Chemical technology principles for selective bioconjugation of proteins and antibodies.

Proteins are multifunctional large organic compounds that constitute an essential component of a living system. Hence, control over their bioconjugation impacts science at the chemistry-biology-medicine interface. A chemical toolbox for their precision engineering can boost healthcare and open a gateway for directed or precision therapeutics. Such a chemical toolbox remained elusive for a long time due to the complexity presented by the large pool of functional groups. The precise single-site modification of a protein requires a method to address a combination of selectivity attributes. This review focuses on guiding principles that can segregate them to simplify the task for a chemical method. Such a disintegration systematically employs a multi-step chemical transformation to deconvolute the selectivity challenges. It constitutes a disintegrate (DIN) theory that offers additional control parameters for tuning precision in protein bioconjugation. This review outlines the selectivity hurdles faced by chemical methods. It elaborates on the developments in the perspective of DIN theory to demonstrate simultaneous regulation of reactivity, chemoselectivity, site-selectivity, modularity, residue specificity, and protein specificity. It discusses the progress of such methods to construct protein and antibody conjugates for biologics, including antibody-fluorophore and antibody-drug conjugates (AFCs and ADCs). It also briefs how this knowledge can assist in developing small molecule-based covalent inhibitors. In the process, it highlights an opportunity for hypothesis-driven routes to accelerate discoveries of selective methods and establish new targetome in the precision engineering of proteins and antibodies.

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.

Human Behavior-Inspired Linchpin-Directed Catalysis for Traceless Precision Labeling of Lysine in Native Proteins.

The complex social ecosystem regulates the spectrum of human behavior. However, it becomes relatively easier to understand if we disintegrate the contributing factors, such as locality and interacting partners. Interestingly, it draws remarkable similarity with the behavior of a residue placed in a social setup of functional groups in a protein. Can it inspire principles for creating a unique environment for the precision engineering of proteins? We demonstrate that localization-regulated interacting partner(s) could render precise and traceless single-site modification of structurally diverse native proteins. The method targets a combination of high-frequency Lys residues through an array of reversible and irreversible reactions. However, excellent simultaneous control over chemoselectivity, site selectivity, and modularity ensures that the user-friendly protocol renders acyl group installation, including post-translational modifications (PTMs), on a single Lys. Besides, it offers a chemically orthogonal handle for the installation of probes. Also, a purification protocol integration delivers analytically pure single-site tagged protein bioconjugates. The precise labeling of a surface Lys residue ensures that the structure and enzymatic activities remain conserved post-bioconjugation. For example, the precise modification of insulin does not affect its uptake and downstream signaling pathway. Further, the method enables the synthesis of homogeneous antibody-fluorophore and antibody-drug conjugates (AFC and ADC; K183 and K249 labeling). The trastuzumab-rhodamine B conjugate displays excellent serum stability along with antigen-specific cellular imaging. Further, the trastuzumab-emtansine conjugate offers highly specific antiproliferative activity toward HER-2 positive SKBR-3 breast cancer cells. This work validates that disintegrate theory can create a comprehensive platform to enrich the chemical toolbox to meet the technological demands at the chemistry, biology, and medicine interface.

Development and characterization of mouse monoclonal antibodies to canine morbillivirus.

Canine morbillivirus is a highly contagious multi-host pathogen with high morbidity and mortality. Timely diagnosis is of utmost importance to effectively control such a dreadful disease. Monoclonal antibodies (mAbs) serve as a high throughput diagnostics and applied tools for research and development (R&D). In the present study, a total of six mouse monoclonal antibodies were developed. All the mAbs generated belonged to IgG class. Of the six mAbs, two of them, namely CD-2F8 and CD-3D8 were directed against the nucleocapsid protein of CDV as determined in western blotting. The reactivity of all the mAbs was checked in indirect-ELISA and cell-ELISA using different morbilliviruses. The mAbs could broadly be categorized as; CDV specific (CD-3D8 and CD-2F8), cross-reactive to PPR virus (CD-AB3 and CD-4D6) and cross-reactive to both PPR virus and measles virus (CD-5D10 and CD-6E5). The characterized mAbs were used for antigenic profiling of CDV, PPR virus and measles virus. Based on the reactivity pattern; a close antigenic relationship was found among CDV and PPR virus as compared to measles virus. A pair of CDV specific mAbs namely CD-2F8 and CD-3D8 were identified which did not cross-react with measles and PPR viruses and thus could be used for diagnostic applications.

Replication competence of canine distemper virus in cell lines expressing signaling lymphocyte activation molecule (SLAM) of goat, sheep and dog origin.

Cellular receptors play an important role in entry and cell to cell spread of morbillivirus infections. The cells expressing SLAM and Nectin-4 have been used for successful and efficient isolation of canine distemper virus (CDV) in high titre. There are several methods for generation of cells expressing receptor molecules. Here, we have used a comparatively cheaper and easily available method, pcDNA 3.1 (+) for engineering Vero cells to express SLAM gene of goat, sheep and dog origin (Vero/Goat/SLAM (VGS), Vero/Sheep/SLAM (VSS) and Vero/Dog/SLAM (VDS), respectively). The generated cell lines were then compared to test their efficacy to support CDV replication. CDV could be grown in high titre in the cells expressing SLAM and a difference of log two could be recorded in virus titre between VDS and native Vero cells. Also, CDV could be grown in a higher titre in VDS as compared to VGS and VSS. The finding of this study supports the preferential use of SLAM expressing cells over the native Vero cells by CDV. Further, the higher titre of CDV in cells expressing dog-SLAM as compared to the cells expressing SLAM of non-CDV hosts (i.e. goat and sheep) points towards the preferential use of dog SLAM by the CDV and may be a plausible reason for differential susceptibility of small ruminants and Canines to CDV.

Reactivity and Selectivity Principles in Native Protein Bioconjugation.

Are chemical methods capable of precisely engineering the native proteins? Is it possible to develop platforms that can empower the regulation of chemoselectivity, site-selectivity, modularity, protein-specificity, and site-specificity? This account delineates our research journey in the last ten years on the developments revolving around these questions. It will range from the realization of chemoselective and site-selective labeling of reactivity hotspots to modular linchpin directed modification (LDM®) platform and site-specific Gly-tag® technology. Also, we outline a few biotechnology tools, including Maspecter®, that accelerated the detailed analysis of the bioconjugates and rendered a powerful toolbox for homogeneous antibody-drug conjugates (ADCs).

Comparing the efficiency of different Escherichia coli strains in producing recombinant capsid protein of porcine circovirus type 2.

The present study was aimed at comparing different E. coli strains in expressing the capsid protein of Porcine Circovirus 2 (PCV2). Full length capsid protein could be expressed only in Rosetta-gami 2 (DE3) pLysS strain using pET32b (+) vector. This confirmed that only those strains which possess tRNAs for rare codons can express the full length capsid protein. Purification of full length capsid protein could not be achieved even after several attempts using native and denaturing conditions. Subsequently, an attempt was made for expression of N-terminal truncated capsid protein using the same expression system. Truncated capsid protein was successfully expressed, purified and characterized by western blotting. The truncated capsid protein was also shown to be efficacious in testing serum samples using an optimized indirect ELISA, wherein a diagnostic sensitivity of 88.89% and specificity of 90.82% was obtained as compared to commercially available GreenSpring® porcine circovirus (PCV2) ELISA test kit. Thus, the expressed truncated capsid protein appears to be a promising diagnostic agent for PCV2. The comparative analysis suggests that cluster of arginine residues at N-terminal of capsid protein not only affects its expression in some E. coli strains but also its purification by Ni-NTA chromatography, when expressed as a histidine tagged fusion protein.

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.

Single-Site Labeling of Native Proteins Enabled by a Chemoselective and Site-Selective Chemical Technology.

Chemical biology research often requires precise covalent attachment of labels to the native proteins. Such methods are sought after to probe, design, and regulate the properties of proteins. At present, this demand is largely unmet due to the lack of empowering chemical technology. Here, we report a chemical platform that enables site-selective labeling of native proteins. Initially, a reversible intermolecular reaction places the "chemical linchpins" globally on all the accessible Lys residues. These linchpins have the capability to drive site-selective covalent labeling of proteins. The linchpin detaches within physiological conditions and capacitates the late-stage installation of various tags. The chemical platform is modular, and the reagent design regulates the site of modification. The linchpin is a multitasking group and facilitates purification of the labeled protein eliminating the requirement of additional chromatography tag. The methodology allows the labeling of a single protein in a mixture of proteins. The precise modification of an accessible residue in protein ensures that their structure remains unaltered. The enzymatic activity of myoglobin, cytochrome C, aldolase, and lysozyme C remains conserved after labeling. Also, the cellular uptake of modified insulin and its downstream signaling process remain unperturbed. The linchpin directed modification (LDM) provides a convenient route for the conjugation of a fluorophore and drug to a Fab and monoclonal antibody. It delivers trastuzumab-doxorubicin and trastuzumab-emtansine conjugates with selective antiproliferative activity toward Her-2 positive SKBR-3 breast cancer cells.

Aldehydes can switch the chemoselectivity of electrophiles in protein labeling.

We show that the chemoselectivity of an electrophile in protein labeling can be promiscuous. An aldehyde enables switching of chemoselectivity of an epoxide and a sulfonate ester along with an enhanced rate of reaction. The chemical technology renders single-site installation of diverse probes on a protein and delivers analytically pure tagged proteins.

Site-Selective Labeling of Native Proteins by a Multicomponent Approach.

Chemical functionalization of proteins is an indispensable tool. Yet, selective labeling of native proteins has been an arduous task. The limited success of chemical methods allows N-terminus protein labeling, but the examples with side-chain residues are rare. Herein, we surpass this challenge through a multicomponent transformation that operates under physiological conditions in the presence of a protein, aldehyde, acetylene, and Cu-ligand complex. The methodology results in the labeling of a single lysine residue in nine distinct proteins.

Twisted amide electrophiles enable cyclic peptide sequencing.

There is an ever-increasing interest in synthetic methods that not only enable peptide macrocyclization, but also facilitate downstream application of the synthesized molecules. We have found that aziridine amides are stereoelectronically attenuated in a macrocyclic environment such that non-specific interactions with biological nucleophiles are reduced or even shut down. The electrophilic reactivity, revealed at high pH, enables peptide sequencing by mass spectrometry, which will further broaden the utility of aziridine amide-containing libraries of macrocycles.

Ordered immobilization of serine proteases enabled by a linchpin directed modification platform.

We report a chemoselective and site-selective precision engineering of lysine in proteases. The mild and physiological reaction conditions keep their auto-degradation under control. Furthermore, it enables single-site ordered immobilization, enhancing protein digestion and peptide mapping efficiency.

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.

Disintegrate (DIN) Theory Enabling Precision Engineering of Proteins.

The chemical toolbox for the selective modification of proteins has witnessed immense interest in the past few years. The rapid growth of biologics and the need for precision therapeutics have fuelled this growth further. However, the broad spectrum of selectivity parameters creates a roadblock to the field's growth. Additionally, bond formation and dissociation are significantly redefined during the translation from small molecules to proteins. Understanding these principles and developing theories to deconvolute the multidimensional attributes could accelerate the area. This outlook presents a disintegrate (DIN) theory for systematically disintegrating the selectivity challenges through reversible chemical reactions. An irreversible step concludes the reaction sequence to render an integrated solution for precise protein bioconjugation. In this perspective, we highlight the key advancements, unsolved challenges, and potential opportunities.

Optimization of lateral flow assay for Canine morbillivirus detection and the application of the strip as sample substitute.

Canine distemper is an emerging disease, caused by the Canine morbillivirus (CDV) of the Paramyxoviridae family. The virus has evolved as a multi-host pathogen as it affects many wildlife animal species. The development of specific and sensitive diagnostic tests is the need for a control program. Several diagnostic tests are available for the detection of CDV antigen and antibody. Lateral flow assay (LFA) is the most promising point of care diagnostic test because of its specificity, easy use, and instant result. This study was designed to develop a lateral flow assay using the in-house developed monoclonal antibody (mAb) against the nucleocapsid protein (N) of the 'CDV/dog/bly/Ind/2018' isolate, which represents the circulating strains of India. The two mAbs included in the study showed high binding affinity in indirect ELISA and dot blot assay. Out of two, one mAb was selected due to its comparatively higher binding affinity in LFA format, and less non-specific binding to the biological matrix and buffer components. The limit of detection was found to be 106.5 TCID50/ml with the assay run time of 5 min. The fresh clinical samples collected on the spot were distinctly detected by the LFA, whereas the stored samples with a reduced titre of the virus showed inconsistent results. Moreover, the blood samples showed a clear distinction of positive and negative than the swab and tissue homogenates. The RNA extraction from the strip was successful with the some modifications in the Trizol RNA extraction method and the N and H gene fragments were amplified. Therefore, the study concludes that the LFA is suitable for CDV antigen detection in the field conditions and the strips can be used as the sample substitute for molecular study.

Comparative pathogenicity of BA.2.12, BA.5.2 and XBB.1 with the Delta variant in Syrian hamsters.

Omicron variant is evolving into numerous sub variants with time and the information on the characteristics of these newly evolving variants are scant. Here we performed a pathogenicity evaluation of Omicron sub variants BA.2.12, BA.5.2 and XBB.1 against the Delta variant in 6-8-week-old Syrian hamster model. Body weight change, viral load in respiratory organs by real time RT-PCR/titration, cytokine mRNA quantification and histopathological evaluation of the lungs were performed. The intranasal infection of the BA.2.12, BA.5.2 and XBB.1 variants in hamster model resulted in body weight loss/reduced weight gain, inflammatory cytokine response and interstitial pneumonia with lesser severity compared to the Delta variant infection. Among the variants studied, BA.2.12 and XBB.1 showed lesser viral shedding through the upper respiratory tract, whereas the BA.5.2 showed comparable viral RNA shedding as that of the Delta variant. The study shows that the Omicron BA.2 sub variants may show difference in disease severity and transmissibility amongst each other whereas the overall disease severity of the Omicron sub variants studied were less compared to the Delta variant. The evolving Omicron sub variants and recombinants should be monitored for their properties.

Bending rigid molecular rods: formation of oligoproline macrocycles.

Bent but not broken: cyclic oligoprolines are accessed in a reaction that effectively bends rigid oligoproline peptides (see scheme; TBDMS=tert-butyldimethylsilyl). The stitching is accomplished during macrocyclization enabled by aziridine aldehydes and isocyanides. Molecular modeling studies suggest that electrostatic attraction between the termini of the linear peptide is pivotal for macrocyclization. The macrocycles were studied by circular dichroism with a polyproline II structure being observed in larger macrocycles.

Protein inspired chemically orthogonal imines for linchpin directed precise and modular labeling of lysine in proteins.

We report a chemoselective, site-selective, and modular technology for precision engineering of high-frequency lysine residues in native proteins. It enables a unique, unexplored reactivity landscape on the protein surface to facilitate their single-site modification. Further, the method presents bond-architecture flexibility and enables orthogonal tagging with probes of interest.