Natural Protein-protein Interactions Research Articles

Design of Synthetic Polymer Nanoparticles That Capture and Neutralize Target Molecules

Protein affinity reagents that specifically and strongly bind to target molecules are widely used in disease detection, diagnosis, and therapy. Although antibodies and their fragments are the gold standard in protein-protein inhibitors (PPIs), synthetic polymers such as linear polymers, dendrimers, and nanoparticles as cost-effective PPIs have attracted great attention as alternatives to antibodies. These polymers exhibit high affinity to the target by imitating natural protein-protein interactions. However, only a few in vivo applications have been reported. Here, our recent advances in the development of synthetic polymers for in vivo application are reviewed. Poly(N-isopropylacrylamide) (pNIPAm) was used as a model of synthetic affinity reagents. Incorporation of both sulfated carbohydrate and hydrophobic monomers into lightly crosslinked pNIPAm nanoparticles (NPs) captured and neutralized vascular endothelial growth factor (VEGF) and inhibited tumor growth upon intravenous injection into tumor-bearing mice. Modification of a liposome with the pNIPAm-based linear polymer increased the polymer circulation time after intravenous injection and improved the affinity for the target. The pNIPAm-based NPs delivered by oral administration captured the target small molecules and inhibited their absorption from the intestine. Our recent findings provide useful information for the design of synthetic polymers that capture target molecules in vivo.

Engineered pH-Sensitive Protein G/IgG Interaction.

While natural protein-protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate that a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G/human IgG Fc domain (referred to as PrG/hIgG), when incorporated with histidine and glutamic acid on PrG (PrG-EHHE), showed a switch in binding affinity by 50-fold when the pH was altered from mild acidic to mild basic. The wild-type (WT) interface showed a negligible switch. The overall binding affinity under mild acidic pH for PrG-EHHE outperformed the wild-type PrG (PrG-WT) interaction. The new reagent PrG-EHHE can be revolutionary in IgG purification, since the standard method of using an extreme acidic pH for elution can be circumvented.

Large-scale survey and database of high affinity ligands for peptide recognition modules.

Many proteins involved in signal transduction contain peptide recognition modules (PRMs) that recognize short linear motifs (SLiMs) within their interaction partners. Here, we used large‐scale peptide‐phage display methods to derive optimal ligands for 163 unique PRMs representing 79 distinct structural families. We combined the new data with previous data that we collected for the large SH3, PDZ, and WW domain families to assemble a database containing 7,984 unique peptide ligands for 500 PRMs representing 82 structural families. For 74 PRMs, we acquired enough new data to map the specificity profiles in detail and derived position weight matrices and binding specificity logos based on multiple peptide ligands. These analyses showed that optimal peptide ligands resembled peptides observed in existing structures of PRM‐ligand complexes, indicating that a large majority of the phage‐derived peptides are likely to target natural peptide‐binding sites and could thus act as inhibitors of natural protein–protein interactions. The complete dataset has been assembled in an online database (http://www.prm‐db.org) that will enable many structural, functional, and biological studies of PRMs and SLiMs.

Host-Guest Protein Assembly for Affinity Purification of Methyllysine Proteomes.

Protein-protein interactions drive self-assembly of biomacromolecules and thus enable important physiological functions at a cellular level. Supramolecular chemists have developed artificial host-guest interactions that are similar with, yet distinct from and orthogonal to, the natural protein-protein interactions. For instance, cucurbit[n]urils are synthetic receptors that can specifically recognize proteins with N-terminal aromatic residues with high affinities, yet this interaction can be reversed by the competition of small molecules such as amantadine. Herein, we develop a site-specific, oriented protein-display method by combining the host-guest interaction based on cucurbit[7]uril and a covalent protein-peptide reaction. A methyllysine-binding protein HP1β chromodomain (CD) is immobilized via host-guest interactions and used as the "bait" to capture methyllysine proteomes from cancer cells. The captured "fish"-methyllysine-containing proteins-can be released via competitive displacement by amantadine in a nondenaturing and traceless manner. This affinity purification method found 73 novel methyllysine sites from 101 identified sites among 66 methylated proteins from 255 HP1β CD-binding proteins in cancer cells via subsequent mass spectrometric analysis. This work thereby presents a new strategy of artificial host-guest protein assembly in affinity purification of methyllysine proteins in coupling to mass spectrometry.

Targeted erythropoietin selectively stimulates red blood cell expansion in vivo

The design of cell-targeted protein therapeutics can be informed by natural protein-protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics.

Metal-Directed Design of Supramolecular Protein Assemblies.

Owing to their central roles in cellular signaling, construction, and biochemistry, protein-protein interactions (PPIs) and protein self-assembly have become a major focus of molecular design and synthetic biology. In order to circumvent the complexity of constructing extensive noncovalent interfaces, which are typically involved in natural PPIs and protein self-assembly, we have developed two design strategies, metal-directed protein self-assembly (MDPSA) and metal-templated interface redesign (MeTIR). These strategies, inspired by both the proposed evolutionary roles of metals and their prevalence in natural PPIs, take advantage of the favorable properties of metal coordination (bonding strength, directionality, and reversibility) to guide protein self-assembly with minimal design and engineering. Using a small, monomeric protein (cytochrome cb562) as a model building block, we employed MDPSA and MeTIR to create a diverse array of functional supramolecular architectures which range from structurally tunable oligomers to metalloprotein complexes that can properly self-assemble in living cells into novel metalloenzymes. The design principles and strategies outlined herein should be readily applicable to other protein systems with the goal of creating new PPIs and protein assemblies with structures and functions not yet produced by natural evolution.

High-affinity small-molecule inhibitors of the menin-mixed lineage leukemia (MLL) interaction closely mimic a natural protein-protein interaction.

The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ∼288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin–MLL interaction with IC50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.

Design of affinity peptides from natural protein ligands: A study of the cardiac troponin complex

We describe a general strategy for the design and discovery of affinity peptides for a protein from its natural ligands. Our approach is guided by protein-protein interactions in natural systems and focuses on the hetero-trimeric complex of cardiac troponin I (cTnI), C (cTnC) and T (cTnT). A key premise of this work is that cTnC and cTnT, owing to their innate ability to bind cTnI, are potential templates for the design and discovery of cTnI-binding peptides. Relying only on the knowledge of primary sequences of cTnC and cTnT, we designed a library of short overlapping peptides that span the entirety of cTnC and cTnT and tested them for binding to cTnI. We were successful in identifying several peptides that display high affinity (1-100 nM) for cTnI. The specific implication of this work is that mimicking natural protein-protein interactions is an excellent starting point for the discovery and rational design of peptide ligands. The knowledge of secondary or tertiary structures of the proteins involved is not a necessary precondition for this approach. Nevertheless, we show that structural information can be used to validate the results of a fragment-based peptide design, and can be potentially beneficial for refining the lead candidates. Our approach is broadly applicable to any protein with at least one natural binding ligand with known primary sequence. For protein targets with multiple natural ligands, this approach can potentially yield several distinct affinity peptides capable of simultaneously binding the target protein via orthogonal modes or at complementary interfaces.

Development of α-helical calpain probes by mimicking a natural protein-protein interaction.

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.

Modulating Proteostasis: Peptidomimetic Inhibitors and Activators of Protein Folding

Protein folding in the cell is a complex process with a fine balance between productive and non-productive folding. To modulate, either up-regulating or down-regulating, the level of one specific protein with multiple approaches is possible, including the modulation of catalysed protein folding, the use of chemical and pharmacological chaperones, alteration of natural protein-protein interactions, the regulation of degradative pathways and manipulation of natural control mechanisms, such as the heat shock response and the unfolded protein response. Errors in proteostasis are linked to a wide range of disease states and many examples exist of the successful manipulation of proteostasis for the partial or complete elimination of the disease phenotype, including for many amyloid based diseases such as Parkinson's and Alzheimer's as well as for 'loss-of-function' diseases such as Fabry's and Gaucher's diseases. This review takes an overview of the different approaches that can be used to alter proteostasis with an emphasis on peptidomimetic inhibitors and activators of protein folding. It covers the modulators available, their mechanisms of action and potential limitations, including the problems of specificity in altering proteostasis.

Phage-Display Methodology for the Study of Protein-Protein Interactions: Overview

INTRODUCTIONIn recent years, phage display has evolved into a powerful tool providing opportunities to define natural protein-protein interactions and to mold novel ligand receptors. The essential advantages of phage-display approaches originate in the incorporation of the protein and genetic components into a single phage particle. By providing a direct physical link between the expressed protein and the encoding genetic information, clones with desirable functional capacities can be efficiently subjected to iterative rounds of selection, followed by amplification of the selected sublibrary. Hence, during library selection (or panning), specific phage clones are progressively enriched on the basis of their specificity and affinity for ligand. Thus, relatively rare ligand-binding clones can be rescued rapidly and efficiently from large libraries. As these expression cloning systems have matured, versatile selection methods have been reported that are based on the functional properties of displayed proteins in diverse immunochemical and biological settings. This article summarizes phage-display methodology, including relevant biology, nucleotide-doping strategies, and considerations for library design.

Structure-Function Studies of Two Synthetic Anti-vascular Endothelial Growth Factor Fabs and Comparison with the Avastin™ Fab

In the quest to discover new research tools and to develop better agents in the fight against cancer, two antibodies, G6 and B20-4, were isolated from synthetic antibody phage libraries. Unlike the AVASTINtrade mark antibody, a recently approved agent for the treatment of patients with colorectal cancer, B20-4 and G6 bind and block both human and murine vascular endothelial growth factor (VEGF). Here we have analyzed and compared the binding epitopes on VEGF for these three antibodies using alanine-scanning mutagenesis and structural analyses. The epitopes recognized by both synthetic antibodies are conserved between human and mouse VEGF, and they match closely to the receptor epitopes both structurally and functionally. In contrast, the Avastin epitope overlaps minimally with the receptor binding surface and centers around a residue that is not conserved in mouse. Our structural and functional analyses elucidate the cross-species reactivity of all three antibodies and emphasize the potential advantages of antibody generation using phage display as the resulting antibodies do not depend on sequence differences across species and preferentially target natural protein-protein interaction surfaces.