Marcos Pires

Blockade of the protein–protein interaction between the transmembrane protein programmed cell death protein 1 (PD-1) and its ligand PD-L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror-image phage display, we developed the first hydrolysis-resistant D-peptide antagonists to target the PD-1/PD-L1 pathway. The optimized compound ^DPPA-1 could bind PD-L1 at an affinity of 0.51 μM in vitro. A blockade assay at the cellular level and tumor-bearing mice experiments indicated that ^DPPA-1 could also effectively disrupt the PD-1/PD-L1 interaction in vivo. Thus D-peptide antagonists may provide novel low-molecular-weight drug candidates for cancer immunotherapy.

Protein chemical synthesis and mirror-image phage display were combined to develop a proteolysis-resistant D-peptide antagonist (^DPPA-1) which targets the immune checkpoint protein PD-L1 (the ligand for PD-1, the programmed cell death protein 1). ^DPPA-1 was found to inhibit the PD-1/PD-L1 protein–protein interaction at the cellular level. IgV=immunoglobulin-like variable.

Despite significant advances in foldamer chemistry, tailored delivery systems based on foldamer architectures, which provide a high level of control over secondary structure, are curiously rare among non-viral technologies for transporting nucleic acids into cells. A potent pH-responsive, bioreducible cell-penetrating foldamer (CPF) was developed through covalent dimerization of a short (8-mer) amphipathic oligourea sequence bearing histidine-type units. This CPF exhibits a high capacity to assemble with pDNA and mediates efficient delivery of nucleic acids into the cell. Furthermore, it does not adversely affect cellular viability and was shown to compare favorably with a cognate peptide transfection agent based on His-rich sequences.

Foldaplex: A pH-responsive cell-penetrating foldamer (CPF) was designed for nucleic acid delivery. Histidine-type residues were introduced along the oligourea sequence to facilitate the release of the cargo into the cytoplasm. Dimerization of the urea-based foldamer sequence through a disulfide bridge enhanced cellular uptake and led to transfection efficiency comparable to that of commercially available transfection agents, as well as negligible cytotoxicity.

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a “clickable” alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl–cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.

Good grip: Alkynyl benziodoxolones (EBX reagents) swiftly and highly selectively react with cysteine residues in cellular lysates and in living cells under physiological conditions. A “clickable” EBX probe allowed identification of the biological targets of natural product curcumin. This new method for cysteine labeling is particularly useful for various chemical proteomics applications.

A flexible synthetic strategy toward the preparation of diverse N-substituted muramyl dipeptides (N-substituted MDPs) from different protected monosaccharides is described. The synthetic MDPs include N-acetyl MDP and N-glycolyl MDP, known NOD2 ligands, and this methodology allows for structural variation at six positions, including the muramic acid, peptide, and N-substituted moieties. The capacity of these molecules to activate human NOD2 in the innate immune response was also investigated. It was found that addition of the methyl group at the C1 position of N-glycolyl MDP significantly enhanced the NOD2 stimulating activity.

A flexible synthetic strategy toward the preparation of diverse N-substituted muramyl dipeptides (N-substituted MDPs), including N-acetyl MDP and N-glycolyl MDP, known NOD2 ligands, from different protected monosaccharides. This methodology allows for structural variation at six positions, including the muramic acid, peptide, and N-substituted moieties. The capacity of these molecules to activate human NOD2 in the innate immune response was also investigated. It was found that addition of the methyl group at the C1 position of N-glycolyl MDP significantly enhanced the NOD2 stimulating activity.

Haptens, such as dinitrophenyl (DNP) are small molecules that induce strong immune responses when attached to proteins or peptides and, as such, have been exploited for diverse applications. We engineered a Methanosarcina barkeri pyrrolysyl-tRNA synthetase (mbPylRS) to genetically encode a DNP-containing unnatural amino acid, N⁶-(2-(2,4-dinitrophenyl)acetyl)lysine (DnpK). Although this moiety was unstable in Escherichia coli, we found that its stability was enhanced in mammalian HEK 293T cells and was able to induce selective interactions with anti-DNP antibodies. The capability of genetically introducing DNP into proteins is expected to find broad applications in biosensing, immunology, and therapeutics.

Something unnatural about it: A dinitrophenyl (DNP)-containing unnatural amino acid was genetically encoded for the preparation of hapten-labeled proteins. This small hapten moiety was able to induce selective interactions with anti-DNP antibodies. The capability of genetically introducing DNP into proteins has potential for applications in biosensing and bioseparation, immunology, and therapeutics.

Gut reaction: There is increasing evidence that the impact on human health and disease of the microbes living in and on us has been underestimated. Several of the small molecules produced by “our” bacteria are structurally highly complex and show unusual biosynthetic pathways or modes of action, as highlighted by the race to elucidate the structure and biosynthesis of colibactin, a genotoxic compound produced by human gut bacteria.

Short α-peptides with less than 10 residues generally display a low propensity to nucleate stable helical conformations. While various strategies to stabilize peptide helices have been previously reported, the ability of non-peptide helical foldamers to stabilize α-helices when fused to short α-peptide segments has not been investigated. Towards this end, structural investigations into a series of chimeric oligomers obtained by joining aliphatic oligoureas to the C- or N-termini of α-peptides are described. All chimeras were found to be fully helical, with as few as 2 (or 3) urea units sufficient to propagate an α-helical conformation in the fused peptide segment. The remarkable compatibility of α-peptides with oligoureas described here, along with the simplicity of the approach, highlights the potential of interfacing natural and non-peptide backbones as a means to further control the behavior of α-peptides.

Conformational symbiosis: Non-natural heterogeneous backbones obtained by fusing peptidomimetic urea-based helical foldamers to either end of an α-peptide segment fold into a helical structure, spanning the entire sequence in an uninterrupted manner. Only a few urea units are sufficient to propagate a helical conformation along short peptide segments.

The prevalence of bioconjugates in the biomedical sciences necessitates the development of novel mechanisms to facilitate their preparation. Towards this end, the translation of the Glaser–Hay coupling to an aqueous environment is examined, and its potential as a bioorthogonal conjugation reaction is demonstrated. This optimized, novel, and aqueous Glaser–Hay reaction is applied towards the development of bioconjugates utilizing protein expressed with an alkynyl unnatural amino acid. Unnatural amino acid technology provides a degree of bioorthognality and specificity not feasible with other methods. Moreover, the scope of the reaction is demonstrated through protein–small molecule couplings, small-molecule–solid-support couplings, and protein–solid-support immobilizations.

A Glaser–Hay bioconjugation has been developed. This new bioconjugation method affords well-defined, linear, highly oxidized bioconjugates. This is the first reported aqueous Glaser–Hay reaction and first utilization of this reaction in the conjugation of proteins, small molecules, and solid supports.