ABSTRACT
The cytosolic fraction of the tumor suppressor p53 activates the apoptotic effector protein BAX to trigger apoptosis. Here we report that p53 activates BAX through a mechanism different from that associated with activation by BH3 only proteins (BIM and BID). We observed that cis-trans isomerization of proline 47 (Pro47) within p53, an inherently rare molecular event, was required for BAX activation. The prolyl isomerase Pin1 enhanced p53-dependent BAX activation by catalyzing cis-trans interconversion of p53 Pro47. Our results reveal a signaling mechanism whereby proline cis-trans isomerization in one protein triggers conformational and functional changes in a downstream signaling partner. Activation of BAX through the concerted action of cytosolic p53 and Pin1 may integrate cell stress signals to induce a direct apoptotic response.
Subject(s)
Apoptosis , Peptidylprolyl Isomerase/physiology , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein/metabolism , Cell Line, Tumor , Humans , Kinetics , NIMA-Interacting Peptidylprolyl Isomerase , Proline/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Stereoisomerism , Tumor Suppressor Protein p53/chemistry , bcl-2-Associated X Protein/chemistryABSTRACT
Intrinsically disordered regions (IDRs) of proteins often regulate function upon post-translational modification (PTM) through interactions with folded domains. An IDR linking two α-helices (α1-α2) of the antiapoptotic protein Bcl-xL experiences several PTMs that reduce antiapoptotic activity. Here, we report that PTMs within the α1-α2 IDR promote its interaction with the folded core of Bcl-xL that inhibits the proapoptotic activity of two types of regulatory targets, BH3-only proteins and p53. This autoregulation utilizes an allosteric pathway whereby, in one direction, the IDR induces a direct displacement of p53 from Bcl-xL coupled to allosteric displacement of simultaneously bound BH3-only partners. This pathway operates in the opposite direction when the BH3-only protein PUMA binds to the BH3 binding groove of Bcl-xL, directly displacing other bound BH3-only proteins, and allosterically remodels the distal site, displacing p53. Our findings show how an IDR enhances functional versatility through PTM-dependent allosteric regulation of a folded protein domain.
Subject(s)
Apoptosis , Gene Expression Regulation , Intrinsically Disordered Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , bcl-X Protein/metabolism , Allosteric Site , Binding Sites , Humans , Intrinsically Disordered Proteins/genetics , Kinetics , Mutation , Protein Binding , Protein Domains , Protein Folding , Protein Processing, Post-Translational , Protein Structure, Secondary , Signal Transduction , bcl-X Protein/geneticsABSTRACT
During apoptotic cell death, cellular stress signals converge at the mitochondria to induce mitochondrial outer-membrane permeabilization (MOMP) through B cell lymphoma-2 (BCL-2) family proteins and their effectors. BCL-2 proteins function through protein-protein interactions, the mechanisms and structural aspects of which are only now being uncovered. Recently, the elucidation of the dynamic features underlying their function has highlighted their structural plasticity and the consequent complex thermodynamic landscape governing their protein-protein interactions. These studies show that canonical interactions involve a conserved, hydrophobic groove, whereas non-canonical interactions function allosterically outside the groove. We review the latest structural advances in understanding the interactions and functions of mammalian BCL-2 family members, and discuss new opportunities to modulate these proteins in health and disease.
Subject(s)
Apoptosis/physiology , Mitochondrial Membranes/metabolism , Proto-Oncogene Proteins c-bcl-2 , Animals , Humans , Permeability , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
Btk is an attractive target for the treatment of a range of Bcell malignancies as well as several autoimmune diseases such as murine lupus and rheumatoid arthritis. Several covalent irreversible inhibitors of Btk are currently in development including ibrutinib which was approved for treatment of B-cell malignancies. Herein, we describe our efforts using X-ray guided structure based design (SBD) to identify a novel chemical series of covalent Btk inhibitors. The resulting pyridine carboxamides were potent and selective inhibitors of Btk having excellent enzymatic and cellular inhibitory activity.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Pyrimidines/pharmacology , Adenine/analogs & derivatives , Administration, Oral , Animals , Caco-2 Cells , Humans , Mice , Molecular Structure , Piperidines , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazoles/pharmacology , Pyridines/administration & dosage , Pyridines/chemical synthesis , Pyridines/chemistry , Pyrimidines/administration & dosage , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Structure-Activity RelationshipABSTRACT
Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Drug Discovery , Protein Kinase Inhibitors/pharmacology , Agammaglobulinaemia Tyrosine Kinase/metabolism , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Understanding signaling and other complex biological processes requires elucidating the critical roles of intrinsically disordered proteins (IDPs) and regions (IDRs), which represent â¼30% of the proteome and enable unique regulatory mechanisms. In this review, we describe the structural heterogeneity of disordered proteins that underpins these mechanisms and the latest progress in obtaining structural descriptions of conformational ensembles of disordered proteins that are needed for linking structure and dynamics to function. We describe the diverse interactions of IDPs that can have unusual characteristics such as "ultrasensitivity" and "regulated folding and unfolding". We also summarize the mounting data showing that large-scale assembly and protein phase separation occurs within a variety of signaling complexes and cellular structures. In addition, we discuss efforts to therapeutically target disordered proteins with small molecules. Overall, we interpret the remodeling of disordered state ensembles due to binding and post-translational modifications within an expanded framework for allostery that provides significant insights into how disordered proteins transmit biological information.
Subject(s)
Intrinsically Disordered Proteins/metabolism , Signal Transduction/physiology , Allosteric Regulation , Intrinsically Disordered Proteins/chemistry , Protein Folding , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Protein Structure, Tertiary , Protein Unfolding , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolismABSTRACT
Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the antiapoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique among BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules BAX and BAK. Structural investigations using NMR spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the antiapoptotic BCL-2 repertoire to sensitize for death receptor-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , Protein Unfolding , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , bcl-X Protein/metabolism , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Humans , Models, Molecular , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Tumor Suppressor Protein p53/chemistry , bcl-X Protein/chemistryABSTRACT
Fatty acids play important signaling roles in biology, albeit typically lacking potency or selectivity, due to their substantial conformational flexibility. While being recognized as having properties of potentially great value as therapeutics, it is often the case that the functionally relevant conformation of the natural fatty acid is not known, thereby complicating efforts to develop natural-product-inspired ligands that have similar functional properties along with enhanced potency and selectivity profiles. In other words, without structural information associated with a particular functional relationship and the hopelessly unbiased conformational preferences of the endogenous ligand, one is molecularly ill-informed regarding the precise ligand-receptor interactions that play a role in driving the biological activity of interest. To address this problem, a molecular strategy to query the relevance of distinct subpopulations of fatty acid conformers has been established through "conformational profiling", a process whereby a unique collection of chiral and conformationally constrained fatty acids is employed to deconvolute beneficial structural features that impart natural-product-inspired function. Using oleic acid as an example because it is known to engage a variety of receptors, including GPR40, GPR120, and TLX, a 24-membered collection of mimetics was designed and synthesized. It was then demonstrated that this collection contained members that have enhanced potency and selectivity profiles, with some being clearly biased for engagement of the GPCRs GPR40 and GPR120 while others were identified as potent and selective modulators of the nuclear receptor TLX. A chemical synthesis strategy that exploited the power of modern technology for stereoselective synthesis was critical to achieving success, establishing a common sequence of bond-forming reactions to access a disparate collection of chiral mimetics, whose conformational preferences are impacted by the nature of stereodefined moieties differentially positioned about the C18 skeleton of the parent fatty acid. Overall, this study establishes a foundation to fuel future programs aimed at developing natural-product-inspired fatty acid mimetics as valuable tools in chemical biology and potential therapeutic leads.
ABSTRACT
It is now widely recognized that intrinsically disordered (or unstructured) proteins (IDPs, or IUPs) are found in organisms from all kingdoms of life. In eukaryotes, IDPs are highly abundant and perform a wide range of biological functions, including regulation and signaling. Despite increased interest in understanding the structural biology of IDPs, questions remain regarding the mechanisms through which disordered proteins perform their biological function(s). In other words, what are the relationships between disorder and function for IDPs? Several excellent reviews have recently been published that discuss the structural properties of IDPs.1-3 Here, we discuss two IDP systems which illustrate features of dynamic complexes. In the first section, we discuss two IDPs, p21 and p27, which regulate the mammalian cell division cycle by inhibiting cyclin-dependent kinases (Cdks). In the second section, we discuss recent results from Follis, Hammoudeh, Metallo and coworkers demonstrating that the IDP Myc can be bound and inhibited by small molecules through formation of dynamic complexes. Previous studies have shown that polypeptide segments of p21 and p27 are partially folded in isolation and fold further upon binding their biological targets. Interestingly, some portions of p27 which bind to and inhibit Cdk2/cyclin A remain flexible in the bound complex. This residual flexibility allows otherwise buried tyrosine residues within p27 to be phosphorylated by nonreceptor tyrosine kinases (NRTKs). Tyrosine phosphorylation relieves kinase inhibition, triggering Cdk2-mediated phosphorylation of a threonine residue within the flexible C-terminus of p27. This, in turn, marks p27 for ubiquitination and proteasomal degradation, unleashing full Cdk2 activity which drives cell cycle progression. p27, thus, constitutes a conduit for transmission of proliferative signals via posttranslational modifications. Importantly, activation of the p27 signaling conduit by oncogenic NRTKs contributes to tumorigenesis in some human cancers, including chronic myelogenous leukemia (CML)9 and breast cancer.10 Another IDP with important roles in human cancer is the proto-oncoprotein, Myc. Myc is a DNA binding transcription factor which critically drives cell proliferation in many cell types and is often deregulated in cancer. Myc is intrinsically disordered in isolation and folds upon binding another IDP, Max and DNA. Follis, Hammoudeh, Metallo and coworkers identified small molecules which bind disordered regions of Myc and inhibit its heterodimerization with Max. Importantly, these small molecules- through formation of dynamic complexes with Myc-have been shown to inhibit Myc function in vitro and in cellular assays, opening the door to IDP-targeted therapeutics in the future. The p21/p27 and Myc systems illustrate, from different perspectives, the role of dynamics in IDP function. Dynamic fluctuations are critical for p21/p27 signaling while the dynamic free state of Myc may represent a therapeutically approachable anticancer target. Herein we review the current state of knowledge related to these two topics in IDP research.
Subject(s)
Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/chemistry , Cyclin-Dependent Kinase Inhibitor p27/chemistry , Signal Transduction , Amino Acid Sequence , Animals , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Humans , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Bruton's tyrosine kinase (BTK) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage. Evidence has shown that inhibition of BTK has clinical benefit for the treatment of a wide array of autoimmune and inflammatory diseases. Previously we reported the discovery of a novel nicotinamide selectivity pocket (SP) series of potent and selective covalent irreversible BTK inhibitors. The top molecule 1 of that series strongly inhibited CYP2C8 (IC50 =100â nM), which was attributed to the bridged linker group. However, our effort on the linker replacement turned out to be fruitless. With the study of the X-ray crystal structure of compound 1, we envisioned the opportunity of removal of this liability via transposition of the linker moiety in 1 from C6 to C5 position of the pyridine core. With this strategy, our optimization led to the discovery of a novel series, in which the top molecule 18 A displayed reduced CYP inhibitory activity and good potency. To further explore this new series, different warheads besides acrylamide, for example cyanamide, were also tested. However, this effort didn't lead to the discovery of molecules with better potency than 18 A. The loss of potency in those molecules could be related to the reduced reactivity of the warhead or reversible binding mode. Further profiling of 18 A disclosed that it had a strong hERG (human Ether-a-go-go Related Gene) inhibition, which could be related to the phenoxyphenyl group.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Cytochrome P-450 CYP2C8 Inhibitors/pharmacology , Cytochrome P-450 CYP2C8/metabolism , Drug Discovery , Protein Kinase Inhibitors/pharmacology , Agammaglobulinaemia Tyrosine Kinase/metabolism , Cytochrome P-450 CYP2C8 Inhibitors/chemical synthesis , Cytochrome P-450 CYP2C8 Inhibitors/chemistry , Dose-Response Relationship, Drug , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/metabolism , Humans , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Deregulation of the c-Myc transcription factor is involved in many types of cancer, making this oncoprotein an attractive target for drug discovery. One approach to its inhibition has been to disrupt the dimeric complex formed between its basic helix-loop-helix leucine zipper (bHLHZip) domain and a similar domain on its dimerization partner, Max. As monomers, bHLHZip proteins are intrinsically disordered (ID). Previously we showed that two c-Myc-Max inhibitors, 10058-F4 and 10074-G5, bound to distinct ID regions of the monomeric c-Myc bHLHZip domain. Here, we use circular dichroism, fluorescence polarization, and NMR to demonstrate the presence of an additional binding site located between those for 10058-F4 and 10074-G5. All seven of the originally identified Myc inhibitors are shown to bind to one of these three discrete sites within the 85-residue bHLHZip domain of c-Myc. These binding sites are composed of short contiguous stretches of amino acids that can selectively and independently bind small molecules. Inhibitor binding induces only local conformational changes, preserves the overall disorder of c-Myc, and inhibits dimerization with Max. NMR experiments further show that binding at one site on c-Myc affects neither the affinity nor the structural changes taking place upon binding to the other sites. Rather, binding can occur simultaneously and independently on the three identified sites. Our results suggest the widespread existence of peptide regions prone to small-molecule binding within ID domains. A rational and generic approach to the inhibition of protein-protein interactions involving ID proteins may therefore be possible through the targeting of ID sequence.
Subject(s)
Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Binding Sites , Drug Discovery , Oxadiazoles , Protein Binding , Spectrum Analysis , Structure-Activity RelationshipABSTRACT
The oncogenic transcription factor c-Myc undergoes coupled binding and folding of its basic-helix-loop-helix-leucine zipper domain (bHLHZip) upon heterodimerization with its partner protein Max. The latter exists in two isoforms: p21, which homodimerizes poorly, and p22, which homodimerizes well. We show that the effect of 10058-F4 (a small-molecule that binds disordered c-Myc monomers and disrupts the c-Myc-Max complex) on both c-Myc-Max heterodimerization and DNA binding is dependent on the nature of the Max isoform. In the presence of p22 Max the effective inhibitor concentration is lower than in the presence of p21 Max, as the p22 Max homodimer formation affects the thermodynamics by competing against the c-Myc-Max heterodimerization event.
Subject(s)
Basic-Leucine Zipper Transcription Factors/chemistry , Protein Binding/drug effects , Proto-Oncogene Proteins c-myc/chemistry , Binding Sites , Chemistry, Pharmaceutical/methods , DNA/chemistry , Dimerization , Drug Design , Humans , Kinetics , Models, Biological , Protein Isoforms , Protein Structure, Tertiary , ThermodynamicsABSTRACT
Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Drug Discovery , Immune System Diseases/drug therapy , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Administration, Oral , Agammaglobulinaemia Tyrosine Kinase/metabolism , Dose-Response Relationship, Drug , Humans , Immune System Diseases/metabolism , Molecular Structure , Piperidines/administration & dosage , Piperidines/chemistry , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Pyrimidines/administration & dosage , Pyrimidines/chemistry , Structure-Activity RelationshipABSTRACT
Compounds that selectively prevent or disrupt the association between the c-Myc oncoprotein and its obligate heterodimeric partner Max (Myc-Max compounds) have been identified previously by high-throughput screening of chemical libraries. Although these agents specifically inhibit the growth of c-Myc-expressing cells, their clinical applicability is limited by their low potency. We describe here several chemical modifications of one of these original compounds, 10058-F4, which result in significant improvements in efficacy. Compared with the parent structure, these analogues show enhanced growth inhibition of c-Myc-expressing cells in a manner that generally correlates with their ability to disrupt c-Myc-Max association and DNA binding. Furthermore, we show by use of a sensitive fluorescence polarization assay that both 10058-F4 and its active analogues bind specifically to monomeric c-Myc. These studies show that improved Myc-Max compounds can be generated by a directed approach involving deliberate modification of an index compound. They further show that the compounds specifically target c-Myc, which exists in a dynamic and relatively unstructured state with only partial and transient alpha-helical content.
Subject(s)
Azepines/pharmacology , Basic-Leucine Zipper Transcription Factors/antagonists & inhibitors , Cell Proliferation/drug effects , Piperidines/pharmacology , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Azepines/chemical synthesis , Azepines/chemistry , Dimerization , Electrophoretic Mobility Shift Assay , Fluorescence , HL-60 Cells , Humans , Immunoblotting , Immunoprecipitation , Molecular Weight , Piperidines/chemical synthesis , Piperidines/chemistry , Rhodanine/chemistryABSTRACT
Under conditions of genotoxic stress, human p53 activates the apoptotic effectors BAX or BAK to result in mitochondrial outer-membrane permeabilization and apoptosis. Antiapoptotic BCL-2 family member BCL-xL opposes this activity by sequestering cytosolic p53 via association with its DNA-binding domain, an interaction enhanced by p53 tetramerization. Here we characterized the BCL-xL-p53 complex by NMR spectroscopy and modulated it through mutagenesis to determine the relative contributions of BCL-xL's interactions with p53 or other BCL-2 family proteins to the BCL-xL-dependent inhibition of UV irradiation-induced apoptosis. Under our experimental conditions, one-third of the antiapoptotic activity of BCL-xL was mediated by p53 sequestration and the remaining two-thirds through sequestration of proapoptotic BCL-2 family members. Our studies define the contributions of cytosolic p53 to UV irradiation-induced apoptosis and provide opportunities to explore its contributions to other p53-dependent apoptotic signaling pathways.
Subject(s)
Tumor Suppressor Protein p53/chemistry , bcl-X Protein/chemistry , Apoptosis/radiation effects , Binding Sites , Cell Nucleus/metabolism , Cytosol/metabolism , Humans , Models, Molecular , Mutagenesis , Nuclear Magnetic Resonance, Biomolecular , Protein Interaction Mapping , Protein Multimerization , Signal Transduction , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/physiology , Ultraviolet Rays , bcl-X Protein/metabolism , bcl-X Protein/physiologyABSTRACT
A three-dimensional pharmacophore model was generated utilizing a set of known inhibitors of c-Myc-Max heterodimer formation. The model successfully identified a set of structurally diverse compounds with potential inhibitory activity against c-Myc. Nine compounds were tested in vitro, and four displayed affinities in the micromolar range and growth inhibitory activity against c-Myc-overexpressing cells. These studies demonstrate the applicability of pharmacophore modeling to the identification of novel and potentially more puissant inhibitors of the c-Myc oncoprotein.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , Models, Molecular , Proto-Oncogene Proteins c-myc/chemistry , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Benzofurans/chemistry , Benzofurans/pharmacology , Cell Line , Cell Proliferation/drug effects , Circular Dichroism , Electrophoretic Mobility Shift Assay , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Knockout Techniques , HL-60 Cells , HMGA1b Protein/biosynthesis , HMGA1b Protein/genetics , Humans , Protein Binding , Protein Multimerization , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Pyridines/chemistry , Pyridines/pharmacology , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Rats , Thiazoles/chemistry , Thiazoles/pharmacologyABSTRACT
The basic-helix-loop-helix-leucine-zipper domains of the c-Myc oncoprotein and its obligate partner Max are intrinsically disordered (ID) monomers that undergo coupled folding and binding upon heterodimerization. We have identified the binding sites and determined the structural means by which two unrelated small molecules, 10058-F4 and 10074-G5, bind c-Myc and stabilize the ID monomer over the highly ordered c-Myc-Max heterodimer. In solution, the molecules bind to distinct regions of c-Myc and thus limit its ability to interact with Max and assume a more rigid and defined conformation. The identification of multiple, specific binding sites on an ID domain suggests that small molecules may provide a general means for manipulating the structure and function of ID proteins, such as c-Myc.