ABSTRACT
The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These 'bi-steric inhibitors' comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Drug Design , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Breast Neoplasms/drug therapy , Cell Line, Tumor , Female , Gene Expression Regulation/drug effects , Humans , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Structure-Activity RelationshipABSTRACT
Current KRASG12C (OFF) inhibitors that target inactive GDP-bound KRASG12C cause responses in less than half of patients and these responses are not durable. A class of RASG12C (ON) inhibitors that targets active GTP-bound KRASG12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRASG12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRASG12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRASG12C-dependent lung cancer.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Drug Synergism , Lung Neoplasms , Mechanistic Target of Rapamycin Complex 1 , Proto-Oncogene Proteins p21(ras) , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Animals , Cell Line, Tumor , Mice , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Xenograft Model Antitumor Assays , Signal Transduction/drug effects , Cyclin D1/metabolism , Cyclin D1/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Female , Bcl-2-Like Protein 11/metabolism , Bcl-2-Like Protein 11/geneticsABSTRACT
RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.
Subject(s)
Xenograft Model Antitumor Assays , Humans , Animals , Mice , Cell Line, Tumor , Proto-Oncogene Proteins p21(ras)/genetics , Female , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacology , Guanosine Triphosphate/metabolism , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mutation , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , MaleABSTRACT
The proteomes of blood plasma and serum represent a potential gold mine of biological and diagnostic information, but challenges such as dynamic range of protein concentration have hampered efforts to unlock this resource. Here we present a method to label and isolate N-terminal peptides from human plasma and serum. This process dramatically reduces the complexity of the sample by eliminating internal peptides. We identify 772 unique N-terminal peptides in 222 proteins, ranging over six orders of magnitude in abundance. This approach is highly suited for studying natural proteolysis in plasma and serum. We find internal cleavages in plasma proteins created by endo- and exopeptidases, providing information about the activities of proteolytic enzymes in blood, which may be correlated with disease states. We also find signatures of signal peptide cleavage, coagulation and complement activation, and other known proteolytic processes, in addition to a large number of cleavages that have not been reported previously, including over 200 cleavages of blood proteins by aminopeptidases. Finally, we can identify substrates from specific proteases by exogenous addition of the protease combined with N-terminal isolation and quantitative mass spectrometry. In this way we identified proteins cleaved in human plasma by membrane-type serine protease 1, an enzyme linked to cancer progression. These studies demonstrate the utility of direct N-terminal labeling by subtiligase to identify and characterize endogenous and exogenous proteolysis in human plasma and serum.
Subject(s)
Blood Proteins/analysis , Proteome/analysis , Proteomics/methods , Blood Proteins/chemistry , Chromatography, Liquid , Humans , Mass Spectrometry , Peptide Fragments/analysis , Peptide Fragments/isolation & purification , Proteome/chemistry , Reproducibility of Results , Staining and Labeling/methodsABSTRACT
Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Mechanistic Target of Rapamycin Complex 1 , Proto-Oncogene Proteins p21(ras)/metabolism , Cell Proliferation , TOR Serine-Threonine Kinases , Mechanistic Target of Rapamycin Complex 2 , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/chemistry , Cell Line, TumorABSTRACT
The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRASG12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRASG12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRASG12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).
Subject(s)
Biological Products , Cyclophilin A , Immunophilins , Molecular Chaperones , Neoplasms , Proto-Oncogene Proteins p21(ras) , Humans , Biological Products/chemistry , Biological Products/pharmacology , Biological Products/therapeutic use , Cysteine/chemistry , Cysteine/genetics , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Signal Transduction , Cyclophilin A/chemistry , Cyclophilin A/metabolism , Immunophilins/chemistry , Immunophilins/metabolism , Neoplasms/drug therapy , Neoplasms/geneticsABSTRACT
Broad-spectrum RAS inhibition holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. However, the impact of inhibiting RAS functions in normal tissues is not known. RMC-7977 is a highly selective inhibitor of the active (GTP-bound) forms of KRAS, HRAS, and NRAS, with affinity for both mutant and wild type (WT) variants. As >90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models, including human and murine cell lines, human patient-derived organoids, human PDAC explants, subcutaneous and orthotopic cell-line or patient derived xenografts, syngeneic allografts, and genetically engineered mouse models. We observed broad and pronounced anti-tumor activity across these models following direct RAS inhibition at doses and concentrations that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS inhibition in the setting of PDAC.
ABSTRACT
The mechanistic target of rapamycin (mTOR) is a kinase whose activity is elevated in hematological malignancies. mTOR-complex-1 (mTORC1) phosphorylates numerous substrates to promote cell proliferation and survival. Eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) are mTORC1 substrates with an integral role in oncogenic protein translation. Current pharmacological approaches to inhibit mTORC1 activity and 4E-BP phosphorylation have drawbacks. Recently we described a series of bi-steric compounds that are potent and selective inhibitors of mTORC1, inhibiting 4E-BP phosphorylation at lower concentrations than mTOR kinase inhibitors (TOR-KIs). Here we report the activity of the mTORC1-selective bi-steric inhibitor, RMC-4627, in BCR-ABL-driven models of B-cell acute lymphoblastic leukemia (B-ALL). RMC-4627 exhibited potent and selective inhibition of 4E-BP1 phosphorylation in B-ALL cell lines without inhibiting mTOR-complex-2 (mTORC2) activity. RMC-4627 suppressed cell cycle progression, reduced survival, and enhanced dasatinib cytotoxicity. Compared to a TOR-KI compound, RMC-4627 was more potent, and its effects on cell viability were sustained after washout in vitro. Notably, a once-weekly, well tolerated dose reduced leukemic burden in a B-ALL xenograft model and enhanced the activity of dasatinib. These preclinical studies suggest that intermittent dosing of a bi-steric mTORC1-selective inhibitor has therapeutic potential as a component of leukemia regimens, and further study is warranted.
ABSTRACT
Shp1, encoded by the gene Ptpn6, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells in vitro. We also generated a novel mouse model to evaluate the impact of global, inducible Ptpn6 deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when Ptpn6 is knocked out from birth. This indicates that acute perturbation of Shp1 in vivo could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.
Subject(s)
Adenocarcinoma/enzymology , Breast Neoplasms/enzymology , Colonic Neoplasms/enzymology , Melanoma, Experimental/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/deficiency , Skin Neoplasms/enzymology , Tumor-Associated Macrophages/enzymology , Adaptive Immunity , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Adenocarcinoma/therapy , Animals , Antigens, Differentiation/metabolism , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Colonic Neoplasms/immunology , Colonic Neoplasms/pathology , Colonic Neoplasms/therapy , Female , Humans , Immunity, Innate , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Melanoma, Experimental/therapy , Mice, Inbred C57BL , Mice, Knockout , Phagocytosis , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Receptors, Immunologic/metabolism , Signal Transduction , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Skin Neoplasms/therapy , THP-1 Cells , Tumor Burden , Tumor Microenvironment , Tumor-Associated Macrophages/immunologyABSTRACT
The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. SIGNIFICANCE: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway-driven cancers.
Subject(s)
Breast Neoplasms/immunology , Immunosuppressive Agents/pharmacology , Macrophages/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Tumor Microenvironment/immunology , Allosteric Regulation , Animals , Apoptosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/prevention & control , Cell Proliferation , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRASG12C). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.
Subject(s)
Biomarkers, Tumor/genetics , Guanosine Triphosphate/metabolism , Mutation , Neoplasms/enzymology , Neoplasms/genetics , Neurofibromin 1/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Genetic Predisposition to Disease , HEK293 Cells , Humans , Mice, Inbred BALB C , Mice, Nude , Mitogen-Activated Protein Kinase Kinases/metabolism , Neoplasms/drug therapy , Neoplasms/pathology , Phenotype , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , SOS1 Protein/metabolism , Signal Transduction , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , raf Kinases/metabolismABSTRACT
We have defined the free-energy profile of the Src SH2 domain using a variety of biophysical techniques. Equilibrium and kinetic experiments monitored by tryptophan fluorescence show that Src SH2 is quite stable and folds rapidly by a two-state mechanism, without populating any intermediates. Native state hydrogen-deuterium exchange confirms this two-state behavior; we detect no cooperative partially unfolded forms in equilibrium with the native conformation under any conditions. Interestingly, the apparent stability of the protein from hydrogen exchange is 2 kcal/mol greater than the stability determined by both equilibrium and kinetic studies followed by fluorescence. Native-state proteolysis demonstrates that this "super protection" does not result from a deviation from the linear extrapolation model used to fit the fluorescence data. Instead, it likely arises from a notable compaction in the unfolded state under native conditions, resulting in an ensemble of conformations with substantial solvent exposure of side chains and flexible regions sensitive to proteolysis, but backbone amides that exchange with solvent approximately 30-fold slower than would be expected for a random coil. The apparently simple behavior of Src SH2 in traditional unfolding studies masks the significant complexity present in the denatured-state ensemble.
Subject(s)
Protein-Tyrosine Kinases/chemistry , src Homology Domains , Animals , CSK Tyrosine-Protein Kinase , Chickens , Deuterium Exchange Measurement , Kinetics , Protein Conformation , Protein Denaturation , Protein Folding , Spectrometry, Fluorescence , Thermodynamics , Tryptophan , Urea/pharmacology , src-Family KinasesABSTRACT
Phi-values, a relatively direct probe of transition-state structure, are an important benchmark in both experimental and theoretical studies of protein folding. Recently, however, significant controversy has emerged regarding the reliability with which phi-values can be determined experimentally: Because phi is a ratio of differences between experimental observables it is extremely sensitive to errors in those observations when the differences are small. Here we address this issue directly by performing blind, replicate measurements in three laboratories. By monitoring within- and between-laboratory variability, we have determined the precision with which folding rates and phi-values are measured using generally accepted laboratory practices and under conditions typical of our laboratories. We find that, unless the change in free energy associated with the probing mutation is quite large, the precision of phi-values is relatively poor when determined using rates extrapolated to the absence of denaturant. In contrast, when we employ rates estimated at nonzero denaturant concentrations or assume that the slopes of the chevron arms (mf and mu) are invariant upon mutation, the precision of our estimates of phi is significantly improved. Nevertheless, the reproducibility we thus obtain still compares poorly with the confidence intervals typically reported in the literature. This discrepancy appears to arise due to differences in how precision is calculated, the dependence of precision on the number of data points employed in defining a chevron, and interlaboratory sources of variability that may have been largely ignored in the prior literature.
Subject(s)
Protein Folding , Fluorometry , Kinetics , Observer Variation , Point Mutation , Proto-Oncogene Proteins c-fyn/chemistry , Proto-Oncogene Proteins c-fyn/genetics , Thermodynamics , src Homology DomainsABSTRACT
Amide hydrogen-deuterium exchange has proven to be a powerful tool for detecting and characterizing high-energy conformations in protein ensembles. Since interactions with ligands can modulate these high-energy conformations, hydrogen exchange appears to be an ideal experimental probe of the physical mechanisms underlying processes like allosteric regulation. The chemical mechanism of hydrogen exchange, however, can complicate such studies. Here, we examine hydrogen exchange rates in a simple model system, the c-Src SH3 domain interacting with a short peptide ligand. Addition of ligand slows the rates of hydrogen exchange at nearly every amide for which we can obtain data. Careful analysis, however, reveals that this slowing is due primarily to a reduction in the population of free protein in the system, and not to any specific property of the complex. We present a method to separate the contributions of free and bound protein to the exchange kinetics that has allowed us to identify the subset of amides where exchange arises directly from the complex. These results demonstrate that the slowing of hydrogen exchange induced by ligand interactions should be interpreted with caution, and more extensive experiments are required to correlate changes in hydrogen exchange with changes in structure or internal dynamics.
Subject(s)
Hydrogen/chemistry , Peptides/chemistry , src Homology Domains , Animals , Binding Sites , Chickens , Ligands , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins pp60(c-src)/chemistryABSTRACT
Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25 degrees C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.