Use Of Phage Display Research Articles

Characterization of mAbs against Klebsiella pneumoniae type 3 fimbriae isolated in a target-independent phage display campaign.

We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of Klebsiella pneumoniae. We report the discovery of monoclonal antibodies (mAbs) binding to type 3 fimbrial proteins, including MrkA. We found that anti-MrkA mAbs were cross-reactive to a diverse panel of K. pneumoniae clinical isolates, representing different O-serotypes. mAbs binding to MrkA have previously been described and have been shown to provide prophylactic protection, although only modest protection when dosed therapeutically in vivo in a murine lung infection model. Here, we used a combination of binding and opsonophagocytic killing studies using a high-content imaging platform to provide a possible explanation for the modest therapeutic efficacy in vivo reported in that model. Our work shows that expression of K. pneumoniae type 3 fimbriae in in vitro culture is not homogenous within a bacterial population. Instead, sub-populations of bacteria that do, and do not, express type 3 fimbriae exist. In a high-content opsonophagocytic killing assay, we showed that MrkA-targeting antibodies initially promote killing by macrophages; however, over time, this effect is diminished. We hypothesize the reason for this is that bacteria not expressing MrkA can evade opsonophagocytosis. Our data support the fact that MrkA is a conserved, immunodominant protein that is antibody accessible on the surface of K. pneumoniae and suggest that additional studies should evaluate the potential of using anti-MrkA antibodies in different stages of K. pneumoniae infection (different sites in the body) as well as against K. pneumoniae biofilms in the body during infection and associated with medical devices.IMPORTANCEThere is an unmet, urgent need for the development of novel antimicrobial therapies for the treatment of Klebsiella pneumoniae infections. We describe the use of phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of K. pneumoniae. We discovered monoclonal antibodies (mAbs) binding to the type 3 fimbrial protein MrkA. The anti-MrkA mAbs were found to be highly cross-reactive, binding to all K. pneumoniae strains tested from a diverse panel of clinical isolates, and were active in an opsonophagocytic killing assay at pM concentrations. MrkA is important for biofilm formation; thus, our data support further exploration of the use of anti-MrkA antibodies for preventing and/or controlling K. pneumoniae in biofilms and during infection.

Use of Phage Display and Other Molecular Display Methods for the Development of Monobodies.

Synthetic binding proteins are human-made binding proteins that use non-antibody proteins as the starting scaffold. Molecular display technologies, such as phage display, enable the construction of large combinatorial libraries and their efficient sorting and, thus, are crucial for the development of synthetic binding proteins. Monobodies are the founding system of a set of synthetic binding proteins based on the fibronectin type III (FN3) domain. Since the original report in 1998, the monobody and related FN3-based systems have steadily been refined, and current methods are capable of rapidly generating potent and selective binding molecules to even challenging targets. The FN3 domain is small (∼90 amino acids) and autonomous and is structurally similar to the conventional immunoglobulin (Ig) domain. Unlike the Ig domain, however, the FN3 lacks a disulfide bond but is highly stable. These attributes of FN3 present unique opportunities and challenges in the design of phage and other display systems, combinatorial libraries, and library sorting strategies. This article reviews key technological innovations in the establishment of our monobody development pipeline, with an emphasis on phage display methodology. These give insights into the molecular mechanisms underlying molecular display technologies and protein-protein interactions, which should be broadly applicable to diverse systems intended for generating high-performance binding proteins.

Novel peptide identified from viable-cell based phage display technique regulates growth cycle of Daphnia magna

Phage display is a widely used technique for selecting specific binding peptides, but presenting antigens in their natural form can be challenging, as protein coating may induce structural changes. In this study, we employed a whole cell-based phage display technique without a coating step to select peptides that bind specifically to Daphnia magna eggs. Boiled eggs were used as a control to ensure that antigens were presented in their natural forms. We identified a peptide, DEP1 (LYALPLSHLKSHGGG), with the highest binding affinity to D. magna eggs. DEP1 did not affect zebrafish eggs, but it inhibited normal hatching and reproductive ability in D. magna eggs, and hindered growth in neonates before their first ecdysis. Morphological analysis revealed that DEP1 caused intestinal damage and tissue abnormalities. Our findings demonstrate that the whole cell-based phage display technique is successful in presenting antigens in their natural form, and that the DEP1 peptide can be applied to regulate the growth cycle of D. magna. These results have implications for the use of phage display in environmental research and the potential use of DEP1 for hazardous organisms in aquatic systems.

Identification and Characterization of MUC5B Binding Peptides by Phage Display

ObjectivesMUC5B plays a multifactorial role in oral health. As a consequence, decreased MUC5B output leads to impaired salivary functions and xerostomia. Synthetic combinatorial technologies have been used to develop functional peptide libraries by phage display e.g. for therapeutic purposes. In this light, our primary aim was to identify peptide sequences with specific selectivity for salivary MUC5B in vitro using phage display. Our secondary aims were to analyze their effect on salivary spinnbarkeit in situ and their effect on acid-induced demineralization in vitro. MethodsMUC5B binding phages were selected by phage display. Peptide affinity to MUC5B was evaluated using MUC5B coated hydroxyapatite (HA) granules. The MUC5B binding peptides (MBPs) were then examined for their effects on salivary spinnbarkeit and protective effect on acid-induced demineralization in vitro. A competitive ELISA was performed to identify the binding epitope on MUC5B using F2, a MUC5B specific antibody. ResultsMBP-12 and MBP-14 displayed the highest affinity to MUC5B. MBP-12 mildly stabilized the spinnbarkeit of serous saliva after overnight incubation and of mucous saliva at all timepoints tested. The addition of MBP-12 to a pellicle of unstimulated saliva on HA discs showed no additive protective effect against acid-induced demineralization. Epitope characterization suggested sulfo-Lewisa SO3–3Gal_1–3GlcNAc (galactose residue) as MBP-12 binding site on MUC5B. ConclusionsThe use of phage display in generating MBPs was successful. Characterization of the MBPs revealed a mild effect on spinnbarkeit in case of mucous saliva. Possibly, combinatorial peptide libraries might contribute to the development of novel formulations to treat xerostomia.

Alternative use of phage display: phage M13 can remain viable in the intestines of poultry without causing damage

Phage display (PD) is a tool for developing new molecules to control pathogens. Peptides selected by PD are commonly synthesised and tested, but the use of phage M13 displaying the selected peptides as a direct biding in the intestinal tract has not yet been tested. This study evaluated whether phage M13 can remain viable in the chicken gastrointestinal tract and whether it causes injury or humoral immune response. We inoculated phage M13 or E. coli ER2738 (ECR) infected with M13 into birds at different ages. We found the virus in faeces at 5 or 13 days after inoculation, just when it infected the ECR. The presence of phage M13 or ECR did not result in gut injuries and had no impacts on weight gain and bird health. Furthermore, the levels of IgY were similar in all treatments, which indicates that the virus can be used in chicken until 42 days without being recognised by the immune system. This work provides a scientific basis for the use of PD as a tool in numerous applications to control different pathogens.

High-performance immune diagnosis of tuberculosis: Use of phage display and synthetic peptide in an optimized experimental design

Immunoassays are practical and cost-effective approaches suitable for large-scale tuberculosis (TB) screening. This study identified new peptide mimotopes of Mycobacterium tuberculosis and applied them in the serodiagnosis of TB. Thereby, linear (X15, X8CX8) and constrained (LX-4 and LX-8) phage display peptide libraries were screened with purified Immunoglobulin G antibodies from TB-positive patients, and eight mimotopes were selected. The mimotope peptides were screened using the SPOT-synthesis technique followed by immunoblotting. Peptides P.Mt.PD.4 and P.Mt.PD.7 demonstrated the highest binding affinity and were chemically synthesized and used as antigens for enzyme-linked immunosorbent assay (ELISA) assays. Experimental designs were used to optimize the assays and to assess each variable's influence. Peptide P.Mt.PD.7 was differentiated between positive and negative samples and achieved 100% sensitivity and specificity when tested on a 100-sera panel. Therefore, the selected peptide was applied to the ELISA assay as a screening method for diagnosing TB represents a potential tool for helping to combat the disease.

Structure-function properties of hypolipidemic peptides.

This review addresses the structure-function properties of hypolipidemic peptides. The cholesterol-lowering peptide (lactostatin: IIAEK) operates via a new regulatory pathway in the calcium-channel-related mitogen-activated protein kinase (MAPK) signaling pathway of cholesterol degradation. The bile acid binding peptide (soystatin, VAWWMY) inhibits the micellar solubility of cholesterol in vitro and cholesterol absorption in vivo. VVYP is the most effective peptide having hypotriglyceridemic action in globin digests. The suppressive effect of globin digest on postprandial hyperlipidemia has been reported in humans. The ability of peptides (KRES, Apolipoprotein A-I mimetic peptides) to interact with lipids, remove LOOH and activate antioxidant enzymes associated with high-density lipoprotein determines their anti-inflammatory and anti-atherogenic properties. The β-conglycinin derived peptides KNPQLR, EITPEKNPQLR, and RKQEEDEDEEQQRE inhibit fatty acid synthase in vitro. These promising findings indicate the need for more conclusive molecular, cellular, and animal and human studies to design innovative new peptides that ameliorate cholesterol and lipid metabolism. PRACTICAL APPLICATIONS: Prevention and amelioration of hypercholesterolemia by dietary regulation are important. Dietary protein and peptides are very useful as regulators of serum cholesterol concentration. Diets low in saturated fat and cholesterol that include soy protein may reduce the risk of heart disease. In Japan, the concept of "food for specified health use" has been introduced for the prevention and treatment of life-style related disease. Thus, peptides derived from food proteins and sources other than food proteins such as peptide-rich functional foods and nutraceutical products, have considerable potential to prevent lifestyle-related diseases, especially hyperlipidemia, as discussed in this review. Furthermore, various strategies have been used for the efficient screening, development, and application of new hypolipidemic peptides. These include the use of phage display (for anti-obesity peptide), peptide mimetics (for anti-atherogenic peptide), and molecular targets such as CYP7A1 (for hypocholesterolemic peptide) and prohibitin (for anti-obesity peptide).

Identification and function probing of an antithrombin IIIβ conformation‐specific antibody

ESSENTIALS: Antithrombin III (AT)β binds heparin with higher affinity than ATα. A conformation-specific antibody against ATβ, TPP2009, was made to investigate ATβ in hemostasis. TPP2009 bound specifically to heparin-ATβ and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATβ in hemostasis. Antithrombin III (AT)β is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. To characterize a conformation-specific antibody against ATβ and begin to investigate the role of ATβ in maintaining hemostasis. Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATβ (ATβ*H) by the use of phage display. SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATβ*H and glycosaminoglycan-associated ATβ on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATβ and heparin. In purified systems with ATβ and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies. TPP2009 specifically targets a unique conformational epitope on ATβ*H and blocks ATβ-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATβ in hemostasis.

Mimtags: The use of phage display technology to produce novel protein-specific probes

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies.

Uses of phage display in agriculture: a review of food-related protein-protein interactions discovered by biopanning over diverse baits.

This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s) contributing most to the allergenic reaction and how it has played a central role in novel approaches to mitigate patient response. Finally, an overview of the use of phage display in identifying the mature seed proteome protection and repair mechanisms is provided. The identification of specific classes of proteins preferentially bound by such protection and repair proteins leads to hypotheses concerning the importance of safeguarding the translational apparatus from damage during seed quiescence and environmental perturbations during germination. These examples, it is hoped, will spur the use of phage display in future plant science examining protein-ligand interactions.

Identification of Human‐Flavonoid Targets Using an Innovative Approach Reveals New Mechanisms Involved in Their Anti‐Inflammatory Activities

Flavonoids are dietary nutraceuticals with anti‐carcinogenic, chemopreventive, and anti‐inflammatory activities. We showed that apigenin, a flavone broadly found in fruits and vegetables, has anti‐inflammatory activity in vitro and in a mouse model of inflammation through the regulation of NF‐κB. However, its molecular mechanisms of action remain unknown. The goal of this study was to identify the human cellular targets of apigenin. We developed and implemented a novel approach coupling phage‐display with next‐generation sequencing (PD‐Seq) to identify human cellular targets of apigenin. Targets were validated using competition and pull down assays, with purified GST and GFP‐tagged proteins and cellular lysates. Development of a FRET‐nanosensor using a newly identified target provided insightful structure‐binding relationships of flavonoids. Molecular cell‐based studies showed that the apigenin target plays central roles in inflammation and reveals novel aspects on how nutraceuticals regulate immunity. Altogether, these results suggest that the innovative use of phage display in combination with next‐generation sequencing provides a robust method to identify novel targets of flavonoids. The findings reveal novel aspects on how nutraceuticals regulate biological activities providing better insights for the rational development of functional foods.Grant Funding Source : This work was supported by Grants RO1HL075040‐01 and NSF‐MCB‐0542244 to AID and by OSUPHPID fellowship to DA

Alternative scaffold proteins

Review is devoted to the challenging direction in modem molecular biology and bioengineering - the properties of alternative scaffold proteins (ASP) and methods for obtaining ASP binding molecules. ASP molecules incorporate conservative protein core and hypervariable regions, providing for the binding function. Structural classification of ASP includes several types which differ also in their molecular targets and potential applications. Construction of artificial binding proteins on the ASP basis implies a combinatorial library design with subsequent selection of specific binders with the use of phage display or the modem cell-free systems. Alternative binding proteins on non-immunoglobulin scaffolds find broad applications in different fields ofbiotechnology and molecular medicine.

Phage display of combinatorial peptide libraries: application to antiviral research.

Given the growing number of diseases caused by emerging or endemic viruses, original strategies are urgently required: (1) for the identification of new drugs active against new viruses and (2) to deal with viral mutants in which resistance to existing antiviral molecules has been selected. In this context, antiviral peptides constitute a promising area for disease prevention and treatment. The identification and development of these inhibitory peptides require the high-throughput screening of combinatorial libraries. Phage-display is a powerful technique for selecting unique molecules with selective affinity for a specific target from highly diverse combinatorial libraries. In the last 15 years, the use of this technique for antiviral purposes and for the isolation of candidate inhibitory peptides in drug discovery has been explored. We present here a review of the use of phage display in antiviral research and drug discovery, with a discussion of optimized strategies combining the strong screening potential of this technique with complementary rational approaches for identification of the best target. By combining such approaches, it should be possible to maximize the selection of molecules with strong antiviral potential.

Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage

BackgroundPhage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display.Methodology/Principal FindingsHere we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient.Conclusions/SignificanceDisplay through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.

Fully in vitro selection of recombinant antibodies

Antibodies are essential for the identification and characterization of proteins. In the current postgenomic era the need for highly specific antibodies has further increased not only for research applications but also because they represent one of the most promising therapeutic options, especially in the field of cancer treatment. One appealing approach for rapid and inexpensive antibody generation is the use of phage display. This technique allows for a fast and animal-free selection of highly functional alternatives to classical antibodies. However, one strong limitation of this recombinant approach has been the difficulty in producing and purifying antigens. These steps have to be adjusted for each new target, are time consuming and sometimes present an insurmountable obstacle. Here we report the development of new antibody selection approach where antigens are produced through in vitro translation and are used directly and without the need for purification. With this approach we were able to rapidly select recombinant antibodies directed against GFP and the mammalian protein tsg101, respectively. We believe that our method greatly facilitates antigen preparation and thus may broaden the use of the recombinant approach for antibody generation, especially since the technique could in the future be adapted to a high-throughput technology, thus further accelerating antibody selection.

Identification and Rational Redesign of Peptide Ligands to CRIP1, A Novel Biomarker for Cancers

Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of cancers. Here we report on the use of phage display in combination with molecular modeling to identify a high-affinity ligand for CRIP1. Panning experiments using a circularized C7C phage library yielded several consensus sequences with modest binding affinities to purified CRIP1. Two sequence motifs, A1 and B5, having the highest affinities for CRIP1, were chosen for further study. With peptide structure information and the NMR structure of CRIP1, the higher-affinity A1 peptide was computationally redesigned, yielding a novel peptide, A1M, whose affinity was predicted to be much improved. Synthesis of the peptide and saturation and competitive binding studies demonstrated approximately a 10–28-fold improvement in the affinity of A1M compared to that of either A1 or B5 peptide. These techniques have broad application to the design of novel ligand peptides.

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.

Specificity Rescue and Affinity Maturation of a Low-Affinity IgM Antibody against Pro-Gastrin-Releasing Peptide using Phage Display and DNA Shuffling

The objectives of the present study were to use phage display to rescue the specificity of an IgM antibody and by the use of DNA shuffling to construct a sublibrary from which mutants with higher affinity could be selected. As a test system, a hybridoma cell line producing low-affinity IgM against recombinant pro-gastrin-releasing peptide (ProGRP) was chosen as starting material for construction of a single-chain Fv (scFv) phage library. One clone, pGRP5, demonstrating affinity for the antigen, was selected for the study. Random mutations were introduced into the scFv sequence by DNA shuffling, and mutants with raised affinity were selected by biopanning against recombinant ProGRP. Clones with affinities improved by approximately two orders of magnitude were selected after the first round of DNA shuffling. An additional 8- to 9-fold rise in affinity was demonstrated in mutants after the second round of mutagenesis. Sequence analysis demonstrated changes primarily in the complementarity-determining region (CDR) H1, CDR L1 and CDR H3 as compared to the original clone. Thus, by the use of phage display in combination with DNA shuffling, the specificity of an IgM antibody was rescued and the affinity was raised almost three orders of magnitude.

Targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-associated virus vectors.

Gene therapy offers an unprecedented opportunity to treat diverse pathologies. Adeno-associated virus (AAV) is a promising gene delivery vector for cardiovascular disease. However, AAV transduces the liver after systemic administration, reducing its usefulness for therapies targeted at other sites. Because vascular endothelial cells (ECs) are in contact with the bloodstream and are heterogeneous between organs, they represent an ideal target for site-specific delivery of biological agents. We isolated human venous EC-targeting peptides by phage display and genetically incorporated them into AAV capsids after amino acid 587. Peptide-modified AAVs transduced venous (but not arterial) ECs in vitro, whereas hepatocyte transduction was significantly lower than with native AAV. Intravenous infusion of engineered AAVs into mice produced reduced vector accumulation in liver measured 1 hour and 28 days after injection and delayed blood clearance rates compared with native AAV. Peptide-modified AAVs produced enhanced uptake of virions in the vena cava with selective transgene expression. Retargeting was dose dependent, and coinfusion of either heparin or free competing peptides indicated that uptake was principally independent of native AAV tropism and mediated via the peptide. AAV tropism can be genetically engineered by use of phage display-derived peptides to generate vectors that are selective for the vasculature.

Use of phage display and polyvalency to design inhibitors of protein-protein interactions.

We describe the synthesis of an inhibitor that interferes with critical protein-protein interactions occurring during the assembly of anthrax toxin. Using a phage display selection strategy, we isolated a peptide directed against the cell binding moiety of the toxin that was able to interfere with binding of the enzymatic moieties. Because the cell binding moiety of the toxin is a heptamer, the peptide can potentially bind up to seven equivalent sites. We synthesized a polyvalent molecule displaying multiple copies of this peptide and showed that it is a much more potent inhibitor than the free peptide. Because little structural knowledge of the interacting proteins was required to synthesize this inhibitor, we believe that this approach may prove useful in the design of inhibitors of protein-protein interactions in other systems.