Discovery of High-Affinity Protein Binding Ligands – Backwards

BACKGROUND: There is a pressing need for high-affinity protein binding ligands for all proteins in the human and other proteomes. Numerous groups are working to develop protein binding ligands but most approaches develop ligands using the same strategy in which a large library of structured ligands is screened against a protein target to identify a high-affinity ligand for the target. While this methodology generates high-affinity ligands for the target, it is generally an iterative process that can be difficult to adapt for the generation of ligands for large numbers of proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a class of peptide-based protein ligands, called synbodies, which allow this process to be run backwards--i.e. make a synbody and then screen it against a library of proteins to discover the target. By screening a synbody against an array of 8,000 human proteins, we can identify which protein in the library binds the synbody with high affinity. We used this method to develop a high-affinity synbody that specifically binds AKT1 with a K(d)<5 nM. It was found that the peptides that compose the synbody bind AKT1 with low micromolar affinity, implying that the affinity and specificity is a product of the bivalent interaction of the synbody with AKT1. We developed a synbody for another protein, ABL1 using the same method. CONCLUSIONS/SIGNIFICANCE: This method delivered a high-affinity ligand for a target protein in a single discovery step. This is in contrast to other techniques that require subsequent rounds of mutational improvement to yield nanomolar ligands. As this technique is easily scalable, we believe that it could be possible to develop ligands to all the proteins in any proteome using this approach

Characterization of a Novel Interaction between Bcl-2 Members Diva and Harakiri

Interactions within proteins of the Bcl-2 family are key in the regulation of apoptosis. The death-inducing members control apoptotic mechanisms partly by antagonizing the prosurvival proteins through heterodimer formation. Structural and biophysical studies on these complexes are providing important clues to understand their function. To help improve our knowledge on protein-protein interactions within the Bcl-2 family we have studied the binding between two of its members: mouse Diva and human Harakiri. Diva has been shown to perform both prosurvival and killing activity. In contrast, Harakiri induces cell death by interacting with antiapoptotic Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Combining the NMR data with the previously reported three-dimensional structure of Diva we find that Harakiri binds to a specific region in Diva. This interacting surface is equivalent to the known binding area of prosurvival Bcl-2 members from the reported structures of the complexes, suggesting that Diva could function at the structural level similarly to the antiapoptotic proteins of the Bcl-2 family. We illustrate this result by building a structural model of the heterodimer using molecular docking and the NMR data as restraints. Moreover, combining circular dichroism and NMR we also show that Harakiri is largely unstructured with residual (13%) α-helical conformation. This result agrees with intrinsic disorder previously observed in other Bcl-2 members. In addition, Harakiri constructs of different length were studied to identify the region critical for the interaction. Differential affinity for Diva of these constructs suggests that the amino acid sequence flanking the interacting region could play an important role in binding

Computational Fragment-Based Binding Site Identification by Ligand Competitive Saturation

Fragment-based drug discovery using NMR and x-ray crystallographic methods has proven utility but also non-trivial time, materials, and labor costs. Current computational fragment-based approaches circumvent these issues but suffer from limited representations of protein flexibility and solvation effects, leading to difficulties with rigorous ranking of fragment affinities. To overcome these limitations we describe an explicit solvent all-atom molecular dynamics methodology (SILCS: Site Identification by Ligand Competitive Saturation) that uses small aliphatic and aromatic molecules plus water molecules to map the affinity pattern of a protein for hydrophobic groups, aromatic groups, hydrogen bond donors, and hydrogen bond acceptors. By simultaneously incorporating ligands representative of all these functionalities, the method is an in silico free energy-based competition assay that generates three-dimensional probability maps of fragment binding (FragMaps) indicating favorable fragment∶protein interactions. Applied to the two-fold symmetric oncoprotein BCL-6, the SILCS method yields two-fold symmetric FragMaps that recapitulate the crystallographic binding modes of the SMRT and BCOR peptides. These FragMaps account both for important sequence and structure differences in the C-terminal halves of the two peptides and also the high mobility of the BCL-6 His116 sidechain in the peptide-binding groove. Such SILCS FragMaps can be used to qualitatively inform the design of small-molecule inhibitors or as scoring grids for high-throughput in silico docking that incorporate both an atomic-level description of solvation and protein flexibility

An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe

A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligand–protein complex is determined. PRE is an isotropic paramagnetic effect observed within 30 Å from the lanthanide ion, and is utilized for the ligand screening in the present study. PCS is an anisotropic paramagnetic effect providing long-range (~40 Å) distance and angular information on the observed nuclei relative to the paramagnetic lanthanide ion, and utilized for the structure determination of the ligand–protein complex. Since a two-point anchored lanthanide-binding peptide tag is utilized for fixing the lanthanide ion to the target protein, this screening method can be generally applied to non-metal-binding proteins. The usefulness of this strategy was demonstrated in the case of the growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain and its low- and high-affinity ligands

Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme

Molecular Design, Functional Characterization and Structural Basis of a Protein Inhibitor Against the HIV-1 Pathogenicity Factor Nef

Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral pathogenicity factor that interacts with host cell factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as “wrapping Nef”, is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors