Picomolar Affinity Research Articles

Abstract LB-325: New synergistic mTOR-based combinations for the treatment of Ewing sarcoma

Abstract Ewing sarcoma (ES), an aggressive bone cancer in children and young adults, is dependent upon the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) cell-signaling axis for malignancy, survival, and metastasis. Individual targeting of mTOR has negligible anti-tumor activity in ES, and single-agent IGF-1R targeting leads to only transient effects that benefit only 10-14% of patients. Co-targeting strategies using IGF-1R Abs combined with mTOR inhibitors were shown in 2012 to increase response rates to 29% but the high toxicity profile limited subsequent clinical development. While an ongoing COG protocol combines IGF-1R Abs with standard-of-care cytotoxic chemotherapy in newly diagnosed ES patients harboring metastatic disease, this combination is unlikely to benefit those who have recurred or developed chemoresistant disease. Herein, we present several highly synergistic drug combinations using novel biologically-targeted therapies aimed at the IGF-1R/mTOR pathway. A dual-specific anti-IGF-1/IGF-2 human monoclonal antibody (m708.5), which binds human IGF-1 and IGF-2 with high pico-molar affinity, potently inhibited phosphorylation of the IGF-1R and the insulin receptor (IR) in an EW5 xenograft animal model of ES. In concert with mTOR inhibitors (e.g., everolimus or ridaforolimus), m708.5 demonstrated striking tumor regression and prolonged mouse survival. Additionally, we expand upon the synergistic role of CRM1 (XPO1) in combating ES tumor growth. A small molecule inhibitor of this protein (KPT-330) was previously shown to attenuate IGF-1-induced activation of IGF-1R when combined with a small-molecule inhibitor of IGF-1R and IR (Linsitinib, OSI906). Our results indicate superior preclinical anti-tumor activity when it is combined with everolimus in our xenograft ES models. Though these combinations have not yet been assessed in the clinical setting, early-phase clinical trials have begun testing KPT-330 in select sarcoma subtypes, and a host of mTOR inhibitors are FDA-approved and widely available. Our data supports several novel drug combinations that could rapidly be explored in the clinic to improve survival of ES patients. SELC & JAL: Co-senior Authors Citation Format: Brian A. Menegaz, Branko Cuglievan, Prasad S. Admane, Feng Yang, Dimitrov Dimiter, Yosef Landesman, Salah-Eddine Lamhamedi Cherradi, Joseph A. Ludwig. New synergistic mTOR-based combinations for the treatment of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-325.

Correlation of CRM1-NES affinity with nuclear export activity.

CRM1 (Exportin1/XPO1) exports hundreds of broadly functioning protein cargoes out of the cell nucleus by binding to their classical nuclear export signals (NESs). The 8- to 15-amino-acid-long NESs contain four to five hydrophobic residues and are highly diverse in both sequence and CRM1-bound structure. Here we examine the relationship between nuclear export activities of 24 different NES peptides in cells and their CRM1-NES affinities. We found that binding affinity and nuclear export activity are linearly correlated for NESs with dissociation constants (Kds) between tens of nanomolar to tens of micromolar. NESs with Kds outside this range have significantly reduced nuclear export activities. These include two unusually tight-binding peptides, one from the nonstructural protein 2 of murine minute virus (MVM NS2) and the other a mutant of the protein kinase A inhibitor (PKI) NES. The crystal structure of CRM1-bound MVM NS2NES suggests that extraordinarily tight CRM1 binding arises from intramolecular contacts within the NES that likely stabilizes the CRM1-bound conformation in free peptides. This mechanistic understanding led to the design of two novel peptide inhibitors that bind CRM1 with picomolar affinity.

The Human Amyloid Precursor Protein Binds Copper Ions Dominated by a Picomolar-Affinity Site in the Helix-Rich E2 Domain.

A manifestation of Alzheimer's disease (AD) is the aggregation in the brain of amyloid β (Aβ) peptides derived from the amyloid precursor protein (APP). APP has been linked to modulation of normal copper homeostasis, while dysregulation of Aβ production and clearance has been associated with disruption of copper balance. However, quantitative copper chemistry on APP is lacking, in contrast to the plethora of copper chemistry available for Aβ peptides. The soluble extracellular protein domain sAPPα (molar mass including post-translational modifications of ∼100 kDa) has now been isolated in good yield and high quality. It is known to feature several copper binding sites with different affinities. However, under Cu-limiting conditions, it binds either Cu(I) or Cu(II) with picomolar affinity at a single site (labeled M1) that is located within the APP E2 subdomain. M1 in E2 was identified previously by X-ray crystallography as a Cu(II) site that features four histidine side chains (H313, H386, H432, and H436) as ligands. The presence of CuII(His)4 is confirmed in solution at pH ≤7.4, while Cu(I) binding involves either the same ligands or a subset. The binding affinities are pH-dependent, and the picomolar affinities for both Cu(I) and Cu(II) at pH 7.4 indicate that either oxidation state may be accessible under physiological conditions. Redox activity was observed in the presence of an electron donor (ascorbate) and acceptor (dioxygen). A critical analysis of the potential biological implications of these findings is presented.

PO-418 ImmTAC® molecules: cancer treatment through soluble, high affinity T cell receptors

Introduction Tumour associated antigen (TAA) peptides presented on cancer cells in complex with human leukocyte antigen (HLA) molecules provide a marker for targeting by cytotoxic T cells. The ability of T cells to identify aberrant peptides presented on malignant cells is achieved through a membrane-anchored a/b heterodimer called the T cell receptor (TCR). Whilst patients frequently mount anti-tumour T cell responses, often these are insufficient to clear the tumour. This can be attributed to the low level expression of tumour peptides on cancer cells and to the low binding affinity of TCRs targeting cancer antigens. To overcome the target recognition limitations of the naturally occurring T cell pool, we have developed a new class of soluble bi-specific molecules called ImmTAC (immune mobilising monoclonal TCRs against cancer) which combine a tumour targeting domain, an affinity-engineered TCR and a T cell-activating effector function, an anti-CD3 scFv. Material and methods T cell clones are isolated from human blood, which harbour wild-type TCRs recognising peptides derived from TAAs. The TCR α- and β- chains are sequenced and a stabilising non-native inter-chain disulphide bond is introduced. The TCR chains are expressed as inclusion bodies in Escherichia coli and are refolded in vitro to produce soluble and stable protein. The binding affinity is determined by surface plasmon resonance (SPR) and is typically in the range ~1–400 mM with half-lives of 1–10 s. For effective tumour targeting, the TCR is engineered using phage display to enhance affinity for cognate antigen by a million-fold or greater. To generate an ImmTAC molecule, the soluble TCR is fused to a humanised anti-CD3 scFv effector function and potency and specificity is assessed in vitro against HLA-relevant antigen positive cancer cell lines and cultured primary cells isolated from normal human tissues. Results and discussions Our lead clinical candidate, IMCgp100, targets the HLA-A*02/gp100280–288 epitope expressed at the surface of melanoma cells. Using directed molecular evolution and phage display targeting the complementarity-determining regions (CDRs) we have produced a bispecific reagent with picomolar binding affinity and a binding half-life of several hours. IMCgp100 has entered pivotal monotherapy trials for the treatment of patients with metastatic uveal melanoma. Conclusion The ImmTAC technology platform is a new class of bi-specific biologic based on T cell receptors. IMCgp100 is the first ImmTAC in clinical trials to treat advanced melanoma.

Breaking the Rules

Breaking the Rules

Engineering Tissue Inhibitor of Metalloproteinases‐1 (TIMP‐1) as a Selective Inhibitor of Matrix Metalloproteinase‐3 (MMP‐3) for Therapeutic Targeting

Matrix metalloproteinases (MMPs) play a pivotal role in progression of several diseases including cancer and fibrosis, which makes these enzymes promising targets for developing protein therapeutics. The challenge in using MMP inhibitors as drugs in MMP‐associated diseases is lack of high selectivity toward specific target MMPs. Therefore, development of selective MMP inhibitors is a critical goal. Tissue inhibitors of metalloproteinases (TIMPs), as natural MMP inhibitors, offer ideal scaffolds for engineering highly selective MMP‐targeted therapeutics. We have previously shown a key role for MMP‐3 in the pathogenesis of pulmonary fibrosis. TIMP‐1 is a natural MMP‐3 inhibitor with picomolar affinity; it also binds to other MMPs with a spectrum of affinities. Engineering of TIMP‐1 for more selective inhibition of MMP‐3 may provide protein therapeutics with applications in treating lung fibrosis.I have used directed evolution and yeast surface display toward development of selective MMP‐3‐targeted biologic agents through protein engineering of TIMP‐1. I have screened a library of TIMP‐1 mutants generated by random mutagenesis of residues comprising the MMP‐interacting loops of TIMP‐1 to select for enhanced MMP‐3 binding on the yeast surface. Diversified residues have been chosen based on structural studies of TIMP‐1/MMP‐3 interactions. TIMP‐1 mutants isolated after rounds of Fluorescent‐Activated Cell Sorting (FACS) showed substantial improvement in MMP‐3 binding compared to the wild‐type TIMP‐1, both in the surface display context and in enzyme inhibition studies using soluble purified proteins.I have also developed a counter‐selection strategy to screen TIMP‐1 mutants that bind selectively to MMP‐3 in the presence of MMP‐10, an MMP with the greatest sequence and structural similarity to MMP‐3. The isolated TIMP‐1 mutants after five rounds of competitive screening showed up to 20‐fold improvement in binding selectivity, highlighting the significant potential of this approach for development of inhibitors with single‐MMP selectivity. In ongoing studies, I have identified some of the key TIMP‐1 mutations responsible for improvements in MMP‐3 affinity and selectivity. I have co‐crystallized one of the top affinity mutants with the catalytic domain of MMP‐3, and have obtained synchrotron diffraction data at a resolution of 2.2 Å; efforts to solve this crystal structure are underway. I anticipate that this structure will offer insights into the structural basis for improvements in selectivity, and will help to guide future library design for refinement of my directed evolution methodology for generating selective engineered TIMPs. This work provides a promising and innovative strategy to develop protein therapeutics based on natural enzyme inhibitors, critically improving selectivity to eliminate offtarget effects.Support or Funding InformationThis project is funded through two grants from Department of Defense (DoD) (ID # W81XWH‐16‐2‐003015), and National Institute of Health (NIH) (R21CA205471).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antitumor Activity of DLX1008, an Anti-VEGFA Antibody Fragment with Low Picomolar Affinity, in Human Glioma Models

Angiogenesis mediated by vascular endothelial growth factor (VEGF) is a hallmark of glioblastoma. Based on the response rate and improved progression-free survival, although not on overall survival, the 149-kDa anti-VEGF-A IgG antibody bevacizumab (Avastin) has been approved in the United States and Japan for recurrent glioblastoma and in Japan for newly diagnosed glioblastoma; however, it is not approved in the EU. Here we characterize the biologic activity of DLX1008, a 26-kDa anti-VEGF-A single-chain antibody fragment that shows 30-fold stronger affinity to human VEGF-A than bevacizumab. The small molecular size of DLX1008 is predicted to result in improved target coverage over bevacizumab. DLX1008 showed superiority to bevacizumab in the inhibition of VEGF-A binding to VEGF receptor (VEGFR) 1 in enzyme-linked immunosorbent assay by a factor of around 10 and comparable efficacy for the inhibition of VEGF-A-stimulated VEGFR2 dimerization. In a tube-formation assay with human cerebral microvascular endothelial cells, DLX1008 was at least as active as bevacizumab. In vivo, DLX1008 delayed growth in a mouse subcutaneous U87 xenograft model (P = 0.0021) and improved survival in a mouse orthotopic U87 xenograft model (P = 0.00026). Given the exceptionally high affinity and small molecular size of DLX1008, these data warrant further clinical development of DLX1008 as an antiangiogenic agent in glioblastoma.

Extreme disorder in an ultrahigh-affinity protein complex.

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

Epitope-Targeted Macrocyclic Peptide Ligand with Picomolar Cooperative Binding to Interleukin-17F.

The IL-17 cytokine family is associated with multiple immune and autoimmune diseases and comprises important diagnostic and therapeutic targets. This work reports the development of epitope-targeted ligands designed for differential detection of human IL-17F and its closest homologue IL-17A. Non-overlapping and unique epitopes on IL-17F and IL-17A were identified by comparative sequence analysis of the two proteins. Synthetic variants of these epitopes were utilized as targets for in situ click screens against a comprehensive library of synthetic peptide macrocycles with 5-mer variable regions. Single generation screens yielded selective binders for IL-17F and IL-17A with low cross-reactivity. Macrocyclic peptide binders against two distinct IL-17F epitopes were coupled using variable length chemical linkers to explore the physical chemistry of cooperative binding. The optimized linker length yielded a picomolar affinity binder, while retaining high selectivity. The presented method provides a rational approach towards targeting discontinuous epitopes, similar to what is naturally achieved by many B cell receptors.

Guest Back-Folding: A Molecular Design Strategy That Produces a Deep-Red Fluorescent Host/Guest Pair with Picomolar Affinity in Water.

One of the major goals of modern supramolecular chemistry, with important practical relevance in many technical fields, is the development of synthetic host/guest partners with ultrahigh affinity and selectivity in water. Currently, most association pairs exhibit micromolar affinity or weaker, and there are very few host/guest systems with Ka > 109 M-1, apparently due to a barrier imposed by enthalpy/entropy compensation. This present study investigated the threading of a water-soluble tetralactam cyclophane by a deep-red fluorescent squaraine guest with flanking polyethylene glycol chains, an association process that is dominated by a highly favorable enthalpic driving force. A squaraine structure was rationally designed to permit guest back-folding as a strategy to greatly expand the hydrophobic surface area that could be buried upon complexation. Guided by computational modeling, an increasing number of N-benzyl groups were appended to the squaraine core, so that, after threading, the aromatic rings could fold back and stack against the cyclophane periphery. The final design iteration exhibited an impressive combination of fluorescence and supramolecular properties, including ratiometric change in deep-red emission, picomolar affinity ( Ka = 5.1 × 1010 M-1), and very rapid threading ( kon = 7.9 × 107 M-1 s-1) in water at 25 °C. Similar excellent behavior was observed in serum solution. A tangible outcome of this study is a new cyclophane/squaraine association pair that will be a versatile platform for many different types of fluorescence-based imaging and diagnostics applications. From a broader perspective, guest back-folding of aromatic groups is a promising new supramolecular stabilization strategy to overcome enthalpy/entropy compensation and produce ultrahigh affinity [2]pseudorotaxane complexes in water and biological media.

Predicting the Effect of Mutations in the KRas/c-Raf-RBD Protein-Protein Interface

KRas is a small GTPase commonly implicated in several difficult-to-treat cancers such as pancreatic ductal adenocarcinoma (PDAC). KRas normally cycles between an active, GTP-bound form and an inactive, GDP-bound form. Active KRas functions by forming protein-protein interactions (PPIs) with multiple effector proteins in order to regulate various important signal transduction pathways. However, when KRas is mutated it is constitutively active which leads to signal transduction pathway dysregulation that subsequently increases and sustains tumorigenicity and invasiveness. KRas has long been considered an “undruggable” target due to its picomolar affinity for its substrate. However, blocking the PPIs between KRas and its effectors eliminates harmful downstream effects. The tightest known binder of KRas is c-Raf, an enzyme in the ERK1/2 pathway. The Ras-binding domain (RBD) is the minimal binding domain of c-Raf that selectively binds to active, GTP-bound KRas. Previous work has measured Kd for various mutations in the KRas/c-Raf-RBD interface [1, 2]. We use OSPREY [3] (Open Source Protein REdesign for You), a state-of-the-art software package for computational structure-based protein design (CSPD), along with K∗ [4], an algorithm that estimates the binding constant for a given protein complex, to computationally predict the effect of these mutations. We compared our computational predictions to the experimental measurements and found that we can accurately predict the effect of these mutations. These results validate the accuracy of CSPD with OSPREY to target protein-protein interfaces and give us confidence that we can accurately redesign the KRas/c-Raf-RBD interface for future work towards targeting “undruggable” proteins. [1] M. Fridman, et al. Journal of Biological Chemistry, 275(39):30363-30371, 2000. [2] C. Kiel, et al. Journal of Biological Chemistry, 284(46):31893-31902, 2009. [3] P. Gainza, et al. Methods in enzymology, 523:87-107, 2013. [4] I. Georgiev, et al. Journal of computational chemistry, 29(10):1527-1542, 2008.

Development of Candidates for Positron Emission Tomography (PET) Imaging of Ghrelin Receptor in Disease: Design, Synthesis, and Evaluation of Fluorine-Bearing Quinazolinone Derivatives.

Molecular imaging with positron emission tomography (PET) is an attractive platform for noninvasive detection and assessment of disease. The development of a PET imaging agent targeting the ghrelin receptor (growth hormone secretagogue receptor type 1a or GHS-R1a) has the potential to lead to the detection and assessment of the higher than normal expression of GHS-R1a in diseases such as prostate, breast, and ovarian cancer. To enable the development of 18F radiopharmaceuticals, we have designed and synthesized three series of quinazolinone derivatives, resulting in the identification of two compound (5i, 17) with subnanomolar binding affinity and one fluorine-bearing compound (10b) with picomolar binding affinity (20 pM), representing the highest binding affinity for GHS-R1a reported to date. Two lead compounds (5b, IC50 = 20.6 nM; 5e, IC50 = 9.3 nM) were successfully 18F-radiolabeled with radiochemical purity of greater than 99%. Molecular modeling studies were performed to shed light on ligand-receptor interactions.

Optimization of Potent and Selective Tricyclic Indole Diazepinone Myeloid Cell Leukemia-1 Inhibitors Using Structure-Based Design

Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, has emerged as an attractive target for cancer therapy. Mcl-1 upregulation is often found in many human cancers and is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here, we describe a series of potent and selective tricyclic indole diazepinone Mcl-1 inhibitors that were discovered and further optimized using structure-based design. These compounds exhibit picomolar binding affinity and mechanism-based cellular efficacy, including growth inhibition and caspase induction in Mcl-1-sensitive cells. Thus, they represent useful compounds to study the implication of Mcl-1 inhibition in cancer and serve as potentially useful starting points toward the discovery of anti-Mcl-1 therapeutics.

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design.

The aim of rational drug design is to develop small molecules using a quantitative approach to optimize affinity. This should enhance the development of chemical compounds that would specifically, selectively, reversibly, and with high affinity interact with a target protein. It is not yet possible to develop such compounds using computational (i.e., in silico) approach and instead the lead molecules are discovered in high-throughput screening searches of large compound libraries. The main reason why in silico methods are not capable to deliver is our poor understanding of the compound structure-thermodynamics and structure-kinetics correlations. There is a need for databases of intrinsic binding parameters (e.g., the change upon binding in standard Gibbs energy (ΔGint), enthalpy (ΔHint), entropy (ΔSint), volume (ΔVintr), heat capacity (ΔCp,int), association rate (ka,int), and dissociation rate (kd,int)) between a series of closely related proteins and a chemically diverse, but pharmacophoric group-guided library of compounds together with the co-crystal structures that could help explain the structure-energetics correlations and rationally design novel compounds. Assembly of these data will facilitate attempts to provide correlations and train data for modeling of compound binding. Here, we report large datasets of the intrinsic thermodynamic and kinetic data including over 400 primary sulfonamide compound binding to a family of 12 catalytically active human carbonic anhydrases (CA). Thermodynamic parameters have been determined by the fluorescent thermal shift assay, isothermal titration calorimetry, and by the stopped-flow assay of the inhibition of enzymatic activity. Kinetic measurements were performed using surface plasmon resonance. Intrinsic thermodynamic and kinetic parameters of binding were determined by dissecting the binding-linked protonation reactions of the protein and sulfonamide. The compound structure-thermodynamics and kinetics correlations reported here helped to discover compounds that exhibited picomolar affinities, hour-long residence times, and million-fold selectivities over non-target CA isoforms. Drug-lead compounds are suggested for anticancer target CA IX and CA XII, antiglaucoma CA IV, antiobesity CA VA and CA VB, and other isoforms. Together with 85 X-ray crystallographic structures of 60 compounds bound to six CA isoforms, the database should be of help to continue developing the principles of rational target-based drug design.

Benzimidazole design, synthesis, and docking to build selective carbonic anhydrase VA inhibitors

The similarity of human carbonic anhydrase (CA) active sites makes it difficult to design selective inhibitors for one or several CA isoforms that are drug targets. Here we synthesize a series of compounds that are based on 5-[2-(benzimidazol-1-yl)acetyl]-2-chloro-benzenesulfonamide (1a) which demonstrated picomolar binding affinity and significant selectivity for CA isoform five A (VA), and explain the structural influence of inhibitor functional groups to the binding affinity and selectivity. A series of chloro-substituted benzenesulfonamides bearing a heterocyclic tail, together with molecular docking, was used to build inhibitors that explore substituent influence on the binding affinity to the CA VA isoform.

The Magic of Crystal Structure-Based Inhibitor Optimization: Development of a Butyrylcholinesterase Inhibitor with Picomolar Affinity and in Vivo Activity.

The enzymatic activity of butyrylcholinesterase (BChE) in the brain increases with the progression of Alzheimer's disease, thus classifying BChE as a promising drug target in advanced Alzheimer's disease. We used structure-based drug discovery approaches to develop potent, selective, and reversible human BChE inhibitors. The most potent, compound 3, had a picomolar inhibition constant versus BChE due to strong cation-π interactions, as revealed by the solved crystal structure of its complex with human BChE. Additionally, compound 3 inhibits BChE ex vivo and is noncytotoxic. In vitro pharmacokinetic experiments show that compound 3 is highly protein bound, highly permeable, and metabolically stable. Finally, compound 3 crosses the blood-brain barrier, and it improves memory, cognitive functions, and learning abilities of mice in a scopolamine model of dementia. Compound 3 is thus a promising advanced lead compound for the development of drugs for alleviating symptoms of cholinergic hypofunction in patients with advanced Alzheimer's disease.

Direct inhibition of RAS: Quest for the Holy Grail?

RAS GTPases (H-, K-, and N-RAS) are the most frequently mutated oncoprotein family in human cancer. However, the relatively smooth surface architecture of RAS and its picomolar affinity for nucleotide have given rise to the assumption that RAS is an "undruggable" target. Recent advancements in drug screening, molecular modeling, and a greater understanding of RAS function have led to a resurgence in efforts to pharmacologically target this challenging foe. This review focuses on the state of the art of RAS inhibition, the approaches taken to achieve this goal, and the challenges of translating these discoveries into viable therapeutics.

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.

Using Tryptophan Mutants To Probe the Structural and Functional Status of BsSCO, a Copper Binding, Cytochrome c Oxidase Assembly Protein from Bacillus subtilis.

The synthesis of cytochrome c oxidase protein from Bacillus subtilis (i.e., BsSCO) binds copper with picomolar affinity, which increases the protein's melting temperature (i.e., TM) by 20 °C. Here two native tryptophans (i.e., W36 and W101) are identified as major contributors to BsSCO's structural form, and their contributions to the stability, intrinsic fluorescence, and copper binding properties of BsSCO are explored. Single mutations of tryptophan to phenylalanine decrease the TM by 10 °C and the folding free energy by 3-4 kcal/mol. A more severe change to alanine (i.e., W36A BsSCO) decreases the TM by 20 °C and the stability by 9 kcal/mol. However, these mutants bind copper with high affinity and assemble cytochrome c oxidase in vivo. Replacing phenylalanine at a position near (∼5 Å) the copper binding site with tryptophan (i.e., F42W) increases the TM of apo-BsSCO by 3 °C but diminishes the effect of copper binding. When both native tryptophans are changed to alanine, apo-BsSCO is unfolded in vitro and is not functional in cytochrome c oxidase assembly in vivo. A double-mutant of BsSCO in which W36A is combined with F42W exhibits a form of metastability. Apo-W36A/F42W BsSCO melts at 37 °C, which upon binding of copper shifts to 65 °C. B. subtilis expressing W36A/F42W BsSCO and grown at 37 °C does not assemble cytochrome c oxidase. However, when these cells are cooled to 25 °C, cytochrome c oxidase activity is recovered. Our results illustrate the subtle relationship between the structural stability and functional properties of BsSCO in the assembly of cytochrome c oxidase.

A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM.

Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).