bioTCIs: Middle-to-Macro Biomolecular Targeted Covalent Inhibitors Possessing Both Semi-Permanent Drug Action and Stringent Target Specificity as Potential Antibody Replacements.

Monoclonal antibody therapies targeting immuno-modulatory targets such as checkpoint proteins, chemokines, and cytokines have made significant impact in several areas, including cancer, inflammatory disease, and infection. However, antibodies are complex biologics with well-known limitations, including high cost for development and production, immunogenicity, a limited shelf-life because of aggregation, denaturation, and fragmentation of the large protein. Drug modalities such as peptides and nucleic acid aptamers showing high-affinity and highly selective interaction with the target protein have been proposed alternatives to therapeutic antibodies. The fundamental limitation of short in vivo half-life has prevented the wide acceptance of these alternatives. Covalent drugs, also known as targeted covalent inhibitors (TCIs), form permanent bonds to target proteins and, in theory, eternally exert the drug action, circumventing the pharmacokinetic limitation of other antibody alternatives. The TCI drug platform, too, has been slow in gaining acceptance because of its potential prolonged side-effect from off-target covalent binding. To avoid the potential risks of irreversible adverse drug effects from off-target conjugation, the TCI modality is broadening from the conventional small molecules to larger biomolecules possessing desirable properties (e.g., hydrolysis resistance, drug-action reversal, unique pharmacokinetics, stringent target specificity, and inhibition of protein-protein interactions). Here, we review the historical development of the TCI made of bio-oligomers/polymers (i.e., peptide-, protein-, or nucleic-acid-type) obtained by rational design and combinatorial screening. The structural optimization of the reactive warheads and incorporation into the targeted biomolecules enabling a highly selective covalent interaction between the TCI and the target protein is discussed. Through this review, we hope to highlight the middle to macro-molecular TCI platform as a realistic replacement for the antibody.

Solvatochromic peptidic binder obtained via extended phage display acts as a fluororeporter for fragment-based drug discovery (FBDD).

We have previously established a selection system to obtain a solvatochromic protein binder from a peptidic fluoroprobe library via the extended T7 phage display. Here, we use the peptidic binder as a fluororeporter in this proof-of-concept study of fragment-based screening approach to drug discovery. The binder is released from the target protein on mixing with an appropriate lead compound, thereby altering its fluorescence color/intensity under 365 nm ultraviolet wavelength irradiation. By this instant screening outcome, the affinity of the lead compound is apparent to the naked eye, and quantified with a portable microvolume fluorophotometer. We envision that our simple and affordable screening system will provide opportunities for early stage drug discovery, especially for non-experts in academia and education because expensive hardware is not required for qualifying the measurements.

Relative Nuclease Resistance of a DNA Aptamer Covalently Conjugated to a Target Protein.

A major obstacle to the therapeutic application of an aptamer is its susceptibility to nuclease digestion. Here, we confirmed the acquisition of relative nuclease resistance of a DNA-type thrombin binding aptamer with a warhead (TBA3) by covalent binding to a target protein in the presence of serum/various nucleases. When the thrombin-inhibitory activity of TBA3 on thrombin was reversed by the addition of the complementary strand, the aptamer was instantly degraded by the nucleases, showing that the properly folded/bound aptamer conferred the resistance. Covalently binding aptamers possessing both a prolonged drug effect and relative nuclease resistance would be beneficial for in vivo translational applications.

A medium-firm drug-candidate library of cryptand-like structures on T7 phage: design and selection of a strong binder for Hsp90.

We designed and synthesized a medium-firm drug-candidate library of cryptand-like structures possessing a randomized peptide linker on the bacteriophage T7. From the macrocyclic library with a 109 diversity, we obtained a binder toward a cancer-related protein (Hsp90) with an antibody-like strong affinity (KD = 62 nM) and the binding was driven by the enthalpy. The selected supramolecular ligand inhibited Hsp90 activity by site-specific binding outside of the well-known ATP-binding pocket on the N-terminal domain (NTD).

Facile and Efficient Chemoenzymatic Semisynthesis of Fc-Fusion Compounds for Half-Life Extension of Pharmaceutical Components.

The formation of Fc-fusions, in which biologically active molecules and the Fc fragment of antibodies are linked to each other, is one of the most efficient and successful half-life extension technologies to be developed and applied to peptide and protein pharmaceuticals thus far. Fc-fusion compounds are generally produced by recombinant methods. However, these cannot be applied to artificial middle molecules, such as peptides with non-natural amino acids, unnatural cyclic peptides, or pharmaceutical oligonucleotides. Here, we developed a simple, efficient, semisynthetic method for Fc-fusion production involving our previously developed enzymatic N-terminal extension reaction (i.e., NEXT-A reaction) and strain-promoted azide-alkyne cycloaddition, achieving quantitative conversion and high selectivity for the N-terminus of the Fc protein. An Fc-fusion compound prepared by this method showed comparable biological activity to that of the original peptide and a long-circulating plasma half-life. Thus, the proposed method is potentially applicable for the conjugation of a wide range of pharmaceutical components.

Mechano-chromic protein-polymer hybrid hydrogel to visualize mechanical strain.

In a proof-of-concept study, a mechano-chromic hydrogel was synthesized here, via chemoenzymatic click conjugation of fluorophore-labeled fibronectin into a synthetic hydrogel co-polymers (i.e., poly-N-isopropylacrylamide/polyethylene glycol). The optical FRET response could be tuned by macroscopic stretch.

Combinatorially Screened Peptide as Targeted Covalent Binder: Alteration of Bait-Conjugated Peptide to Reactive Modifier.

A peptide-type covalent binder for a target protein was obtained by combinatorial screening of fluoroprobe-conjugated peptide libraries on bacteriophage T7. The solvatochromic fluoroprobe works as a bait during the affinity selection process of phage display. To obtain the targeted covalent binder, the bait in the selected consensus peptide was altered into a reactive warhead possessing a sulfonyl fluoride. The reaction efficiency and site/position specificity of the covalent conjugation between the binder and the target protein were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and rationalized by a protein-ligand docking simulation.

Fluorescent "keep-on" type pharmacophore obtained from dynamic combinatorial library of Schiff bases.

We established a novel principle for fluorescence detection of a target protein. A low-molecular-weight fluorescent pharmacophore, as a targeted probe, was selected from a dynamic combinatorial library of Schiff bases. The pharmacophore retains its fluorescence when bound to the hydrophobic site of the target, whereas it loses it because of hydrolysis when unbound. Graphical abstract We describe a novel concept for detection of a target protein (i.e., HSA) by using a keep-on-type fluorescent pharmacophore which is discovered from a dynamic combinatorial library of Schiff bases. When the target is absent, the keep-on-pharmacophore is degraded by hydrolysis, with the result that we can see no fluorescence.

A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry.

To determine the binding-site of a combinatorially-selected peptide possessing a fluoroprobe, a novel cysteine reactive small photo-crosslinker that can be excited by a conventional long-wavelength ultraviolet handlamp (365 nm) was synthesized via Suzuki coupling with three steps. The crosslinker is rationally designed, not only as a bioisostere of the fluoroprobe, but as a caged-fluorophore, and the photo-crosslinked target protein became fluorescent with a large Stokes-shift. By introducing the crosslinker to a designated sulfhydryl (SH) group of a combinatorially-selected peptide, the protein-binding site of the targeted peptide was deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/fluorescence imaging followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis.

Selection of Color-Changing and Intensity-Increasing Fluorogenic Probe as Protein-Specific Indicator Obtained via the 10BASE(d)-T.

To obtain a molecular probe for specific protein detection, we have synthesized fluorogenic probe library of vast diversity on bacteriophage T7 via the gp10 based-thioetherificaion (10BASE(d)-T). A remarkable color-changing and turning-on probe was selected from the library, and its physicochemical properties upon target-specific binding were obtained. Combination analyses of fluorescence emission titration, isothermal titration calorimetry (ITC), and quantitative saturation-transfer difference (STD) NMR measurements, followed by in silico docking simulation, rationalized the most plausible geometry of the ligand-protein interaction.

Unexpectedly fast transfer of positron-emittable artificial substrate into N-terminus of peptide/protein mediated by wild-type L/F-tRNA-protein transferase.

This article demonstrates the fastest enzymatic introduction of a positron emission tomography (PET) probe into acceptor peptides/proteins. It is site-specifically introduced at the basic N-terminus of the acceptors by using L/F-transferase in combination with aminoacyl-tRNA synthetase, namely the NEXT-A/PET reaction. Estimated from kinetic analysis, the transfer efficiency of O-(2-fluoromethyl)-L-tyrosine as an artificial amino acid PET probe mediated by the wild-type transferase is almost as good as that of the natural substrate, phenylalanine.

Pharmacophore generation from a drug-like core molecule surrounded by a library peptide via the 10BASEd-T on bacteriophage T7.

We have achieved site-specific conjugation of several haloacetamide derivatives into designated cysteines on bacteriophage T7-displayed peptides, which are fused to T7 capsid protein gp10. This easiest gp10 based-thioetherification (10BASEd-T) undergoes almost quantitatively like a click reaction without side reaction or loss of phage infectivity. The post-translational modification yield, as well as the site-specificity, is quantitatively analyzed by a fluorescent densitometric analysis after gel electrophoresis. The detailed structure of the modified peptide on phage is identified with tandem mass spectrometry. Construction of such a peptide-fused phage library possessing non-natural core structures will be useful for future drug discovery. For this aim, we propose a novel concept of pharmacophore generation from a drug-like molecule (i.e., salicylic acid) conjugated with surrounding randomized peptides. By using the hybrid library, streptavidin-specific binders are isolated through four rounds of biopanning.

Fission yeast Ubr1 ubiquitin ligase influences the oxidative stress response via degradation of active Pap1 bZIP transcription factor in the nucleus.

Cells adapt to oxidative stress by transcriptional activation of genes encoding antioxidants and proteins of other protective roles. A bZIP transcription factor, Pap1, plays a critical role in this process and overexpression of Pap1 confers resistance to various oxidants and drugs in fission yeast. Pap1 temporarily enters the nucleus upon oxidative stress but returns to the cytoplasm once cells adapt to the stress, suggesting that cellular localization regulates Pap1 function. We report here an additional regulatory mechanism that Ubr1 ubiquitin ligase-dependent degradation lowered the Pap1 protein levels. ubr1 cells were causally resistant to hydrogen peroxide because of the increment of Pap1 levels. Pap1 was preferentially degraded in the nucleus where Ubr1 was consistently enriched. Proteolysis was critical to downregulate Pap1 especially when its activation persisted, as constitutively nuclear Pap1 severely inhibited growth in ubr1 mutants. Inactive mutations in the bZIP DNA binding domain stabilized Pap1 but rescued the lethality caused by constitutively active Pap1 in ubr1 mutants. These findings indicate that either nuclear export or Ubr1-mediated proteolysis must be operative to prevent uncontrolled Pap1 function. Coincidental dysfunction in both inhibitory pathways causes lethality because of prolonged activation of Pap1. Ubr1 is a critical regulator for the homeostasis of oxidative stress response.

Inhibition of thrombin activity by a covalent-binding aptamer and reversal by the complementary strand antidote.

Alleviating the potential risk of irreversible adverse drug effects has been an important and challenging issue for the development of covalent drugs. Here we created a DNA-aptamer-type covalent drug by introducing a sulfonyl fluoride warhead at appropriate positions of the thrombin binding aptamer to create weaponized covalent drugs. We showed the de-activation of thrombin by the novel modality, followed by its re-activation by the complementary strand antidote at an arbitrary time. We envision that such on-demand reversal of covalent drugs will alleviate the major concern of potentially irreversible ADEs and accelerate the translational application of covalent aptamer drugs.

Direct screening of a target-specific covalent binder: stringent regulation of warhead reactivity in a matchmaking environment.

A peptide-type covalent binder for a target protein was obtained by direct and stringent screening of a warhead-modified peptide library on the robust T7 phage. The aryl fluorosulfate (fosylate) warhead was activated only in a matchmaking microenvironment created between the target protein and an appropriate peptide during the reactivity/affinity-based co-selection process of extended phage display.

Strategic design to create HER2-targeting proteins with target-binding peptides immobilized on a fibronectin type III domain scaffold.

Tumor-binding peptides such as human epidermal growth factor receptor 2 (HER2)-binding peptides are attractive therapeutic and diagnostic options for cancer. However, the HER2-binding peptides (HBPs) developed thus far are susceptible to proteolysis and lose their affinity to HER2 in vivo. In this report, a method to create a HER2-binding fluctuation-regulated affinity protein (HBP-FLAP) consisting of a fibronectin type III domain (FN3) scaffold with a structurally immobilized HBP is presented. HBPs were selected by phage-library screening and grafted onto FN3 to create FN3-HBPs, and the HBP-FLAP with the highest affinity (HBP sequence: YCAHNM) was identified after affinity maturation of the grafted HBP. HBP-FLAP containing the YCAHNM peptide showed increased proteolysis-resistance, binding to HER2 with a dissociation constant (K D) of 58 nM in ELISA and 287 nM in biolayer interferometry and specifically detects HER2-expressing cancer cells. In addition, HBP-FLAP clearly delineated HER2-expressing tumors with a half-life of 6 h after intravenous injection into tumor-bearing mice. FN3-based FLAP is an excellent platform for developing target-binding small proteins for clinical applications.

Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system.

Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically aminoacylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA(Pyl) was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.

18F-Containing Positron Emission Tomography Probe Conjugation Methodology for Biologics as Specific Binders for Tumors.

Molecular imaging can be used to evaluate the spatial-time change of the molecular biological phenomenon of the cell-molecule level in living bodies. Molecular imaging technology is expected to be applied in the fields of drug development, clinical diagnosis, and life science research. Specifically, positron emission tomography (PET) is a powerful non-invasive imaging technology for investigating physiological parameters in living animals using compounds labeled with PET radioisotopes as molecular probes. This review summarizes and compares various 18F-conjugation techniques that employ the chemical and enzymatic reactions of different types of tumor-targeting biological molecules such as peptides, proteins, antibodies, and nucleic acids.

Transglutaminase-mediated N- and C-terminal fluorescein labeling of a protein can support the native activity of the modified protein.

Fluorescein and its analogs are among the best fluorophores to label proteins and the labeling generally involves chemical modification of a translated protein. Using this methodology, labeling at a specific position remains difficult. It is known that the guinea pig liver transglutaminase (TGase)-catalyzed enzymatic modification method can allow terminal-specific fluorophore labeling of a protein by monodansylcadaverine. However, native activity of the fluorescent protein has not been investigated so far, nor has direct comparison between the chemical modification and the TGase-catalyzed modification been attempted. Therefore, we compared the possibility of fluorescein labeling via chemical labeling and via TGase-catalyzed modification. The latter method was found to be very practical and overcame some of the problems associated with the specificity of the former; fluorescein was covalently attached only to the N- or C-terminal site of glutathione S-transferase when the reaction was catalyzed by TGase and the resulting labeled protein completely retained its native activity. The TGase-mediated labeling occurred not only at room temperature but also at 4 degrees C to the same extent, which is more desirable for preventing the inactivation of proteins.