Modular Synthesis of Peptide-Based Single- and Multimodal Targeted Molecular Imaging Agents.

A practical, modular synthesis of targeted molecular imaging agents (TMIAs) containing near-infrared dyes for optical molecular imaging (OMI) or chelated metals for magnetic resonance imaging (MRI) and single-photon emission correlation tomography (SPECT) or positron emission tomography (PET) has been developed. In the method, imaging modules are formed early in the synthesis by attaching imaging agents to the side chain of protected lysines. These modules may be assembled to provide a given set of single- or dual-modal imaging agents, which may be conjugated in the last steps of the synthesis under mild conditions to linkers and targeting groups. A key discovery was the ability of a metal such as gadolinium, useful in MRI, to serve as a protecting group for the chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). It was further discovered that two lanthanide metals, La and Ce, can double as protecting groups and placeholder metals, which may be transmetalated under mild conditions by metals used for PET in the final step. The modular method enabled the synthesis of discrete targeted probes with two of the same or different dyes, two same or different metals, or mixtures of dyes and metals. The approach was exemplified by the synthesis of single- or dual-modal imaging modules for MRI-OMI, PET-OMI, and PET-MRI, followed by conjugation to the integrin-seeking peptide, c(RGDyK). For Gd modules, their efficacy for MRI was verified by measuring the NMR spin-lattice relaxivity. To validate functional imaging of TMIAs, dual-modal agents containing Cy5.5 were shown to target A549 cancer cells by confocal fluorescence microscopy.

Rational design of thioamide peptides as selective inhibitors of cysteine protease cathepsin L.

Aberrant levels of cathepsin L (Cts L), a ubiquitously expressed endosomal cysteine protease, have been implicated in many diseases such as cancer and diabetes. Significantly, Cts L has been identified as a potential target for the treatment of COVID-19 due to its recently unveiled critical role in SARS-CoV-2 entry into the host cells. However, there are currently no clinically approved specific inhibitors of Cts L, as it is often challenging to obtain specificity against the many highly homologous cathepsin family cysteine proteases. Peptide-based agents are often promising protease inhibitors as they offer high selectivity and potency, but unfortunately are subject to degradation in vivo. Thioamide substitution, a single-atom O-to-S modification in the peptide backbone, has been shown to improve the proteolytic stability of peptides addressing this issue. Utilizing this approach, we demonstrate herein that good peptidyl substrates can be converted into sub-micromolar inhibitors of Cts L by a single thioamide substitution in the peptide backbone. We have designed and scanned several thioamide stabilized peptide scaffolds, in which one peptide, RS 1A, was stabilized against proteolysis by all five cathepsins (Cts L, Cts V, Cts K, Cts S, and Cts B) while inhibiting Cts L with >25-fold specificity against the other cathepsins. We further showed that this stabilized RS 1A peptide could inhibit Cts L in human liver carcinoma lysates (IC50 = 19 µM). Our study demonstrates that one can rationally design a stabilized, specific peptidyl protease inhibitor by strategic placement of a thioamide and reaffirms the place of this single-atom modification in the toolbox of peptide-based rational drug design.

Rosetta Machine Learning Models Accurately Classify Positional Effects of Thioamides on Proteolysis.

Thioamide substitutions of the peptide backbone have been shown to stabilize therapeutic and imaging peptides toward proteolysis. In order to rationally design thioamide modifications, we have developed a novel Rosetta custom score function to classify thioamide positional effects on proteolysis in substrates of serine and cysteine proteases. Peptides of interest were docked into proteases using the FlexPepDock application in Rosetta. Docked complexes were modified to contain thioamides parametrized through the creation of custom atom types in Rosetta based on ab intio simulations. Thioamide complexes were simulated, and the resultant structural complexes provided features for machine learning classification as the decomposed values of the Rosetta score function. An ensemble, majority voting model was developed to be a robust predictor of previously unpublished thioamide proteolysis holdout data. Theoretical control simulations with pseudo-atoms that modulate only one physical characteristic of the thioamide show differential effects on prediction accuracy by the optimized voting classification model. These pseudo-atom model simulations, as well as statistical analyses of the full thioamide simulations, implicate steric effects on peptide binding as being primarily responsible for thioamide positional effects on proteolytic resistance.

Studies of Thioamide Effects on Serine Protease Activity Enable Two-Site Stabilization of Cancer Imaging Peptides.

Thioamide substitutions in peptides can be used as fluorescence quenchers in protease sensors and as stabilizing modifications of hormone analogs. To guide these applications in the context of serine proteases, we here examine the cleavage of several model substrates, scanning a thioamide between the P3 and P3' positions, and identify perturbing positions for thioamide substitution. While all serine proteases tested were affected by P1 thioamidation, certain proteases were also significantly affected by other thioamide positions. We demonstrate how these findings can be applied by harnessing the combined P3/P1 effect of a single thioamide on kallikrein proteolysis to protect two key positions in a neuropeptide Y-based imaging probe, increasing its serum half-life to >24 h while maintaining potency for binding to Y1 receptor expressing cells. Such stabilized peptide probes could find application in imaging cell populations in animal models or even in clinical applications such as fluorescence-guided surgery.

Fluorescent Probes for Studying Thioamide Positional Effects on Proteolysis Reveal Insight into Resistance to Cysteine Proteases.

Thioamide substitutions of the peptide backbone have been shown to reduce proteolytic degradation, and this property can be used to generate competitive protease inhibitors and to stabilize peptides toward degradation in vivo. Here, we present a straightforward sensor design that allows a systematic study of the positional effects of thioamide substitution by using real-time fluorescence. Thioamide scanning in peptide substrates of five papain family cysteine proteases demonstrates that a thioamide at or near the scissile bond can slow proteolysis in all cases, but that the magnitude of the effects varies with position and protease in spite of high sequence homology. Mechanistic investigation of papain proteolysis reveals that the thioamide effects derive from reductions in both affinity (KM ) and turnover number (kcat ). Computational modeling allows these effects to be understood based on disruption of key enzyme-substrate hydrogen bonds, providing a model for future rational use of thioamides to confer cysteine protease resistance.

Improving the Fluorescent Probe Acridonylalanine Through a Combination of Theory and Experiment.

Acridonylalanine (Acd) is a useful fluorophore for studying proteins by fluorescence spectroscopy, but it can potentially be improved by being made longer wavelength or brighter. Here, we report the synthesis of Acd core derivatives and their photophysical characterization. We also performed ab initio calculations of the absorption and emission spectra of Acd derivatives, which agree well with experimental measurements. The amino acid aminoacridonylalanine (Aad) was synthesized in forms appropriate for genetic incorporation and peptide synthesis. We show that Aad is a superior FRET acceptor to Acd in a peptide cleavage assay, and that Aad can be activated by an aminoacyl tRNA synthetase for genetic incorporation. Together, these results show that we can use computation to design enhanced Acd derivatives which can be used in peptides and proteins.

Thioamide Substitution Selectively Modulates Proteolysis and Receptor Activity of Therapeutic Peptide Hormones.

Peptide hormones are attractive as injectable therapeutics and imaging agents, but they often require extensive modification by mutagenesis and/or chemical synthesis to prevent rapid in vivo degradation. Alternatively, the single-atom, O-to-S modification of peptide backbone thioamidation has the potential to selectively perturb interactions with proteases while preserving interactions with other proteins, such as target receptors. Here, we use the validated diabetes therapeutic, glucagon-like peptide-1 (GLP-1), and the target of clinical investigation, gastric inhibitory polypeptide (GIP), as proof-of-principle peptides to demonstrate the value of thioamide substitution. In GLP-1 and GIP, a single thioamide near the scissile bond renders these peptides up to 750-fold more stable than the corresponding oxopeptides toward cleavage by dipeptidyl peptidase 4, the principal regulator of their in vivo stability. These stabilized analogues are nearly equipotent with their parent peptide in cyclic AMP activation assays, but the GLP-1 thiopeptides have much lower ß-arrestin potency, making them novel agonists with altered signaling bias. Initial tests show that a thioamide GLP-1 analogue is biologically active in rats, with an in vivo potency for glycemic control surpassing that of native GLP-1. Taken together, these experiments demonstrate the potential for thioamides to modulate specific protein interactions to increase proteolytic stability or tune activation of different signaling pathways.

Thieme Chemistry Journals Awardees - Where Are They Now? Improved Fmoc Deprotection Methods for the Synthesis of Thioamide-Containing Peptides and Proteins.

Site-selective incorporation of thioamides into peptides and proteins provides a useful tool for a wide range of applications. Current incorporation methods suffer from low yields as well as epimerization. Here, we describe how the use of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) rather than piperidine in fluorenylmethyloxycarbonyl (Fmoc) deprotection reduces epimerization and increases yields of thioamide-containing peptides. Furthermore, we demonstrate that the use of DBU avoids byproduct formation when synthesizing peptides containing side-chain thioamides.