Xiaozhou Luo

Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis

Significance The identification of new antibiotics with novel mechanisms of action has become a pressing need considering the growing threat of drug-resistant infections. We have identified auranofin, an FDA-approved drug, as having potent bactericidal activity against Gram-positive pathogenic bacteria. Auranofin inhibits an enzyme, thioredoxin reductase, not targeted by other antibiotics, and thus retains efficacy against many clinically relevant drug-resistant strains, including in a mouse model of infection. Because auranofin is an approved drug, its route to the clinic may be expedited with reduced cost. Our work suggests that auranofin is a candidate for drug repurposing in antibacterial therapy. Infections caused by antibiotic-resistant bacteria are a rising public health threat and make the identification of new antibiotics a priority. From a cell-based screen for bactericidal compounds against Mycobacterium tuberculosis under nutrient-deprivation conditions we identified auranofin, an orally bioavailable FDA-approved antirheumatic drug, as having potent bactericidal activities against both replicating and nonreplicating M. tuberculosis. We also found that auranofin is active against other Gram-positive bacteria, including Bacillus subtilis and Enterococcus faecalis, and drug-sensitive and drug-resistant strains of Enterococcus faecium and Staphylococcus aureus. Our biochemical studies showed that auranofin inhibits the bacterial thioredoxin reductase, a protein essential in many Gram-positive bacteria for maintaining the thiol-redox balance and protecting against reactive oxidative species. Auranofin decreases the reducing capacity of target bacteria, thereby sensitizing them to oxidative stress. Finally, auranofin was efficacious in a murine model of methicillin-resistant S. aureus infection. These results suggest that the thioredoxin-mediated redox cascade of Gram-positive pathogens is a valid target for the development of antibacterial drugs, and that the existing clinical agent auranofin may be repurposed to aid in the treatment of several important antibiotic-resistant pathogens.

An immunosuppressive antibody-drug conjugate.

We have developed a novel antibody-drug conjugate (ADC) that can selectively deliver the Lck inhibitor dasatinib to human T lymphocytes. This ADC is based on a humanized antibody that selectively binds with high affinity to CXCR4, an antigen that is selectively expressed on hematopoietic cells. The resulting dasatinib-antibody conjugate suppresses T-cell-receptor (TCR)-mediated T-cell activation and cytokine expression with low nM EC50 and has minimal effects on cell viability. This ADC may lead to a new class of selective immunosuppressive drugs with improved safety and extend the ADC strategy to the targeted delivery of kinase inhibitors for indications beyond oncology.

Recombinant thiopeptides containing noncanonical amino acids

Significance Thiopeptides are a subclass of ribosomally synthesized natural products with complex structures and potent antimicrobial activities. Here we describe a general strategy that allows the incorporation of noncanonical amino acids into thiopeptides by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin-producing strain of Bacillus cereus. We show that thiocillin variants harboring a noncanonical amino acid with bioorthogonal chemical reactivity can be further modified to create probes for biological studies. This work should significantly enhance our ability to manipulate the structures and properties of ribosomally produced natural products by recombinant methods. Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus. We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists

Significance Many therapeutic proteins suffer from short plasma half-lives and, as a consequence, require frequent injections to be therapeutically effective; this in turn can adversely affect patient compliance and quality of life. In contrast, therapeutic antibodies typically have half-lives of weeks in humans. Consequently, there is considerable interest in generating functional antibodies with agonist or antagonist activities. Based on the structure of a natural bovine antibody with an ultralong, well-folded heavy-chain complementarity-determining region, we have developed a strategy for the generation of functional human antibody–hormone chimeras with biological activities comparable to native hormones and significantly enhanced pharmacological properties. This approach likely provides a general, relatively straightforward platform for generating antibody agonists and antagonists for a range of therapeutic applications. On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

Enhancing protein stability with extended disulfide bonds

Significance This work describes a facile system for incorporating noncanonical amino acids containing long side-chain thiols using an expanded genetic code. These amino acids begin to overcome the distance and geometric constraints of the cysteine disulfide and can pair with cysteines to cross-link more remote sites in proteins. To demonstrate this notion, we constructed a library of random β-lactamase mutants containing these noncanonical amino acids and grew them at nonpermissive temperatures. We identified a mutant enzyme that is cross-linked by one such extended disulfide bond that has significantly enhanced thermal stability. This study suggests that an expanded set of amino acid building blocks can provide novel solutions to evolutionary challenges. Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.

Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria.

Tyrosine phosphorylation is a common protein posttranslational modification, which plays a critical role in signal transduction and the regulation of many cellular processes. Using a pro-peptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase/tRNA pair that allows the site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray crystal structure of the synthetase reveals a reconfigured substrate binding site formed by non-conservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site whose corresponding kinase is unknown and determined the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Engineering a long-acting, potent GLP-1 analog for microstructure-based transdermal delivery

Significance Many therapeutic peptides suffer from short plasma half-lives and, as a consequence, require frequent injections to be therapeutically effective; this in turn can adversely affect patient compliance. Here, we describe the development of a novel peptide engineering strategy that incorporates a serum protein binding motif into a covalent side-chain staple. This approach was used to generate stapled long-acting glucagon-like peptide-1 analogs with potency comparable to exendin-4 and significantly enhanced pharmacokinetic properties. Administration by a dissolvable microstructure-based transdermal system resulted in sustained therapeutic blood concentrations with glucose lowering activity in guinea pigs. This approach likely provides a general, straightforward platform for generating stapled long-acting peptide hormones for a range of therapeutic applications. Antidiabetic treatments aiming to reduce body weight are currently gaining increased interest. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist administered twice daily via s.c. injection, improves glycemic control, often with associated weight reduction. To further improve the therapeutic efficacy of exendin-4, we have developed a novel peptide engineering strategy that incorporates a serum protein binding motif onto a covalent side-chain staple and applied to the peptide to enhance its helicity and, as a consequence, its potency and serum half-life. We demonstrated that one of the resulting peptides, E6, has significantly improved half-life and glucose tolerance in an oral glucose tolerance test in rodents. Chronic treatment of E6 significantly decreased body weight and fasting blood glucose, improved lipid metabolism, and also reduced hepatic steatosis in diet-induced obese mice. Moreover, the high potency of E6 allowed us to administer this peptide using a dissolvable microstructure-based transdermal delivery system. Pharmacokinetic and pharmacodynamic studies in guinea pigs showed that a single 5-min application of a microstructure system containing E6 significantly improved glucose tolerance for 96 h. This delivery strategy may offer an effective and patient-friendly alternative to currently marketed GLP-1 injectables and can likely be extended to other peptide hormones.

Genetic Incorporation of a Reactive Isothiocyanate Group into Proteins

Methods for the site-specific modification of proteins are useful for introducing biological probes into proteins and engineering proteins with novel activities. Herein, we genetically encode a novel noncanonical amino acid (ncAA) that contains an aryl isothiocyanate group which can form stable thiourea crosslinks with amines under mild conditions. We show that this ncAA (pNCSF) allows the selective conjugation of proteins to amine-containing molecular probes through formation of a thiourea bridge. pNCSF was also used to replace a native salt bridge in myoglobin with an intramolecular crosslink to a proximal Lys residue, leading to increased thermal stability. Finally, we show that pNCSF can form stable intermolecular crosslinks between two interacting proteins.

Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer

Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5). We demonstrate that both switch formats can be readily optimized to redirect CAR-T cells (specific for the corresponding FITC or PNE) to Her2-expressing tumor cells, and afford dose-titratable activation of CAR-T cells ex vivo and complete clearance of the tumor in rodent xenograft models. This strategy may facilitate the application of immunotherapy to solid tumors by affording comparable efficacy with improved safety owing to switch-based control of the CAR-T response.

An Epitope‐Specific Respiratory Syncytial Virus Vaccine Based on an Antibody Scaffold

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in children. We have generated an epitope-specific RSV vaccine by grafting a neutralizing epitope (F-epitope) in its native conformation into an immunoglobulin scaffold. The resulting antibody fusion exhibited strong binding affinity to Motavizumab, an RSV neutralizing antibody, and effectively induced potent neutralizing antibodies in mice. This work illustrates the potential of the immunoglobulin molecule as a scaffold to present conformationally constrained B-cell epitopes.