Marcos Pires

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.

Really hits the spot: A novel tag for protein conjugation was engineered based on the crystal structure of a natural substrate for a bacterial transglutaminase. This conformationally locked structural mimic of the native conjugation site ensures site-selective and efficient protein ligation, as demonstrated for the therapeutic antibody cetuximab.

The chemical synthesis of the 184-residue ferric heme-binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α-ketoacid-hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild-type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well-suited for the preparation of hydrophobic protein targets.

It′s all coming together! The 20 kDa heme protein nitrophorin 4 was synthesized by sequential KAHA ligations using a convergent strategy. The folded synthetic protein readily coordinates the heme cofactor, and the holoprotein shows identical biological properties to the wild-type protein for NO binding and nitrite dismutase reactivity.

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML.

Touching from a distance: Small molecules block STAT3 SH2 phosphotyrosine recognition by binding to the distal coiled-coil domain (CCD). Inhibition of this new CCD target site, which was identified by catalytic rhodium labeling, blocks tumor progression in a leukemia mouse model.

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir–Blodgett and Langmuir–Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development.

Understanding the outer membranes of Gram-negative bacteria is important for the development of new antibacterial compounds. However, their structure and dynamics are poorly understood because of their small in vivo size and inaccurate in vitro models. A stable asymmetric model of the outer membrane that can be analyzed by a range of biophysical techniques and accurately imitates the in vivo behavior of natural outer membranes is presented herein.

Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under-utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high-yielding step. When coupled with ligation at Sec, this transformation provides a new approach to programmed ligations at Ser residues. This new reaction is compatible with a wide range of functionality, including the presence of unprotected amino acid side chains and appended glycans. The utility of the methodology is demonstrated in the rapid synthesis of complex glycopeptide fragments of the epithelial glycoproteins MUC5AC and MUC4 and through the total synthesis of the structured, cysteine (Cys)-free protein eglin C.

An operationally simple method for the rapid and chemoselective conversion of selenocysteine (Sec) to serine (Ser) in aqueous media is described. This mechanistically distinct transformation at selenium facilitates the synthesis of complex peptides and proteins, as highlighted in the synthesis of fragments of the epithelial glycoproteins MUC5AC and MUC4 and in the total synthesis of the serine protease inhibitor eglin C.

A supramolecular antibiotic switch is described that can reversibly “turn-on” and “turn-off” its antibacterial activity on demand, providing a proof-of-concept for a way to regulate antibacterial activity of biotics. The switch relies on supramolecular assembly and disassembly of cationic poly(phenylene vinylene) derivative (PPV) with cucurbit[7]uril (CB[7]) to regulate their different interactions with bacteria. This simple but efficient strategy does not require any chemical modification on the active sites of the antibacterial agent, and could also regulate the antibacterial activity of classical antibiotics or photosensitizers in photodynamic therapy. This supramolecular antibiotic switch may be a successful strategy to fight bacterial infections and decrease the emergence of bacterial resistance to antibiotics from a long-term point of view.

A supramolecular antibiotic switch to reversibly “turn-on” and “turn-off” antibacterial activity on demand was developed as a proof-of-concept to regulate antibacterial activity. The switch relies on the supramolecular assembly and disassembly of a poly(phenylene vinylene) derivative (PPV) with cucurbit[7]uril (CB[7]). This strategy does not require any chemical modification on the active sites of the antibacterial agent.