Steven J. Metallo

Intrinsically disordered proteins are potential drug targets

Intrinsically disordered (ID) proteins that lack stable secondary and tertiary structure in substantial regions (or throughout) are prevalent in eukaryotes. They exist as ensembles of rapidly fluctuating structures and many undergo coupled folding and binding reactions. Because ID proteins are overrepresented in major disease pathways they are desirable targets for inhibition; however, the feasibility of targeting proteins without defined structures was unclear. Recently, small molecules have been found that bind to the disordered regions of c-Myc, Abeta, EWS-Fli1, and various peptides. As with structured targets, initial hits were further optimized to increase specificity and affinity. Given the number and biological importance of ID proteins, the ability to inhibit their interactions opens tremendous potential in chemical biology and drug discovery.

Multiple Independent Binding Sites for Small-Molecule Inhibitors on the Oncoprotein c-Myc

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.

Improved low molecular weight Myc-Max inhibitors

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 α-helical content. [Mol Cancer Ther 2007;6(9):2399–408]

Pathological Unfoldomics of Uncontrolled Chaos: Intrinsically Disordered Proteins and Human Diseases

Many biologically important proteins lack stable tertiary and/or secondary structure under physiological conditions in vitro as a whole or in part.1–5 These intrinsically disordered proteins (IDPs), or intrinsically disordered protein regions (IDPRs) of hybrid proteins possessing both structured and disordered domains, do not have unique well-defined 3D structures, existing instead as collapsed or extended dynamically mobile conformational ensembles. Therefore, natural proteins can be found in one of three major protein forms: functional and folded, nonfunctional and misfolded, or functional and intrinsically disordered. Although IDPs and IDPRs are highly dynamic, their structures can be described reasonably well by a rather limited number of lower-energy conformations.6,7 The structural plasticity and conformational adaptability of IDPs/IDPRs and their intrinsic lack of rigid structure leads to a number of exceptional functional advantages, providing them with unique capabilities to act in functional modes not achievable by ordered proteins.5 As a result, intrinsic disorder is a common feature of proteins involved in signaling, regulation, and recognition, and IDPs/IDPRs play diverse roles in modulation and control of their binding partners’ functions and in promoting the assembly of supramolecular complexes. The biological actions of IDPs/IDPRs, which frequently serve as major regulators of their binding partners, are controlled by extensive posttranslational modifications (PTMs), such as phosphorylation, acetylation, ubiquitination, and sumoylation,5 and by alternative splicing.8 In fact, many IDPs/IDPRs are known to contain multiple functional elements that contribute to their ability to be involved in interaction with, regulation of, and control by multiple structurally unrelated partners.9 Given the existence of multiple functions in a single disordered protein, and given that each functional element is typically relatively short, alternative splicing could readily generate sets of protein isoforms with highly diverse regulatory elements.8 The complexity of the disorder-based interactomes is further increased by the capacity of a single IDPR to bind to multiple partners, gaining very different structures in the bound state.10 IDPs can form highly stable complexes or be involved in signaling interactions where they undergo constant “bound–unbound” transitions, thus acting as dynamic and sensitive “on–off” switches. The ability of these proteins to return to highly flexible conformations after the completion of a particular function, and their predisposition to adopt different conformations depending on their environment, are unique physiological properties of IDPs that allow them to exert different functions in different cellular contexts according to a specific conformational state.5 Although the field of protein disorder has started from careful analysis of a very limited number of biologically active proteins without unique structures (which, for a long time, were taken as rare exceptions from the general “one sequence–one unique structure–one unique function” paradigm),1–4 applications of various disorder predictors to different proteomes revealed that IDPs are highly abundant in nature,11–16 and the overall amount of disorder in proteins increases from bacteria to archaea to eukaryota, with over half of all eukaryotic proteins predicted to contain extended IDPRs.11,12,15–17 One explanation for this trend is a change in the cellular requirements for certain protein functions, particularly cellular signaling. In support of this hypothesis, an analysis of a eukaryotic signal protein database indicated that the majority of known signal transduction proteins were predicted to contain significant regions of disorder.18 A detailed study focused on the intricate mechanisms of IDP regulation inside the cell was recently conducted by Gsponer et al.19 These authors grouped all the Saccharomyces cerevisiae proteins into three classes according to their predicted disorder propensities and evaluated the correlations between intrinsic disorder and the various regulation steps of protein synthesis and degradation.19 Although the transcriptional rates of mRNAs encoding IDPs and ordered proteins were comparable, IDP-encoding transcripts were generally less abundant than transcripts encoding ordered proteins because of increased decay rates of IDP mRNAs.19 Also, IDPs were found to be less abundant than ordered proteins because of lower rates of protein synthesis and shorter protein half-lives.19 Curiously, IDPs were shown to be substrates of twice as many kinases as ordered proteins. Furthermore, the vast majority of kinases whose substrates were IDPs were either regulated in a cell-cycle-dependent manner or activated upon exposure to specific stimuli or stress.19 Similar regulation trends were also found in proteomes of Schizosaccharomyces pombe and Homo sapiens,19 suggesting that both unicellular and multicellular organisms use evolutionarily conserved mechanisms to regulate the availability of their IDPs. This tight regulation is directly related to the major roles of IDPs/IDPRs in signaling, where it is crucial for a given protein to be available in appropriate amounts and not to be present longer than needed.19 It was also pointed out5 that although the abundance of many IDPs may be closely regulated, some disordered proteins could be present in cells in large amounts or/and for long periods of time, either due to specific PTMs or via interactions with other factors. These events could promote changes in cellular localization of IDPs or protect them from degradation.3,20–23 Taken together, these data highlight that the chaos seemingly associated with highly flexible and promiscuous IDPs/IDPRs is under tight control.24

Structural Rationale for the Coupled Binding and Unfolding of the c-Myc Oncoprotein by Small Molecules

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.

What's in a name? Why these proteins are intrinsically disordered: Why these proteins are intrinsically disordered.

“What’s in a name? That which we call a rose By any other name would smell as sweet.” From “Romeo and Juliet”, William Shakespeare (1594) This article opens a series of publications on disambiguation of the basic terms used in the field of intrinsically disordered proteins. We start from the beginning, namely from the explanation of what the expression “intrinsically disordered protein” actually means and why this particular term has been chosen as the common denominator for this class of proteins characterized by broad structural, dynamic and functional characteristics.

Discovery of novel Myc-Max heterodimer disruptors with a three-dimensional pharmacophore model.

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.

Small-molecule perturbation of competing interactions between c-Myc and Max

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.

Distribution of labor among bZIP segments in the control of DNA affinity and specificity

BACKGROUND The basic region-leucine zipper (bZIP) family of proteins use an atypically simple motif for DNA recognition, yet family members discriminate differently between target sites that differ only in half-site spacing. Two such sites are the cAMP-response element (CRE) and the AP-1 target site. Fos/Jun prefers the AP-1 site (ATGACTCAT), while CRE-BP1 prefers CRE (ATGACGTCAT), and GCN4 binds both sites with equal affinity. We therefore asked what determines the relative specificity for CRE and AP-1 sites in bZIP proteins. RESULTS Here we show that CRE/AP-1 specificity in CRE-BP1 is encoded within the spacer and basic segments of the bZIP element. Of these two regions, the basic segment is the more important. This specificity is in part achieved at the expense of affinity. CONCLUSIONS The small size and simplicity of the bZIP recognition helix was already unusual; our findings show that the information that determines the target site specificity of members of the bZIP family of proteins is even more condensed than expected. These results suggest that it may be possible to design surprisingly small proteins that bind DNA with high sequence specificity, although it may be more difficult to achieve high-affinity binding in small proteins.

Oriented Protein Adsorption to Gold Nanoparticles through a Genetically Encodable Binding Motif

Simple, stable, and specific methods for immobilizing proteins on gold surfaces are needed for the development of applications that rely on the oriented attachment of proteins to gold surfaces. We report a direct, stable, genetically encodable method for the oriented chemisorption of proteins to gold nanoparticles (Au NPs) through the tetracysteine motif (C-C-P-G-C-C) while simultaneously suppressing protein physisorption. Mutants of ubiquitin (Ub) and enhanced green fluorescent protein (eGFP) containing the tetracysteine motif were produced and displayed stronger adsorption to the NPs than did native proteins. An eGFP mutant with a dicysteine motif (G-C-C) did not show a significant improvement in binding to Au NPs compared to that of the wild-type protein. The binding of the proteins to Au NPs of various sizes (14, 18, 28, and 39 nm) was explored. The small Ub tetracysteine mutant stabilized several sizes of Au NPs, and the eGFP tetracysteine mutant clearly had the strongest chemisorption to the 18 nm NPs. The control of binding orientation for proteins bearing a tetracysteine motif was demonstrated through the enhanced specific binding of protein-NP conjugates to immobilized targets.