Portability of a Small-Molecule Binding Site between Disordered Proteins.

Intrinsically disordered proteins (IDPs) are important in both normal and disease states. Small molecules can be targeted to disordered regions, but we currently have only a limited understanding of the nature of small-molecule binding sites in IDPs. Here, we show that a minimal small-molecule binding sequence of eight contiguous residues derived from the Myc protein can be ported into a different disordered protein and recapitulate small-molecule binding activity in the new context. We also find that the residue immediately flanking the binding site can have opposing effects on small-molecule binding in the different disordered protein contexts. The results demonstrate that small-molecule binding sites can act modularly and are portable between disordered protein contexts but that residues outside of the minimal binding site can modulate binding affinity.

Drugging the "Undruggable" MYCN Oncogenic Transcription Factor: Overcoming Previous Obstacles to Impact Childhood Cancers.

Effective treatment of pediatric solid tumors has been hampered by the predominance of currently "undruggable" driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance in vivo, the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein-protein or protein-DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this "undruggable" transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.

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.

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.

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.

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 alpha-helical content.

Characterization of the Binding of Small Molecules to Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) comprise a large fraction of eukaryotic proteomes. IDPs are prevalent in cellular regulation, signaling networks, and disease pathways. The abundance and activity of IDPs is tightly controlled at multiple levels, and their dysregulation is associated with disease. Because of the importance of IDPs in both normal and disease states of the cell, IDPs are attractive targets for modulation by small molecules both to understand their biology and to provide potential drug leads. Multiple screens have successfully identified small molecules that bind to IDPs. Here, we describe how surface plasmon resonance, NMR, and fluorescence methods can be used to characterize the direct binding affinity between small molecules and IDPs. We describe how these techniques can contribute to identifying previously unknown small-molecule binding sites on IDPs.

Promotion of opsonization by antibodies and phagocytosis of Gram-positive bacteria by a bifunctional polyacrylamide.

This paper describes the application of a bifunctional polyacrylamide (pA-V-F) presenting both vancomycin and fluorescein groups, to modify the surfaces of multiple species of Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis) to control molecular recognition of these surfaces. The vancomycin groups allowed the specific recognition of a structural component of the bacterial cell wall: peptides terminated in D-Ala-D-Ala. The fluorescein groups allowed the imaging of binding of polymer to the surfaces of bacteria by fluorescence, and are representative, low molecular weight haptens; their recognition by anti-fluorescein antibodies provides proof-of-principle that bifunctional polymers can be used to introduce haptens onto the surface of the bacteria. Flow cytometry revealed that polymer-labeled S. aureus and S. pneumoniae were opsonized by anti-fluorescein antibodies approximately 20-fold more than were untreated bacteria; nearly all ( approximately 92%) polymer-labeled S. aureus, and a large (76%) fraction of polymer-labeled S. pneumoniae were opsonized. The bound antibodies then promoted phagocytosis of the bacteria by cultured J774 macrophage-like cells. Flow cytometry revealed that macrophages ingested S. aureus decorated with the polymer-antibody complexes approximately 2-fold more efficiently than S. aureus in control groups, in spite of the high background (caused by efficient antibody-independent ingestion of S. aureus by macrophages). This paper, thus, demonstrates the ability of a bifunctional polymer to carry out three distinct functions based on polyvalent molecular recognition: (i) recognition of the surface of Gram-positive bacteria, (ii) modification of this surface to generate specific binding sites recognized by an antibody, and (iii) promotion of phagocytosis of the opsonized bacteria.

A high-throughput screening assay for fungicidal compounds against Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus that causes meningitis worldwide, particularly in human immunodeficiency virus (HIV)-infected individuals. Although amphotericin B is the "gold standard" treatment for cryptococcal meningitis, the toxicity and inconvenience of intravenous injection emphasize a need for development of new anticryptocccal drugs. Recent data from humans and animal studies suggested that a nutrient-deprived host environment may exist in cryptococcal meningitis. Thus, a screening assay for identifying fungicidal compounds under nutrient-deprived conditions may provide an alternative strategy to develop new anticryptococcal drugs for this disease. A high-throughput fungicidal assay was developed using a profluorescent dye, alamarBlue, to detect residual metabolic activity of C. neoformans under nutrient-limiting conditions. Screening the Library of Pharmacologically Active Compounds (LOPAC) with this assay identified a potential chemical scaffold, 10058-F4, that exhibited fungicidal activity in the low micromolar range. These results thus demonstrate the feasibility of this alamarBlue-based assay for high-throughput screening of fungicidal compounds under nutrient-limiting conditions for new anticryptococcal drug development.

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.

Single enantiomer of YK-4-279 demonstrates specificity in targeting the oncogene EWS-FLI1.

Oncogenic fusion proteins, such as EWS-FLI1, are excellent therapeutic targets as they are only located within the tumor. However, there are currently no agents targeted toward transcription factors, which are often considered to be 'undruggable.' A considerable body of evidence is accruing that refutes this claim based upon the intrinsic disorder of transcription factors. Our previous studies show that RNA Helicase A (RHA) enhances the oncogenesis of EWS-FLI1, a putative intrinsically disordered protein. Interruption of this protein-protein complex by small molecule inhibitors validates this interaction as a unique therapeutic target. Single enantiomer activity from a chiral compound has been recognized as strong evidence for specificity in a small molecule-protein interaction. Our compound, YK-4-279, has a chiral center and can be separated into two enantiomers by chiral HPLC. We show that there is a significant difference in activity between the two enantiomers. (S)-YK-4-279 is able to disrupt binding between EWS-FLI1 and RHA in an immunoprecipitation assay and blocks the transcriptional activity of EWS-FLI1, while (R)-YK-4-279 cannot. Enantiospecific effects are also established in cytotoxicity assays and caspase assays, where up to a log-fold difference is seen between (S)-YK-4-279 and the racemic YK-4-279. Our findings indicate that only one enantiomer of our small molecule is able to specifically target a protein-protein interaction. This work is significant for its identification of a single enantiomer effect upon a protein interaction suggesting that small molecule targeting of intrinsically disordered proteins can be specific. Furthermore, proving YK-4-279 has only one functional enantiomer will be helpful in moving this compound towards clinical trials.

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.

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.

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.

Using bifunctional polymers presenting vancomycin and fluorescein groups to direct anti-fluorescein antibodies to self-assembled monolayers presenting d-alanine-d-alanine groups.

This paper describes the synthesis of bifunctional polyacrylamides containing pendant vancomycin (Van) and fluorescein groups, and the use of these polymers to direct antibodies against fluorescein to self-assembled monolayers (SAMs) presenting d-alanine-d-alanine (dAdA) groups. These polymers bind biospecifically to these SAMs via interactions between the dAdA and Van groups and serve as a molecular bridge between the anti-fluorescein antibodies and the SAM. The binding events were characterized using surface plasmon resonance spectroscopy and fluorescence microscopy. The paper demonstrates that polyvalent, biospecific, noncovalent interactions between a polymer and a surface can be used to tailor the properties of the surface in molecular recognition. It also represents a first step toward the design of polymers that direct arbitrarily chosen antibodies to the surfaces of cells.

Arrays of self-assembled monolayers for studying inhibition of bacterial adhesion.

This paper describes a simple and convenient method for the rapid screening of potential inhibitors of bacterial adhesion and for the quantitative evaluation of the efficacy of the inhibitors using arrays of self-assembled monolayers (SAMs) of alkanethiolates on gold that are presented on a 96-well microtiter plate. The SAMs present mixtures of alpha-D-mannopyranoside (a ligand that promotes the adhesion of uropathogenic Escherichia coli by binding to the FimH proteins on the tip of type 1 pili), and tri(ethylene glycol) moieties (organic groups that resist nonspecific adsorption of proteins and cells). The SAMs provide surfaces for studies of adhesion of uropathogenic E. coli to specific ligands; they also provide excellent resistance to nonspecific adhesion. Using arrays of mannoside-presenting SAMs, inhibitors of bacterial adhesion were easily screened by observing the number of bacteria that adhered to the surface of the SAMs in the presence of inhibitor. The potency of the inhibitor was quantified by measuring the percentage of inhibition as a function of the concentration of the inhibitor. The properties of SAMs, when combined with the convenience and standardization of a microtiter plate, make arrays of SAMs a versatile tool that can be applied to high-throughput screening of inhibitors of bacterial, viral, and mammalian cell adhesion and of strongly binding ligands for proteins.

Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.

A solid-object printer was used to produce masters for the fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS). The printer provides an alternative to photolithography for applications where features of > 250 microm are needed. Solid-object printing is capable of delivering objects that have dimensions as large as 250 x 190 x 200 mm (x, y, z) with feature sizes that can range from 10 cm to 250 microm. The user designs a device in 3-D in a CAD program, and the CAD file is used by the printer to fabricate a master directly without the need for a mask. The printer can produce complex structures, including multilevel features, in one unattended printing. The masters are robust and inexpensive and can be fabricated rapidly. Once a master was obtained, a PDMS replica was fabricated by molding against it and used to fabricate a microfluidic device. The capabilities of this method are demonstrated by fabricating devices that contain multilevel and tall features, devices that cover a large area (approximately 150 cm2), and devices that contain nonintersecting, crossing channels.