Nitidine chloride induces cardiac hypertrophy in mice by targeting autophagy-related 4B cysteine peptidase.

Nitidine chloride (NC) is a standard active component from the traditional Chinese medicine Zanthoxylum nitidum (Roxb.) DC. (ZN). NC has shown a variety of pharmacological activities including anti-tumor activity. As a number of anti-tumor drugs cause cardiotoxicity, herein we investigated whether NC exerted a cardiotoxic effect and the underlying mechanism. Aqueous extract of ZN (ZNE) was intraperitoneally injected into rats, while NC was injected into beagles and mice once daily for 4 weeks. Cardiac function was assessed using echocardiography. We showed that both ZNE administered in rats and NC administered in mice induced dose-dependent cardiac hypertrophy and dysfunction, whereas administration of NC at the middle and high dose caused death in Beagles. Consistently, we observed a reduction of cardiac autophagy levels in NC-treated mice and neonatal mouse cardiomyocytes. Furthermore, we demonstrated that autophagy-related 4B cysteine peptidase (ATG4B) may be a potential target of NC, since overexpression of ATG4B reversed the cardiac hypertrophy and reduced autophagy levels observed in NC-treated mice. We conclude that NC induces cardiac hypertrophy via ATG4B-mediated downregulation of autophagy in mice. Thus, this study provides guidance for the safe clinical application of ZN and the use of NC as an anti-tumor drug.

Sinigrin in combination with artesunate provides protection against lethal murine malaria via falcipain-3 inhibition and immune modulation.

Plant-derived antimalarials are indispensable for malaria treatment and a platform for new drugs. The present study explores sinigrin, for malaria using in vitro, in silico and in vivo strategies and the immune response generated after administration. The compound exhibited promising activity against chloroquine (CQ)-resistant (RKL-9) IC50 5.14 µg/mL and CQ-sensitive (3D7) IC50 5.47 µg/mL strains of P. falciparum and was safe in both in vitro (CC50 > 640 µg/mL) and in vivo (LD50 > 2 g/kg) toxicity studies. In addition, virtual screening showed hydrogen bonding, hydrophobic and van der Waals interactions with amino acid residues of 3BPM (falcipain-3). In vivo studies revealed promising antimalarial activity of sinigrin (200 mg/kg) with 87.44% chemo-suppression on day 5 and significantly (p < 0.0001) enhanced the mean survival time (21 ± 4.74 days) in contrast to the infected control (5.4 ± 1.14 days). In combination therapy, sinigrin (100 mg/kg and 200 mg/kg) augmented the efficacy of artesunate (AS 50 mg/kg) with 100% survival and no recrudescence. These observations are further corresponded and supported by DLC, NO production, cytokine analysis, biochemical and histopathological studies. Treatment with the combination resulted in a regulated interplay of immune cells and cytokines aiding in parasite clearance in addition to its specific inhibitory activity. We report the antimalarial activity of sinigrin first time with best D-score against falcipain-3. These findings highlight sinigrin as a HIT molecule, which may potentially be used in drug and vaccine development approaches.

Targeting both BDNF/TrkB pathway and delta-secretase for treating Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia, and no disease-modifying therapeutic agents are currently available. BDNF/TrkB signaling is impaired in AD and is associated with prominent delta-secretase (δ-secretase, also known as asparaginyl endopeptidase or legumain) activation, which simultaneously cleaves both APP and Tau and promotes Aß production and neurofibrillary tangles (NFT) pathologies. Here we show that the optimized δ-secretase inhibitor (#11a) or TrkB receptor agonist (CF3CN) robustly blocks δ-secretase activity separately, and their combination synergistically blunts δ-secretase, exhibiting promising therapeutic efficacy in 3xTg AD mouse model. The optimal δ-secretase inhibitor reveals demonstrable brain exposure and oral bioavailability, suppressing APP N585 and Tau N368 cleavage by δ-secretase. Strikingly, CF3CN treatment evidently escalates BDNF levels. Both #11a and CF3CN display strong in vivo PK/PD properties and ability to suppress δ-secretase activity in the brain. Orally administrated CF3CN strongly activates TrkB that triggers active Akt to phosphorylate δ-secretase T322, preventing its proteolytic activation and mitigating AD pathologies. #11a or CF3CN significantly diminishes AD pathogenesis and improves cognitive functions with the combination exhibiting the maximal effect. Thus, our data support that these derivatives are strong pharmaceutical candidates for the treatment of AD.

Pharmacological inhibition of asparaginyl endopeptidase by δ-secretase inhibitor 11 mitigates Alzheimer's disease-related pathologies in a senescence-accelerated mouse model.

BACKGROUND: Currently, there is no cure for Alzheimer's disease (AD). Therapeutics that can modify the early stage of AD are urgently needed. Recent studies have shown that the pathogenesis of AD is closely regulated by an endo/lysosomal asparaginyl endopeptidase (AEP). Inhibition of AEP has been reported to prevent neural degeneration in transgenic mouse models of AD. However, more than 90% of AD cases are age-related sporadic AD rather than hereditary AD. The therapeutic efficacy of AEP inhibition in ageing-associated sporadic AD remains unknown. METHODS: The senescence-accelerated mouse prone 8 (SAMP8) was chosen as an approximate model of sporadic AD and treated with a selective AEP inhibitor,: δ-secretase inhibitor 11. Activation of AEP was determined by enzymatic activity assay. Concentration of soluble amyloid ß (Aß) in the brain was determined by ELISA. Morris water maze test was performed to assess the learning and memory-related cognitive ability. Pathological changes in the brain were explored by morphological and western blot analyses. RESULTS: The enzymatic activity of AEP in the SAMP8 mouse brain was significantly higher than that in the age-matched SAMR1 mice. The half maximal inhibitory concentration (IC50) for δ-secretase inhibitor 11 to inhibit AEP in vitro is was around 150 nM. Chronic treatment with δ-secretase inhibitor 11 markedly decreased the brain AEP activity, reduced the generation of Aß1-40/42 and ameliorated memory loss. The inhibition of AEP with this reagent not only reduced the AEP-cleaved tau fragments and tau hyperphosphorylation, but also attenuated neuroinflammation in the form of microglial activation. Moreover, treatment with δ-secretase inhibitor 11 prevented the synaptic loss and alleviated dendritic disruption in SAMP8 mouse brain. CONCLUSIONS: Pharmacological inhibition of AEP can intervene and prevent AD-like pathological progress in the model of sporadic AD. The up-regulated AEP in the brain could be a promising target for early treatment of AD. The δ-secretase inhibitor 11 can be used as a lead compound for translational development of AD treatment.

Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain.

Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.

Mass spectrometry reveals potential of ß-lactams as SARS-CoV-2 Mpro inhibitors.

The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some ß-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.

The SUMO pathway in pancreatic cancer: insights and inhibition.

An urgent medical need to develop novel treatment strategies for patients with pancreatic ductal adenocarcinoma (PDAC) exists. However, despite various efforts in the histopathological and molecular subtyping of PDAC, novel targeted or specific therapies have not been established. Posttranslational modifications (PTMs) with ubiquitin-like proteins, including small ubiquitin-like modifiers (SUMOs), mediate numerous processes that can contribute to the fitness and survival of cancer cells. The contribution of SUMOylation to transcriptional control, DNA repair pathways, mitotic progression, and oncogenic signalling has been described. Here we review functions of the SUMO pathway in PDAC, with a special focus on its connection to an aggressive subtype of the disease characterised by high MYC activity, and discuss SUMOylation inhibitors under development for precise PDAC therapies.

Esomeprazole inhibits the lysosomal cysteine protease legumain to prevent cancer metastasis.

Legumain is a newly discovered lysosomal cysteine protease that can cleave asparagine bonds and plays crucial roles in regulating immunity and cancer metastasis. Legumain has been shown to be highly expressed in various solid tumors, within the tumor microenvironment and its levels are directly related to tumor metastasis and poor prognosis. Therefore, legumain presents as a potential cancer therapeutic drug target. In this study, we have identified esomeprazole and omeprazole as novel legumain small molecule inhibitors by screening an FDA approved-drug library. These compounds inhibited enzyme activity of both recombinant and endogenous legumain proteins with esomeprazole displaying the highest inhibitory effect. Further molecular docking analysis also indicated that esomeprazole, the S- form of omeprazole had the most stable binding to legumain protein compared to R-omeprazole. Transwell assay data showed that esomeprazole and omeprazole reduced MDA-MB-231 breast cancer cell invasion without effecting cell viability. Moreover, an in vivo orthotopic transplantation nude mouse model study showed that esomeprazole reduced lung metastasis of MDA-MB-231 breast cancer cells. These results indicated that esomeprazole has the exciting potential to be used in anti-cancer therapy by preventing cancer metastasis via the inhibition of legumain enzyme activity. Graphical abstract.

Combining fragment docking with graph theory to improve ligand docking for homology model structures.

Computational protein-ligand docking is well-known to be prone to inaccuracies in input receptor structures, and it is challenging to obtain good docking results with computationally predicted receptor structures (e.g. through homology modeling). Here we introduce a fragment-based docking method and test if it reduces requirements on the accuracy of an input receptor structures relative to non-fragment docking approaches. In this method, small rigid fragments are docked first using AutoDock Vina to generate a large number of favorably docked poses spanning the receptor binding pocket. Then a graph theory maximum clique algorithm is applied to find combined sets of docked poses of different fragment types onto which the complete ligand can be properly aligned. On the basis of these alignments, possible binding poses of complete ligand are determined. This docking method is first tested for bound docking on a series of Cytochrome P450 (CYP450) enzyme-substrate complexes, in which experimentally determined receptor structures are used. For all complexes tested, ligand poses of less than 1 Å root mean square deviations (RMSD) from the actual binding positions can be recovered. Then the method is tested for unbound docking with modeled receptor structures for a number of protein-ligand complexes from different families including the very recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease. For all complexes, poses with RMSD less than 3 Å from actual binding positions can be recovered. Our results suggest that for docking with approximately modeled receptor structures, fragment-based methods can be more effective than common complete ligand docking approaches.

In silico Potential of Approved Antimalarial Drugs for Repurposing Against COVID-19.

Although the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is wreaking havoc and resulting in mortality and morbidity across the planet, novel treatments are urgently needed. Drug repurposing offers an innovative approach in this context. We report here new findings on the in silico potential of several antimalarial drugs for repurposing against COVID-19. We conducted analyses by docking the compounds against two SARS-CoV-2-specific targets: (1) the receptor binding domain spike protein and (2) the main protease of the virus (MPro) using the Schrödinger software. Importantly, the docking analysis revealed that doxycycline (DOX) showed the most effective binding to the spike protein of SARS-CoV-2, whereas halofantrine and mefloquine bound effectively with the main protease among the antimalarial drugs evaluated in the present study. The in silico approach reported here suggested that DOX could potentially be a good candidate for repurposing for COVID-19. In contrast, to decipher the actual potential of DOX and halofantrine against COVID-19, further in vitro and in vivo studies are called for. Drug repurposing warrants consideration as a viable research and innovation avenue as planetary health efforts to fight the COVID-19 continue.

Topological analysis of SARS CoV-2 main protease.

There is an urgent necessity of effective medication against severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), which is producing the COVID-19 pandemic across the world. Its main protease (Mpro) represents an attractive pharmacological target due to its involvement in essential viral functions. The crystal structure of free Mpro shows a large structural resemblance with the main protease of SARS CoV (nowadays known as SARS CoV-1). Here, we report that average SARS CoV-2 Mpro is 1900% more sensitive than SARS CoV-1 Mpro in transmitting tiny structural changes across the whole protein through long-range interactions. The largest sensitivity of Mpro to structural perturbations is located exactly around the catalytic site Cys-145 and coincides with the binding site of strong inhibitors. These findings, based on a simplified representation of the protein as a residue network, may help in designing potent inhibitors of SARS CoV-2 Mpro.

Potential covalent drugs targeting the main protease of the SARS-CoV-2 coronavirus.

MOTIVATION: Since December 2019, the newly identified coronavirus SARS-CoV-2 has caused a massive health crisis worldwide and resulted in over 70 000 COVID-19 infections so far. Clinical drugs targeting SARS-CoV-2 are urgently needed to decrease the high fatality rate of confirmed COVID-19 patients. Traditional de novo drug discovery needs more than 10 years, so drug repurposing seems the best option currently to find potential drugs for treating COVID-19. RESULTS: Compared with traditional non-covalent drugs, covalent drugs have attracted escalating attention recent years due to their advantages in potential specificity upon careful design, efficiency and patient burden. We recently developed a computational protocol named as SCAR (steric-clashes alleviating receptors) for discovering covalent drugs. In this work, we used the SCAR protocol to identify possible covalent drugs (approved or clinically tested) targeting the main protease (3CLpro) of SARS-CoV-2. We identified 11 potential hits, among which at least six hits were exclusively enriched by the SCAR protocol. Since the preclinical or clinical information of these identified drugs is already available, they might be ready for being clinically tested in the treatment of COVID-19. CONTACT: senliu.ctgu@gmail.com.

δ-secretase in neurodegenerative diseases: mechanisms, regulators and therapeutic opportunities.

Mammalian asparagine endopeptidase (AEP) is a cysteine protease that cleaves its protein substrates on the C-terminal side of asparagine residues. Converging lines of evidence indicate that AEP may be involved in the pathogenesis of several neurological diseases, including Alzheimer's disease, Parkinson's disease, and frontotemporal dementia. AEP is activated in the aging brain, cleaves amyloid precursor protein (APP) and promotes the production of amyloid-ß (Aß). We renamed AEP to δ-secretase to emphasize its role in APP fragmentation and Aß production. AEP also cleaves other substrates, such as tau, α-synuclein, SET, and TAR DNA-binding protein 43, generating neurotoxic fragments and disturbing their physiological functions. The activity of δ-secretase is tightly regulated at both the transcriptional and posttranslational levels. Here, we review the recent advances in the role of δ-secretase in neurodegenerative diseases, with a focus on its biochemical properties and the transcriptional and posttranslational regulation of its activity, and discuss the clinical implications of δ-secretase as a diagnostic biomarker and therapeutic target for neurodegenerative diseases.

In vitro and in silico approaches of antibiofilm activity of 1-hydroxy-1-norresistomycin against human clinical pathogens.

In the present study, an attempt has been made to explore the antibiofilm activity of bioactive compound 1-hydroxy-1-norresistomycin (HNM) derived from coral mucus associated actinomycete Streptomyces variabilis. Initially, different concentration of HNM inhibited the biofilm formation of human clinical pathogens Escherichia coli, Vibrio cholerae and Staphylococcus aureus was determined using crystal-violet staining assay. The light microscopic analysis showed that HNM reduced the biofilm formation and adherence of bacterial cells on the surface of coverslip. HNM also damages the 3D architecture with reduced thickness as well as cell aggregation of biofilm forming bacteria analysed by confocal laser scanning microscopy (CLSM). In addition, HNM also demonstrated the efficiency in inhibiting theoretical adhesion by altering the surface hydrophobicity that can potentially hamper cellular adhesion and prevent biofilm formation. Furthermore, the molecular docking showed the significant interaction between HNM and key biofilm forming proteins proved an excellent antibiofilm activity of HNM. Together, these results suggest that the HNM can serve as potential antibiofilm agent in controlling the infections of E. coli, V. cholerae and S. aureus.

Virtual design of novel Plasmodium falciparum cysteine protease falcipain-2 hybrid lactone-chalcone and isatin-chalcone inhibitors probing the S2 active site pocket.

We report computer-aided design of new lactone-chalcone and isatin-chalcone (HLCIC) inhibitors of the falcipain-2 (PfFP-2). 3D models of 15 FP-2:HLCIC1-15 complexes with known observed activity (IC50exp) were prepared to establish a quantitative structure-activity (QSAR) model and linear correlation between relative Gibbs free energy of enzyme:inhibitor complex formation (ΔΔGcom) and IC50exp: pIC50exp = -0.0236 × ΔΔGcom+5.082(#); R2 = 0.93. A 3D pharmacophore model (PH4) derived from the QSAR directed our effort to design novel HLCIC analogues. During the design, an initial virtual library of 2621440 HLCIC was focused down to 18288 drug-like compounds and finally, PH4 screened to identify 81 promising compounds. Thirty-three others were added from an intuitive substitution approach intended to fill better the enzyme S2 pocket. One hundred and fourteen theoretical IC50 (IC50pre) values were predicted by means of (#) and their pharmacokinetics (ADME) profiles. More than 30 putative HLCICs display IC50pre 100 times superior to that of the published most active training set inhibitor HLCIC1.

Drug combination studies of curcumin and genistein against rhodesain of Trypanosoma brucei rhodesiense.

Curcumin and genistein are two natural products obtained from Curcuma longa L. and soybeans, endowed with many biological properties. Within the last years they were shown to possess also a promising antitrypanosomal activity. In the present paper, we investigated the activity of both curcumin and genistein against rhodesain, the main cysteine protease of Trypanosoma brucei rhodesiense; drug combination studies, according to Chou and Talalay method, allowed us to demonstrate a potent synergistic effect for the combination curcumin-genistein. As a matter of fact, with our experiments we observed that the combination index of curcumin-genistein is < 1 for the reduction from 10 to 90% of rhodesain activity.

Allosteric Site Inhibitor Disrupting Auto-Processing of Malarial Cysteine Proteases.

Falcipains are major haemoglobinases of Plasmodium falciparum required for parasite growth and development. They consist of pro- and mature domains that interact via 'hot-spot' interactions and maintain the structural integrity of enzyme in zymogen state. Upon sensing the acidic environment, these interactions dissociate and active enzyme is released. For inhibiting falcipains, several active site inhibitors exist, however, compounds that target via allosteric mechanism remains uncharacterized. Therefore, we designed and synthesized six azapeptide compounds, among which, NA-01 & NA-03 arrested parasite growth by specifically blocking the auto-processing of falcipains. Inhibitors showed high affinity for enzymes in presence of the prodomain without affecting the secondary structure. Binding of NA-03 at the interface induced rigidity in the prodomain preventing structural reorganization. We further reported a histidine-dependent activation of falcipain. Collectively, for the first time we provide a framework for blocking the allosteric site of crucial haemoglobinases of the human malaria parasite. Targeting the allosteric site could provide high selectivity and less vulnerable to drug resistance.

Lysophosphatidic acid induces neuronal cell death via activation of asparagine endopeptidase in cerebral ischemia-reperfusion injury.

Lysophosphatidic acid (LPA), an extracellular signaling molecule, influences diverse biological events, including the pathophysiological process induced after ischemic brain injury. However, the molecular mechanisms mediating the pathological change after ischemic stroke remain elusive. Here we report that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is regulated by LPA during stroke. AEP proteolytically cleaves tau and generates tauN368 fragments, triggering neuronal death. Inhibiting the generation of LPA reduces the expression of AEP and tauN368, and alleviates neuronal cell death. Together, this evidence indicates that the LPA-AEP pathway plays a key role in the pathophysiological process induced after ischemic stroke. Inhibition of LPA could be a useful therapeutic for treating neuronal injury after stroke.

Proteolytic effects of gingipains on trefoil factor family peptides.

OBJECTIVES: The present study was aimed to determine whether trefoil factor family (TFF) peptides which were generally considered to be resistant to proteolysis could be digested by gingipains, a major proteinases produced by Porphyromonas gingivalis. MATERIALS AND METHODS: Recombinant human TFF1, TFF2, and TFF3 peptides were used as substrates. Gingipains including arginine gingipain (RgpB) and lysine gingipain (Kgp) were used as enzymes. Trypsin was used as a control protease. Matrix-assisted laser desorption/ionization with time-of-flight / time-of-flight (MALDI-TOF/TOF) and liquid chromatography mass spectrometry (LC-MS) were used for analyzing peptide mass signals and amino acid sequences of digested TFF peptides. RESULTS: MALDI-TOF/TOF analyses demonstrated that Kgp, RgpB, and trypsin were able to cleave TFF1 and TFF2 peptides, resulting in different patterns of digested fragments. However, impurity in recombinant TFF3 peptide substrates affected the interpretations of enzymatic reaction by MALDI-TOF/TOF. LC-MS analyses demonstrated that identified fragments of TFF1, TFF2, and TFF3 from digestion by gingipains were similar to those by trypsin. CONCLUSIONS: Using MALDI-TOF/TOF and LC-MS, the present study provides new information that gingipains containing trypsin-like activities are able to digest TFF peptides. CLINICAL RELEVANCE: The proteolytic effects of gingipains on TFF peptides may be responsible for reduction of salivary TFF peptides in chronic periodontitis patients. Further investigations to determine the pathological effects of gingipains on TFF peptides in saliva and periodontal tissues of patients with chronic periodontitis would be of interest.

Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4.

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC50 values of 12nM and 42nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.