Heterologous expression of a papain-like protease inhibitor (SnuCalCpI17) in the E. coli and its mode of inhibition.

The effect of the Escherichia coli (E. coli) Rosetta (DE3) system on the expression of recombinant papain-like cysteine protease inhibitors (SnuCalCpIs) was evaluated, and the inhibition mode of the expressed inhibitor was determined. SnuCalCpI08 and SnuCalCpI17, which previously had not been expressed in the E. coli BL21 (DE3) system due to rare codons of more than 10%, were successfully expressed in E. coli Rosetta (DE3) since the strain provides tRNAs for six rare codons. Initially, both inhibitors were expressed as inclusion bodies; however, the water solubility of SnuCalCpI17 could be improved by lowering the incubation temperature, reducing the IPTG concentration, and increasing the induction time. In contrast, the other inhibitor could not be solubilized in water. To validate whether the inhibitor was expressed with correct protein folding, a papain inhibition assay was performed with SnuCalCpI17. SnuCalCpI17 showed a half-maximal inhibitory concentration (IC50) of 105.671 ± 9.857 µg/mL and a slow-binding inhibition mode against papain at pH 7.0 with a Kiapp of 75.80 µg/mL. The slow-binding inhibitor has a slow dissociation from the inhibitor-target complex, resulting in a long residence time in vivo, and thus can effectively inhibit the target at doses far below the IC50 of the inhibitor. KEY POINTS: • Propeptide inhibitor (SnuCalCpI17) containing rare codons was expressed in E. coli Rosetta (DE3). • The slow-binding inhibition was shown by plotting the apparent first-order rate constant (kobs). • Protein-protein interaction between SnuCalCpIs and papain was verified by docking simulation.

Depth of the Steroid Core Location Determines the Mode of Na,K-ATPase Inhibition by Cardiotonic Steroids.

Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and -resistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs' steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin's effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).

Mechanistic and biological characterisation of novel N5-substituted paullones targeting the biosynthesis of trypanothione in Leishmania.

Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species.

Assessment of polyphenols in purple and red rice bran: Phenolic profiles, antioxidant activities, and mechanism of inhibition against amylolytic enzymes.

Pigmented Thai rice varieties, including purple (Riceberry) and red (Hommali), are gaining popularity due to their health benefits as a source of polyphenols that may exert a hypoglycemic effect through specific inhibition of amylolytic enzymes. This study determined the free phenolic extract from purple rice bran (PFE) to exhibit notably greater content of phytochemical compounds than did phenolic extracts from red rice bran, whether free (RFE) or bound fractions. This phytochemical content correlated with increased antioxidant activity and strong inhibition capacity against amylolytic enzymes, suppressing the conversion of carbohydrates into glucose. Several polyphenol compounds were identified in pigmented rice bran extracts, including benzoic acid, chlorogenic acid, ferulic acid, apigenin, and rutin; among these, flavonoids exhibited greater effect on inhibition capacity. Mechanistically, PFE was found to act as a competitive and uncompetitive inhibitor of α-amylase and α-glucosidase respectively, while RFE showed respective uncompetitive and competitive inhibitory modes.

In silico and in vitro studies of novel derivatives of tyrosol and raspberry ketone as the mushroom tyrosinase inhibitors.

Tyrosinase is the key enzyme for melanin biosynthesis. Overproduction and deposition of this pigment cause different problems in various industries including agriculture and food. Finding safe tyrosinase inhibitors thus attracts great research interest. The goal of this study is evaluation of inhibitory potencies of some novel synthetic derivatives of tyrosol and raspberry ketone on diphenolase activity of mushroom tyrosinase. The ligands inhibited enzyme activity and compound 4-(2-(4-(hydroxymethyl)-2-methyl-1,3-dioxolan-2-yl)ethyl)phenol (1d) exhibited the most inhibitory potency (77% inhibition, IC50 = 0.32 µmol L-1) via the mixed inhibition mode. This compound was also safe according to the results of in vitro analyses. The enzyme-ligands interactions were theoretically and experimentally investigated using molecular docking and fluorescence quenching approaches, respectively. Modes of quenching and related parameters were also determined and molecular docking data showed that the ligands bind to important sites of the enzyme. These compounds, especially 1d, can be suggested as efficient candidates for further investigations.

Recent advances in ß-lactamase inhibitor chemotypes and inhibition modes.

The effectiveness of ß-lactam antibiotics is increasingly influenced by serine ß-lactamases (SBLs) and metallo-ß-lactamases (MBLs), which can hydrolyze ß-lactam antibiotics. The development of effective ß-lactamase inhibitors is an important direction to extend use of ß-lactam antibiotics. Although six SBL inhibitors have been approved for clinical use, but no MBL inhibitors or MBL/SBL dual-action inhibitors are available so far. Broad-spectrum targeting clinically relevant MBLs and SBLs is currently desirable, while it is not easy to achieve such a purpose owing to structural and mechanistic differences between MBLs and SBLs. In this review, we summarized recent advances of inhibitor chemotypes targeting MBLs and SBLs and their inhibition mechanisms, particularly including lead discovery and structural optimization strategies, with the aim to provide useful information for future efforts to develop new MBL and SBL inhibitors.

Comparative Pharmacokinetics and Pharmacodynamics of a Novel Sodium-Glucose Cotransporter 2 Inhibitor, DWP16001, with Dapagliflozin and Ipragliflozin.

Since sodium-glucose cotransporter 2 (SGLT2) inhibitors reduced blood glucose level by inhibiting renal tubular glucose reabsorption mediated by SGLT2, we aimed to investigate the pharmacokinetics and kidney distribution of DWP16001, a novel SGLT2 inhibitor, and to compare these properties with those of dapagliflozin and ipragliflozin, representative SGLT2 inhibitors. The plasma exposure of DWP16001 was comparable with that of ipragliflozin but higher than that of dapagliflozin. DWP16001 showed the highest kidney distribution among three SGLT2 inhibitors when expressed as an area under curve (AUC) ratio of kidney to plasma (85.0 ± 16.1 for DWP16001, 64.6 ± 31.8 for dapagliflozin and 38.4 ± 5.3 for ipragliflozin). The organic anion transporter-mediated kidney uptake of DWP16001 could be partly attributed to the highest kidney uptake. Additionally, DWP16001 had the lowest half-maximal inhibitory concentration (IC50) to SGLT2, a target transporter (0.8 ± 0.3 nM for DWP16001, 1.6 ± 0.3 nM for dapagliflozin, and 8.9 ± 1.7 nM for ipragliflozin). The inhibition mode of DWP16001 on SGLT2 was reversible and competitive, but the recovery of the SGLT2 inhibition after the removal of SGLT2 inhibitors in CHO cells overexpressing SGLT2 was retained with DWP16001, which is not the case with dapagliflozin and ipragliflozin. In conclusion, selective and competitive SGLT2 inhibition of DWP16001 could potentiate the efficacy of DWP16001 in coordination with the higher kidney distribution and retained SGLT2 inhibition of DWP16001 relative to dapagliflozin and ipragliflozin.

Mode of pancreatic lipase inhibition activity in vitro by some flavonoids and non-flavonoid polyphenols.

Numerous reports have shown plant metabolites as potential inhibitors of pancreatic lipase (PL). The most notable group is plant polyphenols. However, a limited number of reports diagnosed their mode of inhibition delineating conflicting results. To elucidate the kinetic mode of PL inhibition, some selected flavonoid and non-flavonoid polyphenol standards were first screened for their lipase inhibition potency by their half maximal inhibitory concentration (IC50) followed by inhibition kinetic analysis. Of the phenolics tested, only gallic acid (GA) and galloyl moiety containing epicatechin, viz., epigallocatechin (EGC) and epigallocatechin gallate (EGCG) showed, comparative to others, higher PL inhibitions (IC50, 387.2, 237.3, and 391.2µM respectively). Analysis of enzyme inhibition modalities at various substrate concentrations revealed a dose-dependent inhibition of reaction velocity. Inhibitory rates decreased by the order of EGCG>EGC>GA (Ki, 13.29>35.0>44.61µM respectively). The results, when verified by visual inspection of Lineweaver-Burk as well as Dixon plots, showed inhibitions of PL by GA, EGC, and EGCG that were best fit to competitive inhibitions. A role of the galloyl moiety in enzyme-inhibitor binding has been evident from their structural resemblance. Depicting it further, ethyl gallate (EG), showed a similar competitive inhibition, therefore, indicating a galloyl moiety driven competitive inhibition of PL.