Cell surface ß-lactamase recruitment: A facile selection to identify protein-protein interactions.

Protein-protein interactions (PPIs) are central to many cellular processes, and the identification of novel PPIs is a critical step in the discovery of protein therapeutics. Simple methods to identify naturally existing or laboratory evolved PPIs are therefore valuable research tools. We have developed a facile selection that links PPI-dependent ß-lactamase recruitment on the surface of Escherichia coli with resistance to ampicillin. Bacteria displaying a protein that forms a complex with a specific protein-ß-lactamase fusion are protected from ampicillin-dependent cell death. In contrast, bacteria that do not recruit ß-lactamase to the cell surface are killed by ampicillin. Given its simplicity and tunability, we anticipate this selection will be a valuable addition to the palette of methods for illuminating and interrogating PPIs.

Tuning through-space interactions via the secondary coordination sphere of an artificial metalloenzyme leads to enhanced Rh(iii)-catalysis.

We report computationally-guided protein engineering of monomeric streptavidin Rh(iii) artificial metalloenzyme to enhance catalysis of the enantioselective coupling of acrylamide hydroxamate esters and styrenes. Increased TON correlates with calculated distances between the Rh(iii) metal and surrounding residues, underscoring an artificial metalloenzyme's propensity for additional control in metal-catalyzed transformations by through-space interactions.

Antibody-Recruitment as a Therapeutic Strategy: A Brief History and Recent Advances.

Antibodies are a significant and growing sector within the global pharmaceutical industry. The popularity of antibodies as therapeutics derives from - at least in part - evolvable affinity for virtually any disease-relevant cell surface receptor, as well as unique immunotherapeutic mechanisms of action, including neutralization, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC). While advances in the large-scale expression and purification of therapeutic antibodies have been made, these remain costly and laborious tasks. Agents that redirect endogenous antibodies to target a pathogen or malignant cell obviate the need for new antibody discovery and production. Chimeric antibody-recruiting technologies consist of a target cell surface receptor binding domain, and an endogenous antibody-binding domain. By design, these agents bring endogenous antibodies to the surface of a target pathogen or diseased cell, which can result in targeted cytotoxicity by antibody-dependent mechanisms. This review highlights seminal contributions and recent advances in this growing and important therapeutic field.

Computational Hot-Spot Analysis of the SARS-CoV-2 Receptor Binding Domain/ACE2 Complex*.

Infection and replication of SARS CoV-2 (the virus that causes COVID-19) requires entry to the interior of host cells. In humans, a protein-protein interaction (PPI) between the SARS CoV-2 receptor-binding domain (RBD) and the extracellular peptidase domain of ACE2 on the surface of cells in the lower respiratory tract is an initial step in the entry pathway. Inhibition of the SARS CoV-2 RBD/ACE2 PPI is currently being evaluated as a target for therapeutic and/or prophylactic intervention. However, relatively little is known about the molecular underpinnings of this complex. Employing multiple computational platforms, we predicted "hot-spot" residues in a positive-control PPI (PMI/MDM2) and the CoV-2 RBD/ACE2 complex. Computational alanine scanning mutagenesis was performed to predict changes in Gibbs' free energy that are associated with mutating residues at the positive control (PMI/MDM2) or SARS RBD/ACE2 binding interface to alanine. Additionally, we used the Adaptive Poisson-Boltzmann Solver to calculate macromolecular electrostatic surfaces at the interface of the positive-control PPI and SARS CoV-2/ACE2 PPI. Finally, a comparative analysis of hot-spot residues for SARS-CoV and SARS-CoV-2, in complex with ACE2, is provided. Collectively, this study illuminates predicted hot-spot residues, and clusters, at the SARS CoV-2 RBD/ACE2 binding interface, potentially guiding the development of reagents capable of disrupting this complex and halting COVID-19.

Computational Hot-Spot Analysis of the SARS-CoV-2 Receptor Binding Domain / ACE2 Complex.

Infection and replication of SARS CoV-2 (the virus that causes COVID-19) requires entry to the interior of host cells. In humans, a Protein-Protein Interaction (PPI) between the SARS CoV-2 Receptor-Binding Domain (RBD) and the extracellular peptidase domain of ACE2, on the surface of cells in the lower respiratory tract, is an initial step in the entry pathway. Inhibition of the SARS CoV-2 RBD / ACE2 PPI is currently being evaluated as a target for therapeutic and/or prophylactic intervention. However, relatively little is known about the molecular underpinnings of this complex. Employing multiple computational platforms, we predicted hot-spot residues in a positive control PPI (PMI / MDM2) and the CoV-2 RBD/ACE2 complex. Computational alanine scanning mutagenesis was performed to predict changes in Gibbs free energy that are associated with mutating residues at the positive control (PMI/MDM2) or SARS RBD/ACE2 binding interface to alanine. Additionally, we used the Adaptive Poisson-Boltzmann Solver to calculate macromolecular electrostatic surfaces at the interface of the positive control PPI and SARS CoV-2 / ACE2 PPI. Collectively, this study illuminates predicted hot-spot residues, and clusters, at the SARS CoV-2 RBD / ACE2 binding interface, potentially guiding the development of reagents capable of disrupting this complex and halting COVID-19.

Disparities between Antibody Occupancy, Orientation, and Cytotoxicity in Immunotherapy.

We report fusion proteins designed to bind spatially distinct epitopes on the extracellular portion of HER2, a breast cancer biomarker and established therapeutic target, and recruit IgG (either anti-His6 or serum IgG) to the cell surface. When the proteins were incubated with anti-His6 antibody and various concentrations of a single HER2-binding protein His6 fusion, we observed interference and a decrease in antibody recruitment at HER2-binding protein concentrations exceeding ∼30 nM. In contrast, concomitant treatment with two or three distinct HER2-binding protein His6 fusions, and anti-His6 , results in increased antibody recruitment, even at relatively high HER2-binding protein concentration. In some instances, increased antibody recruitment leads to increased antibody-dependent cellular cytotoxicity (ADCC) activity. While a fusion protein consisting of a HER2-binding nanobody and Sac7d, a protein evolved to recognize the Fc domain of IgG, binds IgG from serum, antibody recruitment does not lead to ADCC activity. Rationales for these disparities are provided. Collectively, our findings have implications for the design of efficacious targeted immunotherapeutic biologics, and ensembles thereof.

Asymmetric δ-Lactam Synthesis with a Monomeric Streptavidin Artificial Metalloenzyme.

Reliable design of artificial metalloenzymes (ArMs) to access transformations not observed in nature remains a long-standing and important challenge. We report that a monomeric streptavidin (mSav) Rh(III) ArM permits asymmetric synthesis of α,ß-unsaturated-δ-lactams via a tandem C-H activation and [4+2] annulation reaction. These products are readily derivatized to enantioenriched piperidines, the most common N-heterocycle found in FDA approved pharmaceuticals. Desired δ-lactams are achieved in yields as high as 99% and enantiomeric excess of 97% under aqueous conditions at room temperature. Embedding a Rh cyclopentadienyl (Cp*) catalyst in the active site of mSav results in improved stereocontrol and a 7-fold enhancement in reactivity relative to the isolated biotinylated Rh(III) cofactor. In addition, mSav-Rh outperforms its well-established tetrameric forms, displaying 11-33 times more reactivity.

Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1.

Drugs that block HIV-1 entry are relatively limited. Enfuvirtide is a 36-residue synthetic peptide that targets gp41 and blocks viral fusion. However, Enfuvirtide-resistant HIV has been reported, and this peptide drug requires daily injection. Previously, we have reported helix-grafted display proteins, consisting of HIV-1 gp41 C-peptide helix grafted onto Pleckstrin Homology domains. Some of these biologics inhibit HIV-1 entry with relatively modest and varied potency (IC50 = 190 nM to >1 µM). Here, we report that gp41 C-peptide helix-grafted Sac7d (Sac7d-Cpep) potently suppresses HIV-1 entry in a live virus assay (IC50 = 1.9-12.4 nM). Yeast display sequence optimization of solvent exposed helix residues led to new biologics with improved expression in E. coli (a common biosimilar expression host), with no appreciable change in entry inhibition. Evolved proteins inhibit the entry of a clinically relevant mutant of HIV-1 that is gp41 C-peptide sensitive and Enfuvirtide resistant. Fusion proteins designed for serum stability also potently suppress HIV-1 entry. Collectively, we report several evolved biologics that are functional against an Enfuvirtide-resistant strain and are designed for serum stability.

Structure of HIV TAR in complex with a Lab-Evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.

Natural and lab-evolved proteins often recognize their RNA partners with exquisite affinity. Structural analysis of such complexes can offer valuable insight into sequence-selective recognition that can be exploited to alter biological function. Here, we describe the structure of a lab-evolved RNA recognition motif (RRM) bound to the HIV-1 trans-activation response (TAR) RNA element at 1.80 Å-resolution. The complex reveals a trio of arginines in an evolved ß2-ß3 loop penetrating deeply into the major groove to read conserved guanines while simultaneously forming cation-π and salt-bridge contacts. The observation that the evolved RRM engages TAR within a double-stranded stem is atypical compared to most RRMs. Mutagenesis, thermodynamic analysis and molecular dynamics validate the atypical binding mode and quantify molecular contributions that support the exceptionally tight binding of the TAR-protein complex (KD,App of 2.5 ± 0.1 nM). These findings led to the hypothesis that the ß2-ß3 loop can function as a standalone TAR-recognition module. Indeed, short constrained peptides comprising the ß2-ß3 loop still bind TAR (KD,App of 1.8 ± 0.5 µM) and significantly weaken TAR-dependent transcription. Our results provide a detailed understanding of TAR molecular recognition and reveal that a lab-evolved protein can be reduced to a minimal RNA-binding peptide.

Evaluation of sequence variability in HIV-1 gp41 C-peptide helix-grafted proteins.

Many therapeutically-relevant protein-protein interactions (PPIs) have been reported that feature a helix and helix-binding cleft at the interface. Given this, different approaches to disrupting such PPIs have been developed. While short peptides (<15 amino acids) typically do not fold into a stable helix, researchers have reported chemical approaches to constraining helix structure. However, these approaches rely on laborious, and often expensive, chemical synthesis and purification. Our premise is that protein-based solutions that stabilize a therapeutically-relevant helix offer a number of advantages. In contrast to chemically constrained helical peptides, or minimal/miniature proteins, which must be synthesized (at great expense and labor), a protein can be expressed in a cellular system (like all current protein therapeutics). If selected properly, the protein scaffold can stabilize the therapeutically-relevant helix. We recently reported a protein engineering strategy, which we call "helix-grafted display", and applied it to the challenge of suppressing HIV entry. We have reported helix-grafted display proteins that inhibit formation of an intramolecular PPI involving HIV gp41 C-peptide helix, and HIV gp41 N-peptide trimer, which contain C-peptide helix-binding clefts. Here, we used yeast display to screen a library of grafted C-peptide helices for N-peptide trimer recognition. Using 'hits' from yeast display library screening, we evaluated the effect helix mutations have on structure, expression, stability, function (target recognition), and suppression of HIV entry.

Inside Job: Methods for Delivering Proteins to the Interior of Mammalian Cells.

Currently, 7 of the top 10 selling drugs are biologics, and all of them are proteins. Their large size, structural complexity, and molecular diversity often results in surfaces capable of potent and selective recognition of receptors that challenge, or evade, traditional small molecules. However, most proteins do not penetrate the lipid bilayer exterior of mammalian cells. This severe limitation dramatically limits the number of disease-relevant receptors that proteins can target and modulate. Given the major role proteins play in modern medicine, and the magnitude of this limitation, it is unsurprising that an enormous amount of effort has been dedicated to overcoming this pesky impediment. In this article, we summarize and evaluate current approaches for intracellular delivery of exogenous proteins to mammalian cells and, in doing so, aim to illuminate fertile ground for future discovery in this critical area of research.

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation.

PURPOSE: Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts. PROCEDURES: sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [18F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [18F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[18F]RL-I conjugates. RESULTS: [18F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97-100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [125I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95-99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [18F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [125I]SGMIB-2Rs15d. Uptake of [18F]RL-I-2Rs15d in subcutaneous xenografts was 16-20 %ID/g over 1-3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [18F]RL-I-2Rs15d but not with [18F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [18F]RL-I-2Rs15d and [18F]RL-I-5F7. CONCLUSIONS: Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

Evaluation of Nanobody Conjugates and Protein Fusions as Bioanalytical Reagents.

Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blot are common bioanalytical techniques. Successful execution traditionally requires the use of one or more commercially available antibody-small-molecule dyes or antibody-reporter protein conjugates that recognize relatively short peptide tags (<15 amino acids). However, the size of antibodies and their molecular complexity (by virtue of post-translational disulfide formation and glycosylation) typically require either expression in mammalian cells or purification from immunized mammals. The preparation and purification of chemical dye- or reporter protein-antibody conjugates is often complicated and expensive and not commonplace in academic laboratories. In response, researchers have developed comparatively simpler protein scaffolds for macromolecular recognition, which can be expressed with relative ease in E. coli and can be evolved to bind virtually any target. Nanobodies, a minimalist scaffold generated from camelid-derived heavy-chain IgGs, are one such example. A multitude of nanobodies have been evolved to recognize a diverse array of targets, including a short peptide. Here, this peptide tag (termed BC2T) and BC2 nanobody-dye conjugates or reporter protein fusions are evaluated in ELISA, flow cytometry, and Western blot experiments and compared to analogous experiments using commercially available antibody-conjugate/peptide tag pairs. Collectively, the utility and practicality of nanobody-based reagents in bioanalytical chemistry is demonstrated.

Minimalist Antibodies and Mimetics: An Update and Recent Applications.

The immune system utilizes antibodies to recognize foreign or disease-relevant receptors, initiating an immune response to destroy unwelcomed guests. Because researchers can evolve antibodies to bind virtually any target, it is perhaps unsurprising that these reagents, and their small-molecule conjugates, are used extensively in clinical and basic research environments. However, virtues of antibodies are countered by significant challenges. Foremost among these is the need for expression in mammalian cells (largely due to often necessary post-translational modifications). In response to these challenges, researchers have developed an array of minimalist antibodies and mimetics, which are smaller, more stable, simpler to express in Escherichia coli, and amendable to laboratory evolution and protein engineering. Here we describe these scaffolds and discuss recent applications of minimalist antibodies and mimetics.

Helix-Grafted Pleckstrin Homology Domains Suppress HIV-1 Infection of CD4-Positive Cells.

The size, functional group diversity and three-dimensional structure of proteins often allow these biomolecules to bind disease-relevant structures that challenge or evade small-molecule discovery. Additionally, folded proteins are often much more stable in biologically relevant environments compared to their peptide counterparts. We recently showed that helix-grafted display-extensive resurfacing and elongation of an existing solvent-exposed helix in a pleckstrin homology (PH) domain-led to a new protein that binds a surrogate of HIV-1 gp41, a validated target for inhibition of HIV-1 entry. Expanding on this work, we prepared a number of human-derived helix-grafted-display PH domains of varied helix length and measured properties relevant to therapeutic and basic research applications. In particular, we showed that some of these new reagents expressed well as recombinant proteins in Escherichia coli, were relatively stable in human serum, bound a mimic of pre-fusogenic HIV-1 gp41 in vitro and in complex biological environments, and significantly lowered the incidence of HIV-1 infection of CD4-positive cells.

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription.

Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low µM dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5'-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents-small molecule, nucleic acid, or protein-reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition-and modulation-of disease-relevant RNA.

Scratching the Surface: Resurfacing Proteins to Endow New Properties and Function.

Protein engineering is an emerging discipline that dovetails modern molecular biology techniques with high-throughput screening, laboratory evolution technologies, and computational approaches to modify sequence, structure, and, in some cases, function and properties of proteins. The ultimate goal is to develop new proteins with improved or designer functions for use in biotechnology, medicine, and basic research. One way to engineer proteins is to change their solvent-exposed regions through focused or random "protein resurfacing." In this review we explain what protein resurfacing is, and discuss recent examples of how this strategy is used to generate proteins with altered or broadened recognition profiles, improved stability, solubility, and expression, cell-penetrating ability, and reduced immunogenicity. Additionally we comment on how these properties can be further improved using chemical resurfacing approaches. Protein resurfacing will likely play an increasingly important role as more biologics enter clinical use, and we present some arguments to support this view.

Resurfaced cell-penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery.

By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease-relevant macromolecules that present a challenge to small-molecule reagents. Additionally, high-throughput screening and evolution-based methods often make the discovery of new protein binders simpler than the analogous small-molecule discovery process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease-relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, in our experience, many proteins are not amenable to such extensive mutagenesis. Here, we report that nanobodies-a small and stable protein that can be evolved to recognize virtually any disease-relevant receptor-are amenable to cationic resurfacing, which results in cell internalization. Once internalized, these nanobodies access the cytosol. Polycationic resurfacing does not appreciably alter the structure, expression, and function (target recognition) of a previously reported GFP-binding nanobody, and multiple nanobody scaffolds are amenable to polycationic resurfacing. Given this, we propose that polycationic resurfaced cell-penetrating nanobodies might represent a general scaffold for intracellularly targeted protein drug discovery.