Reduced incretin receptor trafficking upon activation enhances glycemic control and reverses obesity in diet-induced obese mice.

Activation of incretin receptors by their cognate agonist augments sustained cAMP generation both from the plasma membrane as well as from the endosome. To address the functional outcome of this spatiotemporal signaling, we developed a nonacylated glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor dual agonist I-M-150847 that reduced receptor internalization following activation of the incretin receptors. The incretin receptor dual agonist I-M-150847 was developed by replacing the tryptophan cage of exendin-4 tyrosine substituted at the amino terminus with the C-terminal undecapeptide sequence of oxyntomodulin that placed lysine 30 of I-M-150847 in frame with the corresponding lysine residue of GIP. The peptide I-M-150847 is a partial agonist of GLP-1R and GIPR; however, the receptors, upon activation by I-M-150847, undergo reduced internalization that promotes agonist-mediated iterative cAMP signaling and augments glucose-stimulated insulin exocytosis in pancreatic ß cells. Chronic administration of I-M-150847 improved glycemic control, enhanced insulin sensitivity, and provided profound weight loss in diet-induced obese (DIO) mice. Our results demonstrated that despite being a partial agonist, I-M-150847, by reducing the receptor internalization upon activation, enhanced the incretin effect and reversed obesity.NEW & NOTEWORTHY Replacement of the tryptophan cage (Trp-cage) with the C-terminal oxyntomodulin undecapeptide along with the tyrosine substitution at the amino terminus converts the selective glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 to a novel GLP-1R and GIPR dual agonist I-M-150847. Reduced internalization of incretin receptors upon activation by the GLP-1R and GIPR dual agonist I-M-150847 promotes iterative receptor signaling that enhances the incretin effect and reverses obesity.

Protein-Protein Interaction in Multicomponent Reaction Enables Chemoselective, Site-Selective, and Modular Labeling of Native Proteins.

A protein's pool of functionalities presents a formidable challenge for its single-site modification. Here, we report a method to harness protein-protein interaction (PPI) to drive selective modification. It involves the chemoselective reversible generation of reactive intermediates and utilizes PPI-specificity to drive the subsequent site-selective irreversible step. The disintegrate (DIN) theory-driven multicomponent aza-Morita-Baylis-Hillman (aza-MBH) reaction offers homogeneous and modular single-site protein modification capable of late-stage mono- and dual-probe installation.

Traceless cysteine-linchpin enables precision engineering of lysine in native proteins.

The maintenance of machinery requires its operational understanding and a toolbox for repair. The methods for the precision engineering of native proteins meet a similar requirement in biosystems. Its success hinges on the principles regulating chemical reactions with a protein. Here, we report a technology that delivers high-level control over reactivity, chemoselectivity, site-selectivity, modularity, dual-probe installation, and protein-selectivity. It utilizes cysteine-based chemoselective Linchpin-Directed site-selective Modification of lysine residue in a protein (LDMC-K). The efficiency of the end-user-friendly protocol is evident in quantitative conversions within an hour. A chemically orthogonal C-S bond-formation and bond-dissociation are essential among multiple regulatory attributes. The method offers protein selectivity by targeting a single lysine residue of a single protein in a complex biomolecular mixture. The protocol renders analytically pure single-site probe-engineered protein bioconjugate. Also, it provides access to homogeneous antibody conjugates (AFC and ADC). The LDMC-K-ADC exhibits highly selective anti-proliferative activity towards breast cancer cells.

Chemical technologies for precise protein bioconjugation interfacing biology and medicine.

Proteins provide an excellent means to monitor and regulate biological processes. Hence, a precise chemical toolbox for their modification becomes indispensable. In this perspective, this feature article outlines our efforts to establish the core principles of chemoselectivity, site-selectivity, site-specificity, site-modularity, residue-modularity, and protein-specificity. With the knowledge to systematically regulate these parameters, the field has access to technological platforms that can address multiple challenges at the interface of chemistry, biology, and medicine.

Chemical methods for modification of proteins.

The library of chemical reactions for C-C and C-heteroatom bond formation is exceptional. The understanding of reactivity and diverse aspects of selectivity facilitates the functional group transformation of high complexity. However, the same is not valid for proteins as an organic substrate. Gratifyingly, we can translate some of the pre-existing reactions for developing methods for the modification of proteins. Also, there is enormous potential to create a new knowledge domain that will be unique to the densely functionalized architecture of proteins. At the outset, we outlined a few concepts that bridge the gap between chemical reactions with small molecules and proteins. Next, we introduced the key attributes and challenges associated with the selectivity that emerges due to the presence of multiple types and copies of functional groups. The examples with nucleophilic amino acids outline the chemoselectivity-associated features. Gradually, the discussion moves toward the concepts that led to the successful realization of site-selectivity and N-terminus residue-specificity. The attributes of organic chemistry that emerge due to the multifunctional organization of the substrate are marked. The last section overviews the analysis of protein bioconjugates by mass spectrometry. Also, the review outlines the unmet needs and opportunities.

Chemoselective and Site-Selective Lysine-Directed Lysine Modification Enables Single-Site Labeling of Native Proteins.

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single-site labeling of a high-frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent-accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys-selective electrophile connected by a spacer. Consequently, it enables the irreversible single-site labeling of a Lys residue independent of its place in the reactivity order. The user-friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site-selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe-tagged proteins. Besides, the methodology provides access to antibody-drug conjugate (ADC), which exhibits highly selective anti-proliferative activity towards HER-2 expressing SKBR-3 breast cancer cells.

A single amino acid Gly-tag enables metal-free protein purification.

Analytically pure proteins are indispensable for diverse applications, including therapeutics. Here, we report a methodology where a single amino acid, glycine, enables metal-free protein purification. This robust platform is enabled by a Gly-tag resin for site-specific capture, enrichment, and release through chemically triggered C-C bond dissociation by resonance-assisted electron density polarization.

Sensitivity booster for mass detection enables unambiguous analysis of peptides, proteins, antibodies, and protein bioconjugates.

A chemical tag enhances peptide detection by multiple orders in mass spectrometry. The substantial improvement in the peptide mapping along with simplified and enhanced fragmentation pattern enables the unambiguous sequencing of a protein and antibody. The chemoselective sensitivity booster provides a tool for remarkably improved analysis of protein bioconjugates.