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

   https://doi.org/10.1039/D1SC00785H  

   Phan, Hoang Anh; Giannakoulias, Sam G.; Barrett, Taylor M.; Liu, Chunxiao; Petersson, E. James (January 2021, Chemical Science)

   null (Ed.)

   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, R S 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 R S 1A peptide could inhibit Cts L in human liver carcinoma lysates (IC 50 = 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. 

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2. Proteases and nucleases involved in the biphasic digestion process of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae)

   https://doi.org/10.1002/arch.21459  

   Lomate, Purushottam R.; Bonning, Bryony C. (March 2018, Archives of Insect Biochemistry and Physiology)

   Abstract Management of the brown marmorated stink bug,Halyomorpha halys(Hemiptera: Pentatomidae), an invasive, agricultural pest in the United States, has presented significant challenges. This polyphagous insect uses both extra‐oral and gut‐based digestion thwarting protein‐ or nucleotide‐based control strategies. The objective of this study was to biochemically characterize the digestive enzymes (proteases and nucleases) from the saliva, salivary gland and the gut ofH. halys. Enzyme profiles for the two tissues and saliva radically differ: The pH optimum for proteases in the gut was six, with cysteine proteases predominant. In contrast, the alkaline pH optima for protease activity in the salivary gland (8–10) and saliva (7) reflected abundant serine protease and cathepsin activities. RNase enzymes were most abundant in saliva, while dsRNase and DNase activities were higher in the salivary gland and saliva compared to those in the gut. These very different enzyme profiles highlight the biphasic digestive system used by this invasive species for efficient processing of plant nutrients. Knowledge ofH. halysdigestive physiology will allow for counteractive measures targeting digestive enzymes or for appropriate protection of protein‐ or nucleotide‐based management options targeting this pest. 

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3. Activatable Photoacoustic Probe for Imaging Infection: Gold Nanorod Dissociation In Vivo Reports Bacterial Protease Activity

   https://doi.org/10.1021/acsnano.4c17874  

   Retout, Maurice; Lepeintre, Victor; Amer, Lubna; Yim, Wonjun; Jokerst, Jesse V (April 2025, ACS Nano)

   We present a strategy for constructing activatable photoacoustic imaging (PAI) probes for in vivo enzyme activity measurements, based on a dissociation strategy previously applied to in vitro sensing. Unlike conventional nanoparticle aggregation strategies, dissociation minimizes false positives and functions effectively in complex biological environments. Overcoming the challenge of dissociating nanostructure aggregates, which arises from the strong van der Waals forces at short distances, we demonstrate the controlled assembly and dissociation of citrate-capped gold nanorods (AuNRs-citrate) using a diarginine peptide additive and a thiolated polyethylene glycol (HS-PEG-OMe), respectively. This assembly dissociation mechanism enables precise control of the optical and photoacoustic (PA) properties of AuNRs in both in vitro and in vivo settings. Building on these findings, we engineered an enzyme-sensitive PAI probe (AuNRs@RgpB) composed of AuNR assemblies and a PEG-peptide conjugate with a protease-specific cleavage sequence. The probe detects Arg-specific gingipain (RgpB), a protease expressed by Porphyromonas gingivalis associated with periodontal disease and Alzheimer’s disease. Proteolytic cleavage of the peptide sequence triggers AuNR dissociation, resulting in enhanced PA signal output. The probe was designed to be injected intrathecally for preclinical trials to image gingipains and investigate the value of gingipain inhibitors developed for Alzheimer’s disease. The probe’s performance was characterized in vitro using UV−vis spectroscopy and PAI, achieving detection limits of 5 and 20 nM, respectively. In vivo studies involved intracranial injection of AuNRs@ RgpB into RgpB-containing murine models, with PA monitoring over time. RgpB activity produced a four-fold PA signal increase within 2 h, while P. gingivalis-infected mice showed similar signal enhancement. Specificity was confirmed by negligible responses to Fusobacterium nucleatum, a non-RgpB-producing bacterium. Additionally, the system demonstrated utility in drug development by successfully monitoring the inhibition of RgpB activity using RgpB inhibitors (leupeptin and KYT-1) in vivo models. Beyond its immediate application to RgpB detection, this modular approach to plasmonic-based sensing holds significant potential for detecting other proteases, advancing both nanotechnology and protease-targeted diagnostics. 

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4. A Review of Small Molecule Inhibitors and Functional Probes of Human Cathepsin L

   https://doi.org/10.3390/molecules25030698  

   Dana, Dibyendu; Pathak, Sanjai K. (February 2020, Molecules)

   Human cathepsin L belongs to the cathepsin family of proteolytic enzymes with primarily an endopeptidase activity. Although its primary functions were originally thought to be only of a housekeeping enzyme that degraded intracellular and endocytosed proteins in lysosome, numerous recent studies suggest that it plays many critical and specific roles in diverse cellular settings. Not surprisingly, the dysregulated function of cathepsin L has manifested itself in several human diseases, making it an attractive target for drug development. Unfortunately, several redundant and isoform-specific functions have recently emerged, adding complexities to the drug discovery process. To address this, a series of chemical biology tools have been developed that helped define cathepsin L biology with exquisite precision in specific cellular contexts. This review elaborates on the recently developed small molecule inhibitors and probes of human cathepsin L, outlining their mechanisms of action, and describing their potential utilities in dissecting unknown function. 

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5. Data-driven supervised learning of a viral protease specificity landscape from deep sequencing and molecular simulations

   https://doi.org/10.1073/pnas.1805256116  

   Pethe, Manasi A.; Rubenstein, Aliza B.; Khare, Sagar D. (December 2018, Proceedings of the National Academy of Sciences)

   Biophysical interactions between proteins and peptides are key determinants of molecular recognition specificity landscapes. However, an understanding of how molecular structure and residue-level energetics at protein−peptide interfaces shape these landscapes remains elusive. We combine information from yeast-based library screening, next-generation sequencing, and structure-based modeling in a supervised machine learning approach to report the comprehensive sequence−energetics−function mapping of the specificity landscape of the hepatitis C virus (HCV) NS3/4A protease, whose function—site-specific cleavages of the viral polyprotein—is a key determinant of viral fitness. We screened a library of substrates in which five residue positions were randomized and measured cleavability of ∼30,000 substrates (∼1% of the library) using yeast display and fluorescence-activated cell sorting followed by deep sequencing. Structure-based models of a subset of experimentally derived sequences were used in a supervised learning procedure to train a support vector machine to predict the cleavability of 3.2 million substrate variants by the HCV protease. The resulting landscape allows identification of previously unidentified HCV protease substrates, and graph-theoretic analyses reveal extensive clustering of cleavable and uncleavable motifs in sequence space. Specificity landscapes of known drug-resistant variants are similarly clustered. The described approach should enable the elucidation and redesign of specificity landscapes of a wide variety of proteases, including human-origin enzymes. Our results also suggest a possible role for residue-level energetics in shaping plateau-like functional landscapes predicted from viral quasispecies theory. 

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