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The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium ‘Candidatus Liberibacter asiaticus’ ( C Las) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all C Las isolates, as a molecular probe to understand C Las virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in C Las-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1 -expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants. Greening disease threatens the productivity of citrus crops worldwide yet the pathosystem is poorly understood. Here, Clark et al. show that an effector cloned from the associated bacteria can suppress host plant papain-like cysteine proteases' activity, suggesting its probable role in pathogenesis.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged to be the greatest threat to humanity in the modern world and has claimed nearly 2.2 million lives worldwide. The United States alone accounts for more than one fourth of 100 million COVID-19 cases across the globe. Although vaccination against SARS-CoV-2 has begun, its efficacy in preventing a new or repeat COVID-19 infection in immunized individuals is yet to be determined. Calls for repurposing of existing, approved, drugs that target the inflammatory condition in COVID-19 are growing. Our initial gene ontology analysis predicts a similarity between SARS-CoV-2 induced inflammatory and immune dysregulation and the pathophysiology of rheumatoid arthritis. Interestingly, many of the drugs related to rheumatoid arthritis have been found to be lifesaving and contribute to lower COVID-19 morbidity. We also performed in silico investigation of binding of epigallocatechin gallate (EGCG), a well-known catechin, and other catechins on viral proteins and identified papain-like protease protein (PLPro) as a binding partner. Catechins bind to the S1 ubiquitin-binding site of PLPro, which might inhibit its protease function and abrogate SARS-CoV-2 inhibitory function on ubiquitin proteasome system and interferon stimulated gene system. In the realms of addressing inflammation and how to effectively target SARS-CoV-2 mediated respiratory distress syndrome, we review in this article the available knowledge on the strategic placement of EGCG in curbing inflammatory signals and how it may serve as a broad spectrum therapeutic in asymptomatic and symptomatic COVID-19 patients.

Emerging outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is a major threat to public health. The morbidity is increasing due to lack of SARS-CoV-2 specific drugs. Herein, we have identified potential drugs that target the 3-chymotrypsin like protease (3CLpro), the main protease that is pivotal for the replication of SARS-CoV-2. Computational molecular modeling was used to screen 3987 FDA approved drugs, and 47 drugs were selected to study their inhibitory effects on SARS-CoV-2 specific 3CLpro enzyme in vitro. Our results indicate that boceprevir, ombitasvir, paritaprevir, tipranavir, ivermectin, and micafungin exhibited inhibitory effect towards 3CLpro enzymatic activity. The 100 ns molecular dynamics simulation studies showed that ivermectin may require homodimeric form of 3CLpro enzyme for its inhibitory activity. In summary, these molecules could be useful to develop highly specific therapeutically viable drugs to inhibit the SARS-CoV-2 replication either alone or in combination with drugs specific for other SARS-CoV-2 viral targets.Here, the authors identify potential drugs that target 3-chymotrypsin like protease (3CLpro), which is a pivotal protease for the replication of SARS-CoV-2. They found that off-target inhibitors such as ivermectin and micafungin inhibit 3CLpro enzyme activity, suggesting that these molecules could constitute useful therapies to inhibit SARS-CoV-2 replication.

Chikungunya is a mosquito-borne viral disease that causes fever and severe joint pain for which there is no direct acting drug treatments. Vinyl sulfone SGC-NSP2PRO-1 ( 3 ) was identified as a potent inhibitor of the nsP2 cysteine protease (nsP2pro) that reduced viral titer against infectious isolates of Chikungunya and other alphaviruses. The covalent warhead in 3 captured the active site C478 and inactivated nsP2pro with a k inact / K i ratio of 5950 M –1  s –1 . The vinyl sulfone 3 was inactive across a panel of 23 other cysteine proteases and demonstrated remarkable proteome-wide selectivity by two chemoproteomic methods. A negative control analog SGC-NSP2PRO-1N ( 4 ) retained the isoxazole core and covalent warhead but demonstrated > 100-fold decrease in enzyme inhibition. Both 3 and 4 were stable across a wide range of pH in solution and upon prolonged storage as solids. Vinyl sulfone 3 and its negative control 4 will find utility as high-quality chemical probes to study the role of the nsP2pro in cellular studies of alphaviral replication and virulence.

A focused library of 1,2,4-thiadiazolidin-3,5-diones (THIA-1–10), previously characterized as hydrogen sulfide (H2S) donors, was evaluated for inhibitory activity against cysteine proteases. We included two key cysteine proteases aiming at antiviral drug development—SARS-CoV-2 3CLpro (Mpro) and PLpro—alongside reference enzymes Papain and Cathepsin L. The compounds exhibited distinct selectivity profiles and inhibition mechanisms. The ability to act as covalent inhibitors of 3CLpro in the nanomolar range is of particular interest, with compounds THIA-6, -7, and -10 proving to be the most potent inhibitors of the series, and compounds THIA-1, -2, and -8 proving to be the most selective with respect to the other proteases. We explored the molecular bases of the observed activity profile of THIA-1–10 through computational studies, which supported and complemented the experimental findings, paving the way for future structure optimization. The results highlight that inhibitory potency depends not only on electrophilicity but also on the ability to access the catalytic cysteine within the active site. The dual functionality of THIA-1–10 as H2S donors and selective cysteine protease inhibitors underscores its potential as a promising lead for therapeutic development.

Cruzipain (CZP), the major cysteine protease present in T. cruzi, the ethiological agent of Chagas disease, has attracted particular attention as a therapeutic target for the development of targeted covalent inhibitors (TCI). The vast chemical space associated with the enormous molecular diversity feasible to explore by means of modern synthetic approaches allows the design of CZP inhibitors capable of exhibiting not only an efficient enzyme inhibition but also an adequate translation to anti-T. cruzi activity. In this work, a computer-aided design strategy was developed to combinatorially construct and screen large libraries of 1,4-disubstituted 1,2,3-triazole analogues, further identifying a selected set of candidates for advancement towards synthetic and biological activity evaluation stages. In this way, a virtual molecular library comprising more than 75 thousand diverse and synthetically feasible analogues was studied by means of molecular docking and molecular dynamic simulations in the search of potential TCI of CZP, guiding the synthetic efforts towards a subset of 48 candidates. These were synthesized by applying a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) centered synthetic scheme, resulting in moderate to good yields and leading to the identification of 12 hits selectively inhibiting CZP activity with IC50 in the low micromolar range. Furthermore, four triazole derivatives showed good anti-T. cruzi inhibition when studied at 50 μM; and Ald-6 excelled for its high antitrypanocidal activity and low cytotoxicity, exhibiting complete in vitro biological activity translation from CZP to T. cruzi. Overall, not only Ald-6 merits further advancement to preclinical in vivo studies, but these findings also shed light on a valuable chemical space where molecular diversity might be explored in the search for efficient triazole-based antichagasic agents.

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the “lock and key” mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.

Cathepsin K plays a pivotal role in bone resorption and has emerged as a prominent therapeutic target for treating bone-related diseases such as osteoporosis. Despite significant advances in synthetic inhibitor development, none have achieved FDA approval due to safety and efficacy challenges. This review highlights the potential of phytochemicals as alternative inhibitors, emphasizing their natural origin, structural diversity, and minimal adverse effects. Key phytochemicals, including AC-5-1, Cycloaltilisin 6, Cycloaltilisin 7, Nicolaioidesin C, and Panduratin A, were examined for their inhibitory activities against cathepsin K. While these compounds exhibit varying IC50 values, their docking studies revealed significant interactions within Cathepsin K’s active site, particularly involving critical residues such as Cys25 and His162. However, challenges such as lower potency compared to synthetic inhibitors and limited in vivo studies underscore the need for structural optimization and comprehensive preclinical evaluations. This review discusses biological insights, current limitations, and future strategies for advancing phytochemical-based inhibitors toward clinical applications in managing Cathepsin K-associated diseases.

We developed a rational approach to design peptide-based covalent inhibitors and coupled the inhibitors with antibodies for cell-specific delivery. We used this platform to generate antibody–peptide inhibitor conjugates (APICs) that target a family of proteases, the cysteine cathepsins. Our drug design and targeted delivery approach ensure specific inhibition and achieve therapeutic efficacy in different cancer cells and osteoclasts.

The discovery of effective cysteine protease inhibitors with crude protein kiwi extracts (CPKEs) has created novel challenges and prospects for pharmaceutical development. Despite extensive research on CPKEs, limited research has been conducted on treating atopic dermatitis (AD). Therefore, the objective of this work was to investigate the anti-inflammatory effects of CPKEs on TNF-α activation in a HaCaT cell model and in a DNCB (1-chloro-2, 4-dinitrochlorobenzene)-induced atopic dermatitis animal model. The molecular weight of the CPKE was determined using SDS-PAGE under non-reducing (17 kDa and 22 kDa) and reducing conditions (25 kDa, 22 kDa, and 15 kDa), whereas gelatin zymography was performed to examine the CPKE’s inhibitory impact on cysteine protease (actinidin and papain) activity. Moreover, the CPKE remains stable at 60 °C, with pH levels varying from 4 to 11, as determined by the azocasein assay. CPKE treatment decreased the phosphorylation of mitogen-activated protein kinase (MAPK) and Akt, along with the activation of nuclear factor-kappa B (NF-κB)-p65 in tumor necrosis factor-α (TNF-α)-stimulated HaCaT cells. Five-week-old BALB/c mice were treated with DNCB to act as an AD-like animal model. The topical application of CPKE to DNCB-treated mice for three weeks substantially decreased clinical dermatitis severity and epidermal thickness and reduced eosinophil infiltration and mast cells into ear and skin tissues. These findings imply that CPKE derived from kiwifruit might be a promising therapy option for inflammatory skin diseases such as AD.