Search

Dabigatran etexilate (DABE) is a lipophilic double alkyl ester prodrug of dabigatran (DAB) which is a serine protease inhibitor used clinically as an anticoagulant. Recently, translocation of serine protease enzymes, including trypsin, from the gut into the mesenteric lymph and then blood has been associated with organ failure in acute and critical illnesses (ACIs). Delivery of DABE into mesenteric lymph may thus be an effective strategy to prevent organ failure in ACIs. Most drugs access the mesenteric lymph in low quantities following oral administration, as they are rapidly transported away from the intestine via the blood. Here, we examine the potential to deliver DABE into the mesenteric lymph by promoting association with lymph lipid transport pathways via co-administration with a lipid-based formulation (LBF). A series of self-emulsifying LBFs were designed and tested in vitro for their potential to form stable DABE loaded emulsions and keep DABE solubilised and stable over time in simulated gastrointestinal conditions. The LBFs were found to form fine emulsions with a droplet size of 214 ± 30 nm and DABE was stable in the formulation. The stability of DABE in vitro in simulated intestinal conditions, plasma and lymph samples was also evaluated to ensure stability in collected samples and to evaluate whether the prodrug is likely to release active DAB. Ultimately, a highly uniform and stable self-emulsifying Type III A LBF of DABE was chosen for progression into in vivo studies in male Sprague Dawley rats to confirm the lymphatic uptake and plasma pharmacokinetics. Both in vitro and in vivo in plasma and lymph, DABE was rapidly converted to an intermediate and DAB. The main species present in vivo in both plasma and lymph was DAB and mass transport of DABE and DAB in lymph was minimal (∼0.5 % of dose). Importantly, the concentration of DABE in lymph was substantially (20–176 fold) higher than in plasma, supporting that if the prodrug were stable and did not convert to DAB in the intestine, it would be lymphatically transported. Future studies will therefore focus on optimizing the design of the prodrug and formulation to improve stability during absorption and further promote lymphatic uptake. [Display omitted] • Dabigatran etexilate (DABE) was formulated into a novel stable lipid-based formulation to enhance its lymphatic uptake. • The lipid-based formulation provided protection against degradation of DABE in simulated intestinal digestion conditions. • DABE showed some lymphatic uptake following intestinal administration in the lipid-based formulation but this appeared limited by intestinal instability of DABE. [ABSTRACT FROM AUTHOR]

• Confirming that ensitrelvir did not influence coproporphyrin-I (CP-I) concentrations, indicating the absence of OATP1B inhibitory effects. • Demonstrating the utility of a normalization method using pre-dose values to minimize individual variabilities in CP-I concentrations when comparing the CP-I concentrations with different participants. • Proposal of a normalization method for assessing OATP1B inhibitory potential using CP-I in a placebo-controlled first-in-human study. Coproporphyrin-I (CP-I) has been investigated as an endogenous biomarker of organic anion transporting polypeptide (OATP) 1B. Here, we determined the CP-I concentrations in a cocktail drug-drug interaction (DDI) study of ensitrelvir to evaluate the OATP1B inhibitory potential because ensitrelvir had increased plasma concentrations of rosuvastatin in this study, raising concerns about breast cancer resistance protein and OATP1B inhibition. Furthermore, CP-I concentrations were compared between active and placebo groups in a first-in-human (FIH) study of ensitrelvir to verify whether the OATP1B inhibitory potential could be estimated at an early drug development stage. In the cocktail DDI study, CP-I did not differ between with/without administration of ensitrelvir, indicating that ensitrelvir has no OATP1B inhibitory effect. Although there were some individual variabilities in CP-I concentrations among the treatment groups in the FIH study, the normalization of CP-I concentrations with pre-dose values minimized these variabilities, suggesting that this normalized method would be helpful for comparing the CP-I from different participants. Finally, we concluded that CP-I concentrations were not affected by ensitrelvir in the FIH study. These results suggested that the CP-I determination in an FIH study and its normalized method can be useful for an early evaluation of the OATP1B-mediated DDI potential in humans. [Display omitted] [ABSTRACT FROM AUTHOR]

• PCSK9 inhibition dramatically reduces both LDL-C and sdLDL-C to a similar degree. • Plasma 7-ketocholesterol levels were not significantly modulated by PCSK9 inhibition. • Changes in 7-ketocholesterol and VLDL-C after PCKS9 inhibition significantly correlated. • 7-Ketocholesterol may be carried by VLDL and lost during LDL formation. Oxidized forms of cholesterol (oxysterols) are implicated in atherogenesis and can accumulate in the body via direct absorption from food or through oxidative reactions of endogenous cholesterol, inducing the formation of LDL particles loaded with oxidized cholesterol. It remains unknown whether drastic reductions in LDL-cholesterol (LDL-C) are associated with changes in circulating oxysterols and whether small dense LDL (sdLDL) are more likely to carry these oxysterols and susceptible to the effects of PCSK9 inhibition (PCSK9i). We investigate the effect of LDL-C reduction accomplished via PCSK9i on changes in plasma levels of sdLDL-cholesterol (sdLDL-C) and a common, stable oxysterol, 7-ketocholesterol (7-KC), among 134 patients referred to our Preventive Cardiology clinic. Plasma lipid panel, sdLDL-C, and 7-KC measurements were obtained from patients before and after initiation of PCSK9i. The intervention caused a significant lowering of LDL-C (-55.4 %). The changes in sdLDL-C levels (mean reduction 51.4 %) were highly correlated with the reductions in LDL-C levels (R = 0.829, p < 0.001). Interestingly, whereas changes in plasma free 7-KC levels with PCSK9i treatment were much smaller than (-6.6 %) and did not parallel those of LDL-C and sdLDL-C levels, they did significantly correlate with changes in triglycerides and very low-density lipoprotein-cholesterol (VLDL-C) levels (R = 0.219, p = 0.025). Our findings suggest a non-preferential clearance of LDL subparticles as a consequence of LDL receptor upregulation caused by PCSK9 inhibition. Moreover, the lack of significant reduction in 7-KC with PCSK9i suggests that 7-KC may be in part carried by VLDL and lost during lipoprotein processing leading to LDL formation. [ABSTRACT FROM AUTHOR]

Arboviruses are a global concern for a multitude of reasons, including their increased incidence and human mortality. Vectors associated with arboviruses include the mosquito Aedes sp., which is responsible for transmitting the Zika virus. Flaviviruses, like the Zika virus, present only one chymotrypsin-like serine protease (NS3) in their genome. Together with host enzymes, the NS2B co-factor NS3 protease complex are essential for the viral replication cycle by virus polyprotein processing. To search for Zika virus NS2B-NS3 protease (ZIKV Pro) inhibitors, a phage display library was constructed using the Boophilin domain 1 (BoophD1), a thrombin inhibitor from the Kunitz family. A BoophilinD1 library mutated at positions P1–P4′ was constructed, presenting a titer of 2.9x106 (cfu), and screened utilizing purified ZIKV Pro. The results demonstrated at the P1–P4' positions the occurrence of 47% RALHA sequence (mut 12) and 11.8% RASWA sequence (mut14), SMRPT, or KALIP (wt) sequence. BoophD1-wt and mutants 12 and 14 were expressed and purified. The purified BoophD1 wt, mut 12 and 14, presented Ki values for ZIKV Pro of 0.103, 0.116, and 0.101 μM, respectively. The BoophD1 mutant inhibitors inhibit the Dengue virus 2 protease (DENV2) with Ki values of 0.298, 0.271, and 0.379 μM, respectively. In conclusion, BoophD1 mut 12 and 14 selected for ZIKV Pro demonstrated inhibitory activity like BoophD1-wt, suggesting that these are the strongest Zika inhibitors present in the BoophD1 mutated phage display library. Furthermore, BoophD1 mutants selected for ZIKV Pro inhibit both Zika and Dengue 2 proteases making them potential pan-flavivirus inhibitors. [Display omitted] • Using phage display to select inhibitor for Zika protease. • Boophilin mut 12 and mut 14 potential candidates as pan-flavivirus inhibitors. • Phage display and Kunitz domain as a tool to select inhibitors for virus proteases. [ABSTRACT FROM AUTHOR]

• Inclisiran lowers LDL-C by -38 % at 3 months in real-world setting. • Patients who use statin co-medication had a greater LDL-C reduction of -45 %. • Most patients switching from PCSK9 mAbs to inclisiran showed an increase in LDL-C. • Inclisiran has a favourable safety profile in real-world setting at 3 and 9 months follow-up. Inclisiran is the first-in-class small interfering RNA (siRNA) proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitor. In clinical trials inclisiran showed effective and sustained low-density lipoprotein cholesterol (LDL-C) reduction of ± 50 %. As data in clinical setting are scarce, we aim to investigate the efficacy and safety in clinical practice. We describe a registry of consecutive patients who started with inclisiran at a lipid clinic of a university hospital. Patients were eligible if they fulfilled the reimbursement criteria in the Netherlands. Patients were included if they started with inclisiran as first line (group 1) or switched from PCSK9 monoclonal antibody (mAbs) to inclisiran (group 2). LDL-C levels were measured at 3 and 9 months after initiation of inclisiran. Median change of LDL-C levels was calculated on an individual and group level. We analysed 65 patients (36 women), median [25th percentile; 75th percentile] age of 63 [54; 68] years. Of these, 44 patients had both a 3 month and 9 month visit. At 3 months, patients who newly started inclisiran (group 1, n = 45) showed a LDL-C decrease of 38 [-49;-33] %. Patients who used statins as co-medication (n = 15) had a higher median LDL-C decrease compared to those without statin use (n=30; 45 % vs 38 %). However, patients who switched from mAbs to inclisiran (group 2, n = 20) had an increase in LDL-C of 38 [+4; +97] %. Adverse effects associated with inclisiran were mild and consisted of mild injection site reactions. Efficacy was slightly less whereas safety results were similar at 9 months. Our initial experience of inclisiran in a clinical setting showed less reduction in LDL-C levels compared to clinical trials but a similar safety profile. Moreover, patients who switched from PCSK9 mAbs to inclisiran generally showed an increase in LDL-C levels implying that inclisiran is less potent in LDL-C reduction compared to PCSK9 mAbs. [Display omitted] [ABSTRACT FROM AUTHOR]

Matriptase-2 (MT2), encoded by TMPRSS6, is a membraneanchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/-mice revealed that increases in dietary iron to ~0.5% were sufficient to overcome the high hepcidin barrier and to correct iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/-mice was able to further upregulate hepcidin expression to a similar magnitude as in wildtype mice. These results suggest that a lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) infection is associated with severe neurological disorders and congenital malformation. Despite efforts to eradicate the disease, there is still neither vaccine nor approved drugs to treat ZIKV infection. The NS2B-NS3 protease is a validated drug target since it is essential to polyprotein virus maturation. In the present study, we describe an experimental screening of 2,320 compounds from the chemical library of the Muséum National d'Histoire Naturelle of Paris on ZIKV NS2B-NS3 protease. A total of 96 hits were identified with 90% or more of inhibitory activity at 10 μM. Amongst the most active compounds, five were analyzed for their inhibitory mechanisms by kinetics assays and computational approaches such as molecular docking. 2-(3-methoxyphenoxy) benzoic acid (compound 945) show characteristics of a competitive inhibition (K i = 0.49 μM) that was corroborated by its molecular docking at the active site of the NS2B-NS3 protease. Taxifolin (compound 2292) behaves as an allosteric inhibitor whereas 3,8,9-trihydroxy-2-methyl-1H-phenalen-1-one (compound 128), harmol (compound 368) and anthrapurpurin (compound 1499) show uncompetitive inhibitions. These new NS2B-NS3 protease inhibitors are valuable hits to further hit-to-lead optimization. • Experimental screening of 2,320 compounds on ZIKV NS2B-NS3 protease. • Five compounds showed K i values ranging from 0.49 to 10.72 μM. • From the 5 best hits, 1 is competitive, 3 are uncompetitives and 1 is noncompetitive. • Docking approach corroborates experimental inhibition mechanisms of compounds. • This study reveals novel inhibitors of ZIKV NS2B-NS3 with diverse inhibition mechanisms. [ABSTRACT FROM AUTHOR]

Borrelial pathogens are vector-borne etiological agents known to cause Lyme disease, relapsing fever, and Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind components of the human complement system to evade host immunity. One borrelial lipoprotein, BBK32, protects the Lyme disease spirochete from complement-mediated attack via an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical complement pathway, C1r. In addition, the B. miyamotoi BBK32 orthologs FbpA and FbpB also inhibit C1r, albeit via distinct recognition mechanisms. The C1rinhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever–causing spirochetes, remains unknown. Here, we report the crystal structure of the C-terminal domain of Borrelia hermsii FbpC to a limiting resolution of 1.5 Å. We used surface plasmon resonance and assays of complement function to demonstrate that FbpC retains potent BBK32-like anticomplement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. Taken together, these results advance our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveal a surprising plasticity in the structures of borrelial C1r inhibitors. [ABSTRACT FROM AUTHOR]

α-synuclein (aS) is an abundant, neuronal protein that assembles into fibrillar pathological inclusions in a spectrum of neurodegenerative diseases that include Lewy body diseases (LBD) and Multiple System Atrophy (MSA). The cellular and regional distributions of pathological inclusions vary widely between different synucleinopathies contributing to the spectrum of clinical presentations. Extensive cleavage within the carboxy (C)-terminal region of aS is associated with inclusion formation, although the events leading to these modifications and the implications for pathobiology are of ongoing study. aS preformed fibrils can induce prion-like spread of aS pathology in both in vitro and animal models of disease. Using C truncation-specific antibodies, we demonstrated here that prion-like cellular uptake and processing of aS preformed fibrils resulted in two major cleavages at residues 103 and 114. A third cleavage product (122 aS) accumulated upon application of lysosomal protease inhibitors. In vitro, both 1-103 and 1-114 aS polymerized rapidly and extensively in isolation and in the presence of full-length aS. 1-103 aS also demonstrated more extensive aggregation when expressed in cultured cells. Furthermore, we used novel antibodies to aS cleaved at residue Glu114, to assess x-114 aS pathology in postmortem brain tissue from patients with LBD and MSA, as well as three different transgenic aS mouse models of prion-like induction. The distribution of x-114 aS pathology was distinct from that of overall aS pathology. These studies reveal the cellular formation and behavior of aS C-truncated at residues 114 and 103 as well as the disease dependent distribution of x-114 aS pathology. [ABSTRACT FROM AUTHOR]

[Display omitted] • Schistosoma mansoni eggs are enriched in expression of metalloproteases. • Aminopeptidase N1 is the most expressed protease in immature eggs. • Schistosoma mansoni eggs express a variety of leishmanolysin-like peptidases (M8 family) • Alpha-2-macroglobulin is a highly expressed protease inhibitor in schistosome eggs. • Fasciola hepatica eggs express antistasin, an anticoagulant known from leeches. Schistosoma mansoni eggs are the main causative agents of the pathological manifestations of schistosomiasis. The eggs are laid in the host bloodstream, then they migrate through the intestinal wall into the lumen. However, a significant proportion of the eggs become lodged in the liver, where they cause inflammation and fibrosis. In this study, we focus on a specific group of proteins expressed by the egg, namely proteases and their inhibitors. These molecules are often involved in schistosome-host interactions, but are still unexplored in the egg stage. Using RNA-seq and comparative transcriptomics of immature and mature S. mansoni eggs, we mapped the portfolio of proteases and their inhibitors, and determined their gene expression levels. In addition, we compared these data with gene expression of proteases and their inhibitors in Fasciola hepatica eggs. Fasciola hepatica eggs served as a useful comparative model, as they do not migrate through tissues and inflict pathology. We detected transcription of 135 and 117 proteases in S. mansoni and F. hepatica eggs, respectively, with 87 identified as orthologous between the two species. In contrast, we observed only four orthologous inhibitors out of 21 and 16 identified in S. mansoni and F. hepatica eggs, respectively. Among others, we measured high and developmentally regulated levels of expression of metalloproteases in S. mansoni eggs, specifically aminopeptidase N1, endothelin-converting enzyme 1, and several leishmanolysin-like peptidases. We identified highly transcribed protease inhibitors serpin and alpha-2-macroglobulin that are unique to S. mansoni eggs, and antistasin-like inhibitor in F. hepatica eggs. This study provides new insights into the portfolio of proteases and inhibitors expressed by S. mansoni with potential roles in egg tissue migration, stimulation of angiogenesis, and interaction with host blood and immunity. [ABSTRACT FROM AUTHOR]

• We examined LDL-C treatment in Israeli patients with premature ACS. • 34.5% of Arab Israeli patients achieved LDL-C<55mg/dl at 1 year. • 45.3% of Jewish Israeli patients achieved LDL-C<55mg/dl at 1 year. • The use of ezetimibe and PCSK9 inhibitors was trivial in both groups. • MACCE occurred more often among Arab patients. There are significant health gaps between Arabs and Jews in Israel. However, there are limited data on the management and treatment of dyslipidemia among Israeli adults who experience premature acute coronary syndrome (ACS). This study aimed to assess the differences between Arabs and Jews with regard to lipid-lowering therapy administration and low-density lipoprotein cholesterol (LDL-C) levels goal attainment at 1 year post-ACS. This study included patients aged ≤55 years who had been hospitalized for ACS at Meir Medical Center between 2018 and 2019. Outcomes included the rate of use of lipid-lowering medications, LDL-C levels 1 year post-admission, and major adverse cardiovascular and cerebrovascular events (MACCE) during 30 months of follow-up. The study population comprised 687 young adults with a median age of 48.5 years. 81.9% of the Arab patients and 79.8% of the Jewish patients were discharged on high intensity statins. At 1 year of follow-up, the proportions of Arab patients who had LDL-C levels <70 mg/dL and <55 mg/dL were lower than those of Jewish patients (43.8% vs. 58%, p < 0.001 and 34.5% vs. 45.3%, p < 0.001, respectively). At 1 year of follow-up, only 25% and 4% of both groups were treated with ezetimibe and proprotein convertase subtilisin/kexin type 9 inhibitor. The incidence of MACCE was significantly higher among Arab patients. Our study highlighted the need for a more aggressive lipid-lowering strategy in both Arab and Jewish populations. Culturally adapted interventions are required to reduce gaps between Arab and Jewish patients. [ABSTRACT FROM AUTHOR]

Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family. [ABSTRACT FROM AUTHOR]

• Upamostat at specified doses used was well tolerated, without significant side effects. • Entry criteria enhanced the hospitalization rate in the placebo group. • Dose-dependent increased international normalized ratio and partial thromboplastin time and decreased d-dimer levels were observed. • Upamostat speeded resolution and decreased the incidence of new severe symptoms. • No upamostat patients were hospitalized because of COVID-19. We performed a pilot study of upamostat, a serine protease inhibitor, in outpatients with symptomatic COVID-19 before a pivotal trial. SARS-CoV-2 patients with ≥2 moderate-severe symptoms onset within 5 days were randomized to oral upamostat 200 or 400 mg or placebo daily for 14 days. Patients completed COVID-19 symptom questionnaires daily for 28 days, then thrice weekly for 4 weeks, and underwent physical and laboratory examinations periodically. A total of 61 patients enrolled of which 20 received a placebo or upamostat 200 mg daily; 21 received upamostat 400 mg daily. Treatment was well tolerated; only one patient (upamostat 400) reported a drug-related adverse event, mild skin rash; no patient discontinued owing to a drug-related adverse event. The median time to a sustained recovery from severe symptoms was 8, 4, and 3 days for the three treatment groups, respectively. New severe symptoms developed in 20% of the placebo group vs 2.4% in the combined upamostat groups, (P = 0.036). Three placebo patients (15%) versus no upamostat patients were hospitalized for worsening COVID (P = 0.03). The mean d-dimer level remained constant in placebo patients but decreased by 38% and 48% in upamostat 200 and 400 patients, respectively. Upamostat was well tolerated, shortened recovery time, and decreased new severe symptoms and hospitalization. [ABSTRACT FROM AUTHOR]

Global Genomics Group, LLC is the sponsor of the GLOBAL clinical study. Editing support was provided by Susannah Thornhill, Ph.D. (BOLDSCIENCE Ltd.), and was funded by Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA, in accordance with GPP 20. Lipoprotein(a) [Lp(a)] is the most prevalent genetic cause of atherosclerotic coronary artery disease, and proprotein convertase subtilisin/kexin type 9 (PCSK9) is a genetically and clinically validated target. Little is known about the relationship between Lp(a) isoform size, serum levels of PCSK9, and coronary atherosclerosis. We hypothesized that circulating PCSK9 levels are associated with circulating levels of small Lp(a) (≤24 Kringle IV type 2 repeats) and coronary atherosclerosis. The GLOBAL study (NCT01738828) enrolled patients referred for coronary computed tomography angiography (CCTA). Circulating PCSK9 was measured using ELISA (R&D Systems, MN). Circulating Lp(a) (nmol/L) was measured using isoform-independent ELISA. Kringle IV type 2 repeats and the percentage of each of the two Lp(a) isoforms were measured using a validated western blot technique (Northwest Lipid Metabolism and Diabetes Research Laboratories, WA). Circulating levels of small and large Lp(a) particles were calculated based on total Lp(a) and the percent contribution of each isoform. Coronary atherosclerosis was identified and quantified using CCTA in a core laboratory. First, we used linear regression to assess associations between PCSK9 and Lp(a) as continuous variables. Second, we compared serum levels of Lp(a) across PSCK9 quartiles using ANOVA. Third, the prevalence of coronary atherosclerosis was assessed based on serum PCSK9 and Lp(a) quartiles. We enrolled 340 patients: 53% female, mean age 55.6±9.8 years. Increasing levels of circulating PCSK9 were associated with increasing levels of Lp(a) when PCSK9 was assessed as a continuous variable (rho=0.11; p=0.038; Figure, Panel A) and when PCSK9 was assessed by quartiles (16±20.61, 24.05±29.5, 23.75±24.76, and 45±59.3 nmol/L, respectively; ANOVA p=0.043; Figure, Panel D). This association was seen for small Lp(a) particles (rho=0.122; p=0.026; Figure, Panels B and E) but was not seen for large Lp(a) particles (rho=0.055; p=0.32; Figure, Panels C and F). In patients in the highest Lp(a) quartile, the prevalence of coronary atherosclerosis across PCSK9 quartiles was 38.9%, 50.0%, 78.9%, and 77.8% (Figure, Panel G). Increasing levels of circulating PCSK9 are associated with higher levels of circulating Lp(a), driven by the small Lp(a) particles; this is associated with the increasing prevalence of coronary atherosclerosis. These results are consistent with a mechanistic hypothesis that the PCSK9/low-density lipoprotein receptor axis may be involved in the metabolism of small but not large Lp(a) particles, and small Lp(a) particles are associated with coronary atherosclerosis. In patients with elevated Lp(a), high circulating PCSK9 levels are associated with a very high prevalence of atherosclerosis, representing a high-risk group. [ABSTRACT FROM AUTHOR]

Cystic endometrial hyperplasia (CEH)-pyometra (CEH-P) is one of the most common reproductive disorders in bitches, posing a risk to both future fertility and life. The aims of the current study were to elucidate the differential expression patterns of inflammatory mediators at transcript and protein levels in the endometrium and to assess the concentrations of key inflammatory mediators in the peripheral circulation of bitches with different graded CEH-P. A total of 25 client-owned intact mixed breed bitches of 3–10 years presented to the outpatient department of RVP-TVCC of the institute were considered for the study. Of which, 22 cases suggestive of pyometra and 3 cases of CEH obtained during routine elective ovariohysterectomy were subjected to histopathological examination. Uteri were categorized into CEH (n = 3), moderate CEH-P (mCEH-P, n = 9), severe CEH-P (sCEH-P, n = 6) and atrophic pyometra (AT-P, n = 7). A group of age matched (n = 12) bitches without pyometra served as control. Endometrial transcripts such as IL6, IL8, PTGS2, PGFS, and SLPI were expressed differentially in the CEH and CEH-P bitch. In addition, a strong immunoreactivity (IR) of IL6, IL8, PTGS2, and mPGES1 was recorded in the sCEH-P uterus, while expression of IL10 was noticed in AT-P. In circulation, serum IL6 was the most relevant marker with high sensitivity of 96.2% and specificity of 84.6% at a cut off concentration 8.5 pg/mL followed by SLPI with 95.2% sensitivity, and 84.6% specificity at cut off concentration of 1.3 ng/mL. Serum IL10, PGFM and SLPI concentration in the peripheral circulation were 1.5–2.23 fold higher in mCEH-P, 0.87–2.5 fold higher in sCEH-P and 2.9–3.5 fold higher in AT-P than that of control. It is concluded that monitoring the serum concentration of IL6, IL10 and SLPI would be useful adjunct to the established hematobiochemical parameters in the management of pyometra in the bitch with critical illness. • Interleukin 6, 8 and prostaglandin synthases expressed differentially in the endometrium of CEH and CEH-P bitch. • Measurement of IL6 and SLPI concentrations in serum serve as potential biomarker in assessing the severity of CEH-P. • Upregulation of IL10 in the endometrium and serum are associated with chronic inflammation in atrophic pyometra in bitch. [ABSTRACT FROM AUTHOR]

Hepatitis C, a liver inflammation caused by the hepatitis C virus (HCV), is treated with antiviral drugs. In this context, simeprevir (SIM) is an NS3/4A protease inhibitor used in HCV genotypes 1 and 4. It is orally administered and achieves high virological cure rates. Among adverse reactions associated with SIM treatment, photosensitivity reactions have been reported. In the present work, it is clearly shown that SIM is markedly phototoxic, according to the in vitro NRU assay using BALB/c 3T3 mouse fibroblast. This result sheds light on the nature of the photosensitivity reactions induced by SIM in HCV patients, suggesting that porphyrin elevation in patients treated with SIM may not be the only mechanism responsible for SIM-associated photosensitivity. Moreover, lipid photoperoxidation and protein photooxidation assays, using human skin fibroblasts (FSK) and human serum albumin (HSA), respectively, reveal the capability of this drug to promote photodamage to cellular membranes. Also, DNA photo lesions induced by SIM are noticed through comet assay in FSK cells. Photochemical and photobiological studies on the mechanism of SIM-mediated photodamage to biomolecules indicate that the key transient species generated upon SIM irradiation is the triplet excited state. This species is efficiently quenched by oxygen giving rise to singlet oxygen, which is responsible for the oxidation of lipids and DNA (Type II mechanism). In the presence of HSA, the photobehavior is dominated by binding to site 3 of the protein, to give a stable SIM@HSA complex. Inside the complex, quenching of the triplet excited state is less efficient, which results in a longer triplet lifetime and in a decreased singlet oxygen formation. Hence, SIM-mediated photooxidation of the protein is better explained through a radical (Type I) mechanism. [Display omitted] • Simeprevir is photo(geno)toxic to cells as revealed by the 3T3 NRU and comet assays. • Simeprevir is an efficient photosensitizer for singlet oxygen formation. • Photosensitization of lipids and DNA by simeprevir proceeds via Type II mechanism. • Binding of simeprevir to HSA leading to 1:1 complex, occurs at site 3 of the protein. • Simeprevir-mediated photooxidation of proteins takes place by a Type I mechanism. [ABSTRACT FROM AUTHOR]

Serine proteases are an important group of enzymes present in several organisms such as viruses, bacteria and eukaryotes involved in several physiological and pathological processes such as cancer, neurodegeneration, tissue inflammation and infections. Kunitz-type serine protease inhibitors have been studied as therapeutical targets with positive results in many of these diseases. rBmTI-A (recombinant B. microplus Trypsin Inhibitor A) is a Kunitz-BPTI type inhibitor based on the native protein BmTI-A. BmTI-A was extracted from tick larvae and presented inhibitory activity against trypsin, human plasma kallikrein (HuPK), human neutrophil elastase (HNE) and human plasmin. rBmTI-A presented the same inhibitory activities of the BmTI-A and its thermostability has already been demonstrated. In emphysema induced by porcine pancreatic elastase (PPE) and by cigarette smoke animal models, the treatment using rBmTI-A showed a protective effect against the development of pulmonary emphysema and prevented the increase of inflammatory cells. In chronic allergic animal model, rBmTI-A treatment resulted in attenuated bronchial hyperresponsiveness, inflammation, remodeling. These are important physiological results in emphysema and lung inflammatory animal models with rBmTI-A treatment confirming its therapeutical potential. • rBmTI-A is a recombinant Kunitz-BPTI type inhibitor based on BmTI-A native protein. • rBmTI-A inhibit trypsin, human plasma kallikrein, human neutrophil elastase, plasmin. • rBmTI-A attenuated the development of pulmonary emphysema in mice. • rBmTI-A reduced the bronchial inflammation in chronic pulmonary inflammation model. • rBmTI-A inhibited viability of HUVEC cells in in vitro vessel formation assay. [ABSTRACT FROM AUTHOR]

Data on the long-term persistence of HCV resistance-associated substitutions (RASs) after treatment with direct-acting antivirals (DAAs) are limited. This study evaluated the persistence of NS3, NS5A, and NS5B RASs for up to 5 years after the end of treatment (EOT). We included samples from 678 individuals with an HCV genotype (GT) 1 or 3 infection and virologic DAA treatment failure collected in the European Resistance Database. NS3, NS5A, and NS5B were sequenced, and clinical parameters were evaluated. A total of 242 individuals with HCV GT1a (36%), 237 with GT1b (35%), and 199 (29%) with GT3 and a DAA failure were included. After protease inhibitor failure, the frequencies of NS3 RASs were 40–90% after the EOT. NS3 RASs disappeared rapidly in GT1b and GT3 after follow-up month 3 but were stable (≥60%) in GT1a owing to Q80K. The SOF-resistant NS5B RAS S282T was only found in individuals with GT3a. Non-nucleoside NS5B RASs were frequent in GT1 (56–80%) and decreased to 30% in GT1a but persisted in GT1b. NS5A RASs were very common in all GTs after NS5A inhibitor failure (88–95%), and even after follow-up month 24, their frequency was 65% and higher. However, RASs in GT1b had a stable course, whereas RASs in GT1a and GT3 declined slightly after follow-up month 24 (GT1a, 68%; GT1b, 95%; and GT3, 65%), mainly because of the slow decline of high-level resistant Y93H. We found that low-to medium-level RASs persisted, whereas high-level resistant RASs disappeared over time. Different patterns of RAS persistence according to HCV subtype could have implications for retreatment with first-generation DAAs and for global HCV elimination goals. There are little data on the long-term persistence of HCV resistance-associated substitutions (RASs) after DAA treatment failure, and RASs could have an impact on the efficacy of a rescue treatment. Especially in countries with limited availability of VOX/VEL/SOF or G/P/SOF, different patterns of RAS persistence could have implications for retreatment with first-generation DAAs and for global HCV elimination goals. The different patterns of RAS persistence identified in this study can be used to derive general rules regarding the persistence of RASs after DAA failure that could be applied by physicians in less developed countries to plan individualized HCV retreatment. [Display omitted] • Real-world study of the long-term persistence of NS3, NS5A, and NS5B RASs in 678 individuals with GT1 or GT3 infection after DAA failure. • Sequencing showed a rapid decrease of NS3 RASs after FU month 3 and almost no SOF-resistant RASs. • NS5A RASs persisted for more than 2 years, with a tendency to decrease in GT1a and GT3 owing to the loss of Y93H. • Patterns of RAS persistence could have implications for retreatment with first-generation DAAs and for global HCV elimination goals. [ABSTRACT FROM AUTHOR]

Lentinula edodes (Shiitake) is one of the most heavily cultivated mushrooms in the world with proven antioxidant and antibacterial properties, among others. Evidence indicates that the choice of mushroom cultivation technique strongly influences the production of bioactive compounds, but to date the nature of many of these compounds has not been fully established. This work focuses on the proteomic characterization of L. edodes to highlight the main active processes two days after harvest and elucidates the proteins involved in the known antioxidant and antibacterial proprieties of Shiitake fruit bodies cultivated on oak logs. A label-free approach allowed us to identify a total of 2702 proteins which were mainly involved in carbohydrate and protein metabolism, cell growth and replication, indicating that several developmental processes remain active in fruit bodies post-harvest. Proteins with antioxidant activities were identified, indicating the contribution of proteins to the antioxidant properties of L. edodes extracts. Antibacterial assays also reveal the activity of a serine protease inhibitor that strongly accumulates in the post-harvest fruit body grown on oak logs. Overall, this study contributes to the understanding of the impact of the log cultivation method on the production of Shiitake mushrooms richest in high-value bioactive compounds. [ABSTRACT FROM AUTHOR]

We report the impact of gut protease inhibition on the development of Helicoverpa armigera by trypsin inhibitor and the use of molecular modeling to understand the mechanism of trypsin inhibition. Larvae of H. armigera fed on an artificial diet containing 150 and 300 μg/ml SSTI showed a negative impact on the insects' development in terms of mean larval weight, larval fatality, survival rate, and nutritional indices. Prominent physical abnormalities like curled wings, malformed appendages, and small body size were observed during the development. Gene expression studies revealed down regulation in trypsin (HaTry 1, 2, 3, 4, 6, 8) and chymotrypsin (HaChy 1, 2, 3, 4) genes of the larval gut upon treatment of SSTI. Homology modeling has been used to build the three-dimensional structure of SSTI, which showed β-sheets having a stable canonical inhibitory loop (CIL) with conserved lysine residue. Molecular docking studies showed the strong binding of SSTI at the active site of trypsin. Molecular dynamic (MD) simulation revealed the stable interactions of the rigid CIL of SSTI at the active site of trypsin, leading to its destabilization. Conserved lysine63 of the P1 site in SSTI forms a strong hydrogen bonding network with residues Asp189 and Ser190 of trypsin. [Display omitted] • SSTI negatively impacted the growth and development of Helicoverpa armigera. • A decrease in trypsin and chymotrypsin mRNA abundance was observed in SSTI treated insects. • The SSTI structure is stabilized by disulfide bonds and a rigid canonical inhibitory loop (CIL). • SSTI forms a stable interaction at the substrate binding site of trypsin. • CIL strongly interact with Asp189 and Ser190 in substrate binding site of trypsin, leading to inhibition. [ABSTRACT FROM AUTHOR]

• Out of 45917 natural compounds, about thirty phyto components had a higher binding affinity and inhibited Papain-Like Proteases (PLpro). • All the selected compounds obey the ADMET properties. • When compared to remidisevir, ritonavir, and lopinavir, SN00334175 and SN00162745 had better docking scores. • SN00334175/7JN2 and SN00162745/7JN2 demonstrated that these complexes were stabilised by ligand binding interactions with Gly266, Asn267, Tyr268, Tyr273, Thr301, and Asp302, Lys157, Leu162, Asp164, Arg166, Glu167, Pro248, Tyr264, respectively. • Plant Based medicine may possess potent inhibitors against SARS-CoV-2 Coronaviruses (CoVs) are a large group of enveloped positive sense single-stranded RNA viruses that can cause disease to humans. These are zoonotic having potential to cause large-scale outbreaks of infections widely causing morbidity and mortality. Papain-Like Protease (PLpro) is a cysteine protease, essential for viral replication and proliferation, as a highly conserved enzyme it cleaves peptide linkage between Nsp1, Nsp2, Nsp3, and Nsp4. As a valid therapeutic target, it stops viral reproduction and boosts host immune response thereby halting further spread of infection. In the purpose of identifying inhibitors targeting Papain-Like Proteases (PLpro) we initiated a high throughput virtual screening (HTVS) protocol using a SuperNatural Database. The XP docking results revealed that two compounds SN00334175 and SN00162745 exhibited docking scores of -10.58 kcal/mol and -9.93 kcal/mol respectively. The Further PRIME MMGB-SA studies revealed Van der Waal energy and hydrophobic energy terms as major contributors for total binding free energy. The 100 ns molecular dynamics simulation of SN00334175/7JN2 and SN00162745/7JN2 revealed that these complexes were stabilized with ligand binding forming interactions with Gly266, Asn267, Tyr268, Tyr273, Thr301 and Asp302, Lys157, Leu162, Asp164, Arg166, Glu167, Pro248 and Tyr264. [ABSTRACT FROM AUTHOR]

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ~1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants. [ABSTRACT FROM AUTHOR]

Protease inhibitors (PIs) function as a natural adversary to proteolytic enzymes. They can diminish or inhibit the catalytic properties of proteases, which are crucial for various tasks in the physiology and metabolism of cellular forms. Protease Inhibitors are low molecular weight (5–25 kDa) stable proteins. Plants are a fair source of PIs, so foods containing PIs remarkably influence human health. PIs are usually present in storage tissues of the plant, although they are present in other aerial parts as well. In plants, protease inhibitors participate in vital functions such as maintaining physiological homeostasis, mobilization of storage proteins, defense systems, apoptosis, and other processes. In recent years, plant-derived PIs have shown promising results in treating various diseases including inflammatory conditions, osteoporosis, cardiovascular issues, and brain disorders. The primary goal of this review is to provide a comprehensive understanding of the characteristics, applications, and challenges associated with natural protease inhibitors in plants, which draws insights from an extensive examination of 80+ research papers with a focus on their potential in agriculture and medicine. By synthesizing findings from an extensive literature review, this work aims to guide future research directions and innovations in leveraging plant-based PIs for sustainable agricultural practices and advanced therapeutic interventions. [Display omitted] • Protease inhibitors (PIs), having a molecular mass between 5-25KDa, have a widespread prevalence in the plant kingdom. • They play roles in agriculture and therapeutics such as homeostasis, cell signaling, apoptosis, and pathophysiology. • They are classified as serine, cysteine, metalloid, and aspartic protease inhibitors based on their reactive sites. • Various purification methods have evolved and been improvised to customize innumerable applications. [ABSTRACT FROM AUTHOR]

[Display omitted] An uncontrolled activity of neutrophil serine proteases (NSPs) contributes to inflammatory diseases. Cathepsin C (CatC) is known to activate NSPs during neutrophilic differentiation and represents a promising pharmacological target in NSP-mediated diseases. In humans, Papillon-Lefèvre syndrome (PLS) patients have mutations in their CTSC gene, resulting in the complete absence of CatC activity. Despite this, low residual NSP activities are detected in PLS neutrophils (<10% vs healthy individuals) , suggesting the involvement of CatC-independent proteolytic pathway(s) in the activation of proNSPs. This prompted us to characterize CatC-independent NSP activation pathways by blocking proCatC maturation. In this study, we show that inhibition of intracellular CatS almost completely blocked CatC maturation in human promyeloid HL-60 cells. Despite this, NSP activation was not significantly reduced, confirming the presence of a CatC-independent activation pathway involving a CatC-like protease that we termed NSPs-AAP-1. Similarly, when human CD34+ progenitor cells were treated with CatS inhibitors during neutrophilic differentiation in vitro , CatC activity was nearly abrogated but ∼30% NSP activities remained, further supporting the existence of NSPs-AAP-1. Our data indicate that NSPs-AAP-1 is a cysteine protease that is inhibited by reversible nitrile compounds designed for CatC inhibition. We further established a proof of concept for the indirect, although incomplete, inhibition of NSPs by pharmacological targeting of CatC maturation using CatS inhibitors. This emphasizes the potential of CatS as a therapeutic target for inflammatory diseases. Thus, preventing proNSP maturation using a CatS inhibitor, alone or in combination with a CatC/NSPs-AAP-1 inhibitor, represents a promising approach to efficiently control the extent of tissue injury in neutrophil-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

It is well known that aquatic products such as fish and shellfish, when stored for a long period of time under inappropriate conditions, can suffer from muscle softening. This phenomenon is mainly caused by endogenous proteases, which are activated during heating and accelerates the degradation of myofibrillar proteins, directly leading to weaker gels and poorer water retention capacity. This paper reviews the changes in fish proteins during storage after death and the factors affecting protein hydrolysis. A brief overview of the extraction of protease inhibitors, polysaccharides and proteins is given, as well as their mechanism of inhibition of protein hydrolysis in surimi and the current status of their application to improve the properties of surimi. • Myofibrillar proteins play a major role in the formation of surimi gels. • Endogenous enzymes cause protein hydrolysis and thus muscle softening. • The mechanism of protein hydrolysis of surimi by different additives was reviewed. • The effect of different additives on the quality of surimi gels was emphasised. [ABSTRACT FROM AUTHOR]

Cystatins are well known as a vast superfamily of functional proteins participated in the reversible competitive inhibition of cysteine proteases. Currently, increasing evidences point to the extensive phylogenetic diversity and crucial immune roles of type-2 cystatins in the vertebrate species. However, no information is available regarding the homologue in cephalochordate amphioxus, the representative of most basal living chordates, whose immune regulation are still ambiguous. Here, we clearly identified the presence of type-2 cystatin gene in amphioxus Branchiostoma japonicum , termed Bjcystatin-2, which was structurally characterized by typical wedge-shaped cystatin feature. Evolutionary analyses revealed that Bjcystatin-2 is the putative ancestral type-2 cystatin for chordates, with gene diversity emerging through duplication events. The expression of Bjcystatin-2 showed tissue-specific profile and was inducible upon invasive pathogens. Significantly, the recombinant Bjcystatin-2 exhibited not merely cathepsin L inhibitory activity, but also the ability to bind with bacteria and their characteristic molecules. Furthermore, Bjcystatin-2 also showed the capacity to enhance the macrophage-driven bacterial phagocytosis and to attenuate the generation of pro-inflammatory cytokines within macrophages. In summary, these findings demonstrate that Bjcystatin-2 exhibits dual role acting as both a protease inhibitor and an immunoactive molecule, greatly enriching our understanding of immune defense mechanisms of type-2 cystatin within the amphioxus. [ABSTRACT FROM AUTHOR]

In vitro passage experiments are crucial to the development of antiretroviral (ARV) drugs. We created an online database containing data from 102 published studies in which HIV-1 or HIV-2 was cultured with increasing concentrations of the FDA-approved nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), integrase strand transfer inhibitors (INSTIs), protease inhibitors (PIs), capsid inhibitor (CAI) lenacapavir, and nucleoside RT translocation inhibitor (NRTTI) islatravir. We summarized the mutations selected in the subset of passage experiments with NRTIs lamivudine (3TC), emtricitabine (FTC), abacavir (ABC), tenofovir (TFV), and zidovudine (AZT), NNRTIs doravirine (DOR), efavirenz (EFV), and rilpivirine (RPV), INSTIs bictegravir (BIC), cabotegravir (CAB), and dolutegravir (DTG), and PIs atazanavir (ATV), darunavir (DRV), and lopinavir (LPV). Mutations selected in vitro were compared with those selected in persons receiving the same ARV. Twenty-seven studies described 89 experiments of wildtype isolates passaged with 3TC, FTC, ABC, TFV, or AZT; sixteen studies described 89 experiments passaged with EFV, RPV, or DOR; eleven studies described 76 experiments passaged with the INSTIs BIC, CAB, or DTG; six studies described 33 experiments passaged with ATV, LPV, or DRV. With several exceptions, mutations selected in two or more experiments were among the most common mutations selected in persons receiving the same ARV. We created a database of published ARV in vitro selection experiments. Mutations emerging from these experiments generally predict those observed in persons receiving the same ARV. However, there are notable differences in mutation frequencies between in vitro and in vivo settings. • An online database containing in vitro selection experiments of HIV drug resistance mutations from 102 studies. • HIV-1 or HIV-2 was cultured with 14 FDA-approved ARVs. • Mutations selected in vitro were compared with those selected in persons received same antiretrovirals. [ABSTRACT FROM AUTHOR]

Herbs of the genus Juniperus (family Cupressaceae) have been commonly used in ancestral folk medicine known as "Al'Araar" for treatment of rheumatism, diabetes, inflammation, pain, and fever. Bioassay-guided isolation of bioactives from medicinal plants is recognized as a potential approach for the discovery of novel drug candidates. In particular, non-addictive painkillers are of special interest among herbal phytochemicals. The current study aimed to assess the safety of J. thurifera , J. phoenicea , and J. oxycedrus aqueous extracts in oral treatments; validating the traditionally reported anti-inflammatory and analgesic effects. Further phytochemical investigations, especially for the most bioactive species, may lead to isolation of bioactive metabolites responsible for such bioactivities supported with in vitro enzyme inhibition assays. Firstly, the acute toxicity study was investigated following the OECD Guidelines. Then, the antinociceptive, and anti-inflammatory bioactivities were evaluated based on chemical and mechanical trauma assays and investigated their underlying mechanisms. The most active J. thurifera n -butanol fraction was subjected to chromatographic studies for isolating the major anti-inflammatory metabolites. Moreover, several enzymatic inhibition assays (e.g., 5-lipoxygenase, protease, elastase, collagenase, and tyrosinase) were assessed for the crude extracts and isolated compounds. The results showed that acute oral administration of the extracts (300–500 mg/kg, p. o.) inhibited both mechanically and chemically triggered inflammatory edema in mice (up to 70% in case of J. thurifera) with a dose-dependent antinociceptive (tail flick) and anti-inflammatory pain (formalin assay) activities. This effect was partially mediated by naloxone inhibition of the opioid receptor (2 mg/kg, i. p.). In addition, 3-methoxy gallic acid (1), quercetin (2), kaempferol (3), and ellagic acid (4) were successfully identified being involved most likely in J. thurifera extract bioactivities. Nevertheless, quercetin was found to be the most potent against 5-LOX, tyrosinase, and protease with IC 50 of 1.52 ± 0.01, 192.90 ± 6.20, and 399 ± 9.05 μM, respectively. J. thurifera extract with its major metabolites are prospective drug candidates for inflammatory pain supported with inhibition of inflammatory enzymes. Interestingly, antagonism of opioid and non-opioid receptors is potentially involved. [Display omitted] • Juniperus leaves are traditionally used as anti-inflammatory and antinociceptive preparations. • The oral administration inhibited inflammatory edema reached up to 70% in case of J. thurifera. • The naloxone treatment indicated that opioid receptors may play a role in the mechanism of action. • The bioguided chromatographic fractionation led to isolation of 4 compounds from J. thurifera n-butanol fraction. • Quercetin was found to be the most potent against 5-LOX, tyrosinase, and protease. [ABSTRACT FROM AUTHOR]

[Display omitted] • Application of QSAR with Machine Learning techniques on the Ebselen compounds as training data related to their SAR-CoV-2 protein target. • Identify the Ebselen binding conformation using the ligand-binding pathway sampling method based on parallel cascade selection molecular dynamics (LB-PaCS-MD). • Providing new designed compounds which meet both ligand-based drug design and structure-based drug design approaches. The global outbreak of the COVID-19 pandemic caused by the SARS-CoV-2 virus had led to profound respiratory health implications. This study focused on designing organoselenium-based inhibitors targeting the SARS-CoV-2 main protease (Mpro). The ligand-binding pathway sampling method based on parallel cascade selection molecular dynamics (LB-PaCS-MD) simulations was employed to elucidate plausible paths and conformations of ebselen, a synthetic organoselenium drug, within the Mpro catalytic site. Ebselen effectively engaged the active site, adopting proximity to H41 and interacting through the benzoisoselenazole ring in a π-π T -shaped arrangement, with an additional π-sulfur interaction with C145. In addition, the ligand-based drug design using the QSAR with GFA-MLR, RF, and ANN models were employed for biological activity prediction. The QSAR-ANN model showed robust statistical performance, with an r2 training exceeding 0.98 and an RMSE test of 0.21, indicating its suitability for predicting biological activities. Integration the ANN model with the LB-PaCS-MD insights enabled the rational design of novel compounds anchored in the ebselen core structure, identifying promising candidates with favorable predicted IC 50 values. The designed compounds exhibited suitable drug-like characteristics and adopted an active conformation similar to ebselen, inhibiting Mpro function. These findings represent a synergistic approach merging ligand and structure-based drug design; with the potential to guide experimental synthesis and enzyme assay testing. [ABSTRACT FROM AUTHOR]

This exploratory analysis of the double-blind, phase 3, SCORPIO-SR trial assessed the effect of ensitrelvir in preventing post coronavirus disease 2019 (COVID-19) condition (PCC). Patients with mild-to-moderate COVID-19 were randomized (1:1:1) within 120 h of symptom onset; received 5-day oral ensitrelvir 125 mg (375 mg on day 1), 250 mg (750 mg on day 1), or a matching placebo once daily; and were assessed for the severity of typical PCC symptoms using a self-administered questionnaire. In total, 341, 317, and 333 patients were assessed in the ensitrelvir 125-mg, ensitrelvir 250-mg, and placebo groups, respectively (mean age, 35.6–36.5 years; men, 53.3%–58.3%). On days 85, 169, and 337, ensitrelvir 125-mg treatment showed 32.7% (95% confidence interval [CI]: -30.6, 66.1), 21.5% (95% CI: -37.3, 55.6), and 24.6% (95% CI: -43.7, 60.9) reductions versus placebo, respectively, in the risk of any of the 14 acute-phase COVID-19 symptoms (at least one mild, moderate, or severe symptom with general health not returning to the usual level). Ensitrelvir 250-mg treatment showed 10.9% (95% CI: -67.0, 52.8), 9.5% (95% CI: -56.6, 48.0), and 30.6% (95% CI: -36.2, 65.5) risk reductions versus placebo on days 85, 169, and 337, respectively. Risk reductions were observed in any of the 4 neurological symptoms and were more pronounced among patients with high acute-phase symptom scores at baseline and among those with a baseline body mass index ≥25 kg/m2. Ensitrelvir treatment in the acute phase of COVID-19 may reduce the risk of various symptoms associated with PCC. jRCT2031210350. • Our results suggested that ensitrelvir may reduce the risk of persistent and/or late-onset COVID-19 symptoms. • The potential benefit of ensitrelvir was indicated irrespective of risk factors or vaccination status of patients. • The potential benefit was more pronounced among patients with high COVID-19 symptom scores at baseline or BMI of ≥25 kg/m2. • Further research is granted for the effect of ensitrelvir in preventing post COVID-19 condition. [ABSTRACT FROM AUTHOR]

RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins. Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine, offering the opportunity to inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold and a similar active site. The hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. We demonstrate that targeting the active site cysteine of Mpro can have profound allosteric effects, distorting secondary structures to disrupt the active dimeric unit. Acrylamide inhibitors were identified against EV71 3Cpro using qIT. Re-purposing against SARS-CoV-2 Mpro revealed a conformational change which allosterically disrupts dimerization, locking Mpro into its inactive monomeric state. [Display omitted] [ABSTRACT FROM AUTHOR]

• We present a case with FHBL caused by homozygous LOF mutations in PCSK9. • A novel missense mutation in PCSK9 was associated with reduced serum PCSK9 level. • She had no complications, except for mild fatty liver. Here, we present the first case of a Japanese patient with familial hypobetalipoproteinemia (HBL) that is caused by homozygous loss-of-function mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9). A 46-year-old female patient who was born in a consanguineous marriage of parents who were second cousins was referred to our hospital due to decreased low-density lipoprotein (LDL)-cholesterolemia (22 mg/dL). She did not have any secondary HBL causes. Novel homozygous mutations were identified in PCSK9 (c.1133G>A [p.Cys378Tyr]) using panel sequencing. The serum levels of heterodimer PCSK9 and furin-cleaved PCSK9 were extremely low (<32 and 15 ng/mL, respectively), leading to the diagnosis of familial HBL diagnosis caused by loss-of-function mutations in PCSK9. The patient did not exhibit any complications associated with low LDL cholesterol, except for mild fatty liver and reduced serum 25-OH vitamin D level (15.7 ng/mL). Here, we provide a detailed molecular and functional characterization of a novel loss-of-function mutation in PCSK9. [ABSTRACT FROM AUTHOR]

• The virtual lipid visits model led to an early optimization of lipid-lowering therapy. • Intensive lipid-lowering therapy included high use of ezetimibe and PCSK9i. • Lipid targets were achieved in most patients within the first 3 months post-ACS. • LDL-C levels <70 and <55 mg/dL were reached in 95.4% and 81.5% of patients. • High LDL-c control rates meant better outcomes with low MACE and mortality rates. Atherosclerotic cardiovascular disease is a very common condition in routine practice and a leading cause of morbidity and mortality all around the world. To determine the impact of a strategy based on strict control and close follow-up for patients with acute coronary syndrome (ACS) through the use of "post-ACS virtual lipid visits" on lipid-lowering therapy, low-density lipoprotein cholesterol (LDL-c), and outcomes. Prospective study that consecutively included patients with ACS during 2020. All patients were discharged with high-intensity statins, and the lipid profile was determined after 1 month. At this time, patients were contacted by phone, and treatment was adjusted following the therapeutic algorithm of the Spanish Society of Cardiology. These visits were repeated every month until LDL-c reached <55 mg/dL. Patients were then transferred to scheduled conventional outpatient visits. A total of 346 patients (67.3±2.3 years; 68.2% men) were included. Follow-up was 12-24 months (mean, 17.7±3.8; median, 17.3). Definitive lipid-lowering therapy (mean uptitration time, 3.2±2.1 months) consisted of high-intensity statins alone (32.4%), high-intensity statins plus ezetimibe (56.9%), and PCSK9 inhibitors (10.7%). LDL-c decreased from 108.4±40.6 to 48.7±14.4 mg/dL. At baseline, LDL-c was <100 mg/dL, 70 mg/dL, and 55 mg/dL in 44.5%, 17.6%, and 7.2% of patients, respectively. At study end, these percentages were 100%, 95.4%, and 81.5%, respectively. After one year of follow-up, 3-P MACE, 4-P MACE, and cardiovascular mortality were recorded in 3.5%, 4.0% and 1.5% of patients, respectively. At study end, these percentages were 4.0%, 5.2%, and 1.7%, respectively. The implementation of a post-ACS virtual lipid visit model led to early optimization of lipid-lowering therapy, which led to marked improvements in LDL-c control rates and low MACE and mortality rates. [ABSTRACT FROM AUTHOR]

The protease inhibitor α2-macroglobulin (A2M) is a member of the ancient α2-macroglobulin superfamily (A2MF), which also includes structurally related proteins, such as complement factor C3. A2M and other A2MF proteins undergo an extensive conformational change upon cleavage of their bait region by proteases. However, the mechanism whereby cleavage triggers the change has not yet been determined. We have previously shown that A2M remains functional after completely replacing its bait region with glycine and serine residues. Here, we use this tabula rasa bait region to investigate several hypotheses for the triggering mechanism. When tabula rasa bait regions containing disulfide loops were elongated by reducing the disulfides, we found that A2M remained in its native conformation. In addition, cleavage within a disulfide loop did not trigger the conformational change until after the disulfide was reduced, indicating that the introduction of discontinuity into the bait region is essential to the trigger. Previously, A2MF structures have shown that the C-terminal end of the bait region (a.k.a. the N-terminal region of the truncated α chain) threads through a central channel in native A2MF proteins. Bait region cleavage abolishes this plug-in-channel arrangement, as the bait region retracts from the channel and the channel itself collapses. We found that mutagenesis of conserved plug-in-channel residues disrupted the formation of native A2M. These results provide experimental evidence for a structural hypothesis in which retraction of the bait region from this channel following cleavage and the channel's subsequent collapse triggers the conformational change of A2M and other A2MF proteins. [ABSTRACT FROM AUTHOR]

Introduction: Olfactory dysfunction (OD) is highly prevalent amongst type 2 diabetes mellitus (DM2) patients and has many associated health risks. For example, OD can lead to poor nutrition, safety issues related to diminished hazard detection, and increased mortality rates. While limited research exists about therapeutics for DM2-associated OD, recovery of olfactory function is better studied in other pathologic states. The objectives of this scoping review are to synthesize the existing data on interventions for DM2-associated OD and present the evidence for therapies that have been utilized for non-DM2-associated causes of OD. Additionally, the potential therapeutic opportunities for patients with DM2 are explored.Methods: A scoping review was conducted with a medical librarian to identify studies investigating treatments of DM2-related OD. 6 databases were searched (Embase, CINAHL, the Cochrane Library, Google Scholar, OVID Medline, and Web of Science). Studies were eligible if the primary discussion involved treatment of olfactory deficits in the context of DM2. All publication dates were included, and studies published in languages other than English were excluded.Results: 3631 articles were identified; 3 articles met inclusion criteria and underwent full text review. Hyperbaric oxygen (HBO), the DPP-4 inhibitor Linagliptin and the GLP-1 agonists Exenatide and Liraglutide are the only therapeutics that have been used in the context of DM2. Only HBO and GLP-1 agonists produced statistically significant improvements in olfactory identification. The literature regarding non-DM2-associated OD supports interventions such as olfactory training, dietary supplements, and intranasal insulin. Specifically, olfactory training was very effective in many contexts such as post-viral and traumatic OD while being affordable and non-invasive.Conclusion: This scoping review of olfactory rehabilitation options for DM2-induced OD demonstrates a paucity of prospective investigations of plausible therapeutics. Additionally, treatments for OD related to non-DM2-associated etiologies, such as olfactory training, are well-studied, efficacious, and should be investigated in the context of DM2. Future investigation has the potential to enhance the quality of clinical intervention for OD and improve short- and long-term outcomes for DM2 patients. [ABSTRACT FROM AUTHOR]

To evaluate the inhibitory effect of a novel mussel-inspired monomer (N -(3,4-dihydroxyphenethyl)methacrylamide (DMA) on the soluble and matrix-bound proteases. The inhibitory effect of DMA (0, 1, 5, and 10 mM) and 1 mM chlorhexidine (CHX) dissolved in 50% ethanol/water on soluble recombinant human matrix metalloproteinases (rhMMP-2, −8, and −9), as well as cysteine cathepsins (B and K) were evaluated using both fluorometric assay kits and molecular docking. The effect of CHX and DMA on matrix-bound proteases was examined by in situ zymography, and the fluorescence intensity and relative area were calculated by Image J software. All data obtained were analyzed by one-way ANOVA followed by Tukey test (α = 0.05). The anti-proteolytic ability of DMA increased in a dose-dependent manner except that of rhMMP-9. Inhibitory effect of 1 mM DMA against rhMMP-2, − 8, − 9, as well as cathepsin B and K was all significantly lower than 1 mM CHX (p < 0.05). The molecular docking analysis was in good agreement with the experimental results, that the binding energy of DMA was lower than CHX for all proteases. In situ zymography revealed that all DMA- and CHX-treated groups significantly inactivated the matrix-bound proteases, with a dramatic reduction of the fluorescence intensity and relative area compared with the control group (p < 0.05). Under the prerequisite condition that the overall inhibitory performance on matrix-bound proteases was comparable by DMA and CHX, the more selective property of DMA could avoid inducing potential negative effects by suppressing MMP-9 when applied in dental treatment compared with CHX. [Display omitted] • Anti-proteolytic ability of DMA increased in a dose-dependent manner except MMP-9. • The binding mechanism of DMA toward proteases was simulated by molecular docking. • CHX was a broad-spectrum inhibitor, while DMA selectively inhibited MMP-2 and − 8. • DMA manifested comparable inhibitory effect as CHX on matrix-bound proteases. [ABSTRACT FROM AUTHOR]

• The cardiovascular benefit from LDL-C lowering is proportional to the magnitude of reduction. • Evolocumab reduces LDL-C levels by approximately 60% when measured at drug trough. • Time-averaged estimates include both peaks and troughs of drug effect. • Time-averaged LDL-C reduction with evolocumab vs placebo was 65–68%. • Time-averaged measures may better estimate long-term cardiovascular benefit. LDL-C is the pivotal risk factor for atherosclerotic cardiovascular disease, and the benefit from LDL-C lowering is proportional to the magnitude of reduction. Clinical trials demonstrate that evolocumab reduces LDL-C levels by approximately 60% when measured at the trough of drug effect, which may underestimate cumulative LDL-C reduction. We obtained a time-averaged estimate of LDL-C lowering that included both peaks and troughs. Pooled analysis of 5 phase 2 trials included patients with hypercholesterolemia who received placebo or evolocumab (140 mg every 2 weeks [Q2W] or 420 mg monthly [QM]). Percent changes from baseline LDL-C and free serum PCSK9 were averaged across weeks 9–12. In 372 patients, time-averaged percent reduction from baseline in LDL-C with evolocumab vs placebo was 67.6% (95% CI: 63.9–71.3) with Q2W dosing and 65.0% (95% CI: 60.7–69.3) with QM dosing. The time-averaged measure yielded LDL-C reductions for evolocumab that exceeded measurements at the end of dosing intervals and may provide a better estimate of cardiovascular benefit during long-term therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action. [ABSTRACT FROM AUTHOR]

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin.Wedecipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP- 2-inhibitory, MASP-3-inhibitory, and lectin pathway–inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation. [ABSTRACT FROM AUTHOR]

Potato proteins have been shown to be suitable alternatives for animal proteins in food foam formulations but their ability to form and stabilize foam is mechanistically ill-understood. The present study aimed to better understand the foaming properties of a protein fraction isolated from potato trimmings, a by-product from the potato industry. This was achieved by comparing the properties of these in-house isolated proteins to those of commercial and highly purified potato proteins. Protein isolates (53.8 ± 4.7 g/100 g dry weight) were produced from trimmings by alkaline solubilization (pH 9.0) and isoelectric precipitation (pH 4.0). The foaming properties of the in-house isolate at pH 3.0 and 7.0 were better than those of a blend of commercial potato proteins with similar relative amounts of patatin and protease inhibitors as the in-house isolate. Notably, this was the case despite the fact that more proteins were insoluble in the in-house isolate than in the commercial potato protein suspensions. Patatin and protease inhibitors were further shown to both contribute considerably to the formation and stabilization of foams. Interestingly, a beneficial effect on foam stability was found when patatin and protease inhibitors of various colloidal sizes were present. Additionally, impurities in the in-house isolates likely contributed to their superior foaming properties. In conclusion, a protein-rich fraction with good foaming properties can be recovered from potato trimmings. Mechanistic insights were gained into the foaming properties of potato protein (mixtures). This research also highlighted the importance of assessing protein colloidal state when investigating plant protein structure-function relationships. [Display omitted] • Protein isolates with good foaming properties were obtained from potato trimmings. • Patatin and protease inhibitors both contribute to the isolate foaming properties. • Patatin adsorbs slower but is more surface-active than the protease inhibitors. • Assessment of the colloidal state is crucial for understanding foaming properties. • Impurities in such isolates positively affect the foaming and interfacial properties. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the expression of serine protease inhibitor kazal type 1 (SPINK1) and its carcinogenic effect in oral tongue squamous cell carcinoma (OTSCC). Design: Initially, bioinformatics analysis was conducted using data from The Cancer Genome Atlas and Gene Expression Omnibus to compare SPINK1 mRNA expression between malignant and adjacent tissues. Subsequently, the impact of differential expression on survival and other clinical variables was examined. Additionally, histology microarray analysis was performed to assess SPINK1 protein expression in 35 cases of malignant and adjacent tissues. Finally, alterations in SPINK1 expression were evaluated to determine its biological phenotypes in OTSCC, including proliferation, apoptosis, invasion, and metastasis. Results: OTSCC tissues exhibit higher levels of SPINK1 compared to surrounding cancerous tissues. Notably, increased SPINK1 expression correlates with the pathological N stage and independently predicts overall survival among patients with OTSCC. Conclusion: Suppression of SPINK1 inhibited OTSCC cell proliferation, invasion, and motility while promoting apoptosis. These findings suggest that SPINK1 may serve as a prognostic biomarker as well as a potential therapeutic target for managing OTSCC. • High SPINK1 expression in oral tongue squamous cell carcinoma (OTSCC) is related with the pathological N stage. • SPINK1 serves as a crucial independent factor influencing the overall survival of patients with OTSCC. • SPINK1 promotes proliferation, invasion, and motility of OTSCC cells while suppressing apoptosis. [ABSTRACT FROM AUTHOR]

While numerous case reports exist on non-responders to inhibitors of PCSK9 by monoclonal antibodies (PSCK9mAb), to the best of our knowledge, there is no data available on individuals who do not respond to both PSCK9mAb and RNA interference by inclisiran. To describe a unique case with nonresponse to PSCK9mAb and inclisiran in order to outline potential treatment options for such patients. We report a patient with hyperlipidemia, elevated lipoprotein (a) (Lp(a)), subclinical coronary atherosclerosis, and partial statin intolerance who has nonresponse to novel non-statin LDL cholesterol (LDL-C) lowering agents. A 68-year-old female nurse with BMI of 20.3 kg/m2, hypertension, and hyperlipidemia presented to preventive cardiology clinic for consultation. Family history is notable for a stroke at 60 years in her mother. LDL-C was 143 mg/dL on rosuvastatin 5 mg oral daily for five years. She maintained a healthy diet and was an avid runner. In light of high Lp(a) (217 nmol/L), coronary artery calcium score (204 AU), and family history of early atherosclerotic events, we aimed for a LDL-C goal of <70 mg/dL. Maximal statin dosages with different statins and ezetimibe resulted in proximal muscle aches and therefore unachievable threshold LDL-C. Patient was switched to alirocumab. LDL-C levels rose to 162 mg/dL despite six administrations of alirocumab and a change to inclisiran was made (given in office). One month after the first dose, LDL-C was 165 mg/dL and one month after the second dose, LDL-C was 199 mg/dL. There were no other dietary or medication changes. Inclisiran is an siRNA that inhibits hepatic synthesis of PCSK9 while PSCK9mAb inhibits circulating PCSK9. PSCK9mAb nonresponse can be attributed to incorrect injection techniques (unlikely given that our patient works in healthcare). The development of antidrug antibodies has not correlated with LDL response in clinical trials. In the setting of elevated Lp(a) in this case, it is possible that LDL-C reflects cholesterol ester in Lp(a), however, still less of a response than expected. Patient is currently pending genetic testing which may yield more information about lack of response. Lastly, sitosterolemia could be considered based on lack of response to medical therapy in the setting of a plant predominant diet. If the LDL-C continues to remain above threshold possible options include bempedoic acid, evanacumab or apheresis depending on the results of genetic testing. Understanding the mechanism of non-response to both PSCK9mAb and inclisiran is crucial to guide appropriate treatment options for such patients and reduce future atherosclerotic events. [ABSTRACT FROM AUTHOR]

[Display omitted] • The CT001 was encapsulated into CPPMs/HAMs to target cathepsin S in AD. • CPPMs/HAMs displayed a macrophage-mediated controlled release profile. • CPPMs/HAMs reduced inflammation and CTSS in LPS-induced SIM-A9 cells. • CPPMs/HAMs enhanced learning and memory while reducing Aβ accumulation in AD mice. • CPPMs/HAMs can be effective and safe sustained drug-release carriers for AD therapy. The cause of Alzheimer's disease (AD) remains unknown; however, studies have indicated the increased expression of cathepsin S (CTSS) in AD brains. Cathepsin S acts as a β-secretase, prompting the release of amyloid-β peptides and stimulating microglial cell activation, causing microglial migration, and contributing to neuroinflammation and disease progression. In this study, CT001, an inhibitor of the cysteine protease CTSS, was encapsulated in poly(lactic-co-glycolic acid)-polyethylene glycol microspheres combined with hydroxyapatite microspheres (CPPMs/HAMs) to achieve a constant and long-term drug release for AD. The results showed that CPPMs/HAMs had a uniform diameter of approximately 1 μm, enabling macrophage endocytosis and facilitating sustained CT001 release into the bloodstream, ultimately reaching the brain for AD treatment. The developed CPPMs/HAMs demonstrated excellent anti-inflammatory properties and mitigated CTSS expression in an in vitro inflammation microglial model. Intramuscular administration of CPPMs/HAMs improved learning and memory in AD mice during behavioral assessments. Furthermore, CPPMs/HAMs reduced CTSS expression, amyloid-beta accumulation, and deposition in vivo. This study demonstrated the potential of CT001 delivered via CPPMs/HAMs as a promising approach for managing AD in patients. [ABSTRACT FROM AUTHOR]

High expression of ubiquitin-specific protease 10 (USP10) promote the proliferation of hepatocellular carcinoma (HCC), thus the development of USP10 inhibitors holds promise as a novel therapeutic approach for HCC treatment. However, the development of selective USP10 inhibitor is still limited. In this study, we developed a novel USP10 inhibitor for investigating the feasibility of targeting USP10 for the treatment of HCC. Due to high USP10 inhibition potency and prominent selectivity, compound D1 bearing quinolin-4(1 H)-one scaffold was identified as a lead compound. Subsequent research revealed that D1 significantly inhibits cell proliferation and clone formation in HCC cells. Mechanistic insights indicated that D1 targets the ubiquitin pathway, facilitating the degradation of YAP (Yes-associated protein), thereby triggering the downregulation of p53 and its downstream protein p21. Ultimately, this cascade leads to S-phase arrest in HCC cells, followed by cell apoptosis. Collectively, our findings highlight D1 as a promising starting point for USP10-positive HCC treatment, underscoring its potential as a vital tool for unraveling the functional intricacies of USP10. [Display omitted] • A series of quinolin-4(1 H)-one derivatives targeting USP10 was identified through protein structure prediction and virtual screening. • Compound D1 exhibited strong USP10 inhibition and promising selectivity against USP7. • Compound D1 could promote YAP degradation and inhibit the proliferation of HCC cells. [ABSTRACT FROM AUTHOR]

Four undescribed amide alkaloids hongkongensines A-C and 1-(1-oxo-6-hydroxy-2 E ,4 E -dodecadienyl)-piperidine, five known amide alkaloids, and three known neolignans were isolated from the aerial part of Piper hongkongense. The planar structures of these compounds were determined by detailed analyses of HR-ESI-MS and NMR data. The absolute configurations of hongkongensines A-C were elucidated by single-crystal X-ray diffraction analysis and ECD calculations. Moreover, the inhibitory activities of PCSK9 expression in vitro for all compounds were assessed by PCSK9 AlphaLISA screening. Kadsurenone (10) displayed a significant inhibitory activity at 5 μM with an inhibition rate of 51.98%, compared with 55.55% of berberine (BBR 5 μM). [Display omitted] • Four undescribed amide alkaloids were isolated from Piper hongkongense. • Their absolute configurations were elucidated by single-crystal X-ray diffraction and ECD calculations. • Hongkongensine C (3) is a chlorine-containing natural product. • Kadsurenone (10) displayed a significant inhibitory activity of PCSK9 expression. [ABSTRACT FROM AUTHOR]

SERPIN (ser ine p roteinase in hibitors) is an acronym for the superfamily of structurally similar proteins found in animals, plants, bacteria, viruses, and archaea. Over 1500 SERPINs are known in nature, while only 37 SERPINs are found in humans, which participate in inflammation, coagulation, angiogenesis, cell viability, and other pathophysiological processes. Both qualitative or quantitative deficiencies or overexpression and/or abnormal accumulation of SERPIN can lead to diseases commonly referred to as "serpinopathies". Hence, strategies involving SERPIN supplementation, elimination, or correction are utilized and/or under consideration. In this review, we discuss relationships between certain SERPINs and diseases as well as putative strategies for the clinical explorations of SERPINs. [Display omitted] • Serpin is a merge of ser from serine protease and pin from protease inhibitor. • Serpins are extracellular or intracellular, and inhibitors or non-inhibitors. • Loss or gain of serpin function leads to pathologies- "serpinopathies". • Serpins have diagnostic and therapeutic value. [ABSTRACT FROM AUTHOR]

Tomato leaves are by-products of tomato production and a potential source of proteins. Part of these proteins are proteases that carry out important functions for the plants. These endogenous proteases can bring challenges in downstream protein extraction, and can alter protein functionality. The aim of this study was to characterize the endogenous proteases in tomato leaf juice and to investigate the hydrolysis by these proteases on both endogenous leaf proteins and WPI. In addition, we characterized the gelation behavior, rheological properties and structural and thermal properties change of WPI by the juice proteases. 38 different proteases were identified in the juice, along with 4 protease inhibitors. The proteases hydrolyzed both endogenous leaf proteins and WPI, with predominantly an endopeptidase manner for the latter. The protein hydrolysis was positively affected by the incubation time and temperature. The protease activity in juice could not be fully eliminated by heat deactivation or the addition of protease inhibitors. The protein hydrolysis however remained limited (max. 43 %) under all conditions below 60 °C. This hydrolysis resulted in small changes in both secondary structure and thermal properties of WPI. Limited hydrolysis led to WPI gelation at lower protein concentration. Gel formed with WPI after limited hydrolysis had higher gel strength and more brittle structure than the gel with intact proteins. To conclude, the complex and active nature of proteases in tomato leaves poses challenge for downstream protein extraction. Alternatively, juice from tomato leaves could be used as natural gelling agent for WPI. [Display omitted] • Tomato leaves contain a mixture of active proteases. • The proteases were not fully inhibited by heat or addition of protease inhibitors. • Limited hydrolysis resulted in WPI gelation at lower protein concentration. • Gel formed with limited protein hydrolysis was firmer and more brittle. • Tomato leaf juice could be used as natural gelling agent for WPI. [ABSTRACT FROM AUTHOR]

Flaviviruses can cause severe illness in humans. Effective and safe vaccines are available for some species; however, for many flaviviruses disease prevention or specific treatments remain unavailable. The viral replication cycle depends on the proteolytic activity of the NS2B-NS3 protease, which releases functional viral proteins from a non-functional polyprotein precursor, rendering the protease a promising drug target. In this study, we characterised recombinant NS2B-NS3 proteases from ten flaviviruses including three unreported proteases from the Usutu, Kyasanur forest disease and Powassan viruses. All protease constructs comprise a covalent Gly 4 -Ser-Gly 4 linker connecting the NS3 serine protease domain with its cofactor NS2B. We conducted a comprehensive cleavage site analysis revealing areas of high conversion. While all proteases were active in enzymatic assays, we noted a 1000-fold difference in catalytic efficiency across proteases from different flaviviruses. Two bicyclic peptide inhibitors displayed anti-pan-flaviviral protease activity with inhibition constants ranging from 10 to 1000 nM. [Display omitted] • Comprehensive analysis of NS2B-NS3 proteases from ten different flaviviruses. • First disclosure of proteases from Usutu, Kyasanur forest disease, and Powassan viruses. • Robust protocol for the rapid expression of proteases from emerging flaviviruses. • Catalytic efficiency ranges over three orders of magnitude among different flaviviruses. • Development of pan-flavivirus inhibitors with nanomolar activity. [ABSTRACT FROM AUTHOR]