigens , Humans , Mass SpectrometryABSTRACT
Aberrant gene activation driven by the histone acetyltransferases p300 and CREB binding protein (CBP) has been linked to several diseases, including cancers. Because of this, many efforts have been aimed toward the targeting of the closely related paralogues, p300 and CBP, but these endeavors have been exclusively directed toward noncovalent inhibitors. X-ray crystallography of A-485 revealed that both p300 and CBP possess a cysteine (C1450) near the active site, thus rendering covalent inhibition an attractive chemical approach. Herein we report the development of compound 2, an acrylamide-based inhibitor of p300/CBP that forms a covalent adduct with C1450. We demonstrated using mass spectrometry that compound 2 selectively targets C1450, and we also validated covalent binding using kinetics experiments and cellular washout studies. The discovery of covalent inhibitor 2 gives us a unique tool for the study of p300/CBP biology.
ABSTRACT
Peptides presented by the class-I major histocompatibility complex (MHC-I) are important targets for immunotherapy. The identification of these peptide targets greatly facilitates the generation of T-cell-based therapeutics. Herein, we report the capability of proteolysis targeting chimera (PROTAC) compounds to induce the presentation of specific MHC class-I peptides derived from endogenous cellular proteins. Using LC-MS/MS, we identified several BET-derived MHC-I peptides induced by treatment with three BET-directed PROTAC compounds. To understand our ability to tune this process, we measured the relative rate of presentation of these peptides under varying treatment conditions using label-free mass spectrometry quantification. We found that the rate of peptide presentation reflected the rate of protein degradation, indicating a direct relationship between PROTAC treatment and peptide presentation. We additionally analyzed the effect of PROTAC treatment on the entire immunopeptidome and found many new peptides that were displayed in a PROTAC-specific fashion: we determined that these identifications map to the BET pathway, as well as, potential off-target or unique-to-PROTAC pathways. This work represents the first evidence of the use of PROTAC compounds to induce the presentation of MHC-I peptides from endogenous cellular proteins, highlighting the capability of PROTAC compounds for the discovery and generation of new targets for immunotherapy.
Subject(s)
Antigen Presentation , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/immunology , Peptides/chemistry , Peptides/immunology , Proteolysis , Cell Line , HumansABSTRACT
PURPOSE: PARP inhibitors are being developed as therapeutic agents for cancer. More than six compounds have entered clinical trials. The majority of these compounds are ß-nicotinamide adenine dinucleotide (NAD(+))-competitive inhibitors. One exception is iniparib, which has been proposed to be a noncompetitive PARP inhibitor. In this study, we compare the biologic activities of two different structural classes of NAD(+)-competitive compounds with iniparib and its C-nitroso metabolite. EXPERIMENTAL DESIGN: Two chemical series of NAD(+)-competitive PARP inhibitors, iniparib and its C-nitroso metabolite, were analyzed in enzymatic and cellular assays. Viability assays were carried out in MDA-MB-436 (BRCA1-deficient) and DLD1(-/-) (BRCA2-deficient) cells together with BRCA-proficient MDA-MB-231 and DLD1(+/+) cells. Capan-1 and B16F10 xenograft models were used to compare iniparib and veliparib in vivo. Mass spectrometry and the (3)H-labeling method were used to monitor the covalent modification of proteins. RESULTS: All NAD(+)-competitive inhibitors show robust activity in a PARP cellular assay, strongly potentiate the activity of temozolomide, and elicit robust cell killing in BRCA-deficient tumor cells in vitro and in vivo. Cell killing was associated with an induction of DNA damage. In contrast, neither iniparib nor its C-nitroso metabolite inhibited PARP enzymatic or cellular activity, potentiated temozolomide, or showed activity in a BRCA-deficient setting. We find that the nitroso metabolite of iniparib forms adducts with many cysteine-containing proteins. Furthermore, both iniparib and its nitroso metabolite form protein adducts nonspecifically in tumor cells. CONCLUSIONS: Iniparib nonselectively modifies cysteine-containing proteins in tumor cells, and the primary mechanism of action for iniparib is likely not via inhibition of PARP activity.