Protease-inhibitor Complexes Research Articles

Understanding the complex formation of falstatin; an endogenous macromolecular inhibitor of falcipains

Proteolytic activity constitutes a fundamental process essential for the survival of the malaria parasite and is thus highly regulated. Falstatin, a protease inhibitor of Plasmodium falciparum, tightly regulates the activity of cysteine hemoglobinases, falcipain-2 and 3 (FP2, FP3), by inhibiting FP2 through a single surface exposed loop. However, the multimeric nature of falstatin and its interaction with FP2 remained unexplored. Here we report that the N-terminal falstatin region is highly disordered, and needs chaperone activity (heat-shock protein 70, HSP70) for its folding. Protein-protein interaction assays showed a significant interaction between falstatin and HSP70. Further, characterization of the falstatin multimer through a series of biophysical techniques identified the formation of a falstatin decamer, which was extremely thermostable. Computational analysis of the falstatin decamer showed the presence of five falstatin dimers, with each dimer aligned in a head-to-tail orientation. Further, the falstatin C-terminal region was revealed to be primarily involved in the oligomerization process. Stoichiometric analysis of the FP2-falstatin multimer showed the formation of a heterooligomeric complex in a 1:1 ratio, with the participation of ten subunits of each protein. Taken together, our results report a novel protease-inhibitor complex and strengthens our understanding of the regulatory mechanisms of major plasmodium hemoglobinases.

Correlation of Experimental and Calculated Inhibition Constants of Protease Inhibitor Complexes.

Predicting the potency of inhibitors is key to in silico screening of promising synthetic or natural compounds. Here we describe a predictive workflow that provides calculated inhibitory values, which concord well with empirical data. Calculations of the free interaction energy ΔG with the YASARA plugin FoldX were used to derive inhibition constants Ki from PDB coordinates of protease-inhibitor complexes. At the same time, corresponding KD values were obtained from the PRODIGY server. These results correlated well with the experimental values, particularly for serine proteases. In addition, analyses were performed for inhibitory complexes of cysteine and aspartic proteases, as well as of metalloproteases, whereby the PRODIGY data appeared to be more consistent. Based on our analyses, we calculated theoretical Ki values for trypsin with sunflower trypsin inhibitor (SFTI-1) variants, which yielded the more rigid Pro14 variant, with probably higher potency than the wild-type inhibitor. Moreover, a hirudin variant with an Arg1 and Trp3 is a promising basis for novel thrombin inhibitors with high potency. Further examples from antibody interaction and a cancer-related effector-receptor system demonstrate that our approach is applicable to protein interaction studies beyond the protease field.

LRP1 protects against excessive superior mesenteric artery remodeling by modulating angiotensin II-mediated signaling.

Vascular smooth muscle cells (vSMCs) exert a critical role in sensing and maintaining vascular integrity. These cells abundantly express the low-density lipoprotein receptor-related protein 1 (LRP1), a large endocytic signaling receptor that recognizes numerous ligands, including apolipoprotein E-rich lipoproteins, proteases, and protease-inhibitor complexes. We observed the spontaneous formation of aneurysms in the superior mesenteric artery (SMA) of both male and female mice in which LRP1 was genetically deleted in vSMCs (smLRP1-/- mice). Quantitative proteomics revealed elevated abundance of several proteins in smLRP1-/- mice that are known to be induced by angiotensin II-mediated (AngII-mediated) signaling, suggesting that this pathway was dysregulated. Administration of losartan, an AngII type I receptor antagonist, or an angiotensinogen antisense oligonucleotide to reduce plasma angiotensinogen concentrations restored the normal SMA phenotype in smLRP1-/- mice and prevented aneurysm formation. Additionally, using a vascular injury model, we noted excessive vascular remodeling and neointima formation in smLRP1-/- mice that was restored by losartan administration. Together, these findings reveal that LRP1 regulates vascular integrity and remodeling of the SMA by attenuating excessive AngII-mediated signaling.

Anti-Inflammatory Therapy for Acute Coronary Syndromes: Is It Time for a Shift in the Treatment Paradigm?

Anti-Inflammatory Therapy for Acute Coronary Syndromes: Is It Time for a Shift in the Treatment Paradigm?

Production and characterization of mono-PEGylated alpha-1 antitrypsin for augmentation therapy

Alpha-1 antitrypsin (AAT) is an endogenous inhibitor of serine proteases which, in physiological conditions, neutralizes the excess of neutrophil elastase and other serine proteases in tissues and especially the lungs. Weekly intravenous infusion of plasma-purified human AAT is used to treat AAT deficiency-associated lung disease. However, only 2 % of the AAT dose reach the lungs after intravenous infusion. Inhalation of AAT might offer an alternative route of administration. Yet, the rapid clearance of AAT from the respiratory tract results in high and frequent dosing by inhalation and limited efficacy. In the present study, we produced and characterized in vitro a PEGylated version of AAT which could offer a prolonged body residence time and thereby be useful for augmentation therapy by the intravenous and inhalation routes. Two PEGylation reactions – N-terminal and thiol PEGylation – and three polyethylene glycol (PEG) chains – linear 30 kDa, linear 40 kDa and 2-armed 40 kDa – were used. The yields of mono-PEGylated AAT following purification by anion exchange chromatography were 40–50 % for N-terminal PEGylation and 60–70% for thiol PEGylation. The PEG-AAT conjugates preserved the ability to form a protease-inhibitor complex with neutrophil elastase and proteinase 3 as well as the full inhibitory capacity to neutralize neutrophil elastase activity. These results open up interesting prospects for PEGylated AAT to achieve a prolonged half-life and an improved therapeutic efficacy in vivo.

Immunohistochemical evaluation of D2-40, Galectin-3, Maspin and MCM7 expression in palate squamous cell carcinomas.

Squamous cell carcinoma (SCC) is the most frequent cancer in oral cavity and its prognosis has exhibited little improvement in the last decades. Although much less common palate SCCs manifests a higher local aggression invading very quickly the adjacent muscles and jawbones, thus being able frequently to lead to dysfunctions in chewing, swallowing, and speech. To elucidate what underlies such local aggression, we investigated the immunohistochemical expression in palate SCCs of Podoplanin (D2-40), Galectin-3 (Gal-3), mammary serine protease inhibitor (Maspin) and minichromosome maintenance complex component 7 (MCM7), markers that are known to be involved in tumor invasiveness. We found a progressive increase in reactivity for D2-40 and MCM7 from the normal epithelium toward dysplastic epithelium and respectively to SCC, which suggests the intervention of these markers in the early stages of squamous cell carcinogenesis in the palate. The highest D2-40, Gal-3 and MCM7 reactivity was observed in basaloid and in poorly differentiated (G3) palate SCCs, while for Maspin the well-differentiated (G1) palate SCCs were the most reactive. The first three markers mentioned above were most intensely expressed at the invasion front, while the Maspin reactivity was low or absent at this level. Statistically, we found significant stratification on localization, grading, muscle invasion, and survival for all investigated markers, but with very high direct correlations between D2-40, Gal-3, and MCM7 immunoreactive score (IRS) values, while between the Maspin and each of the previous markers there were very high inverse correlations. Overall, all these investigate markers proved to be responsible for the local invasiveness and regional lymph node metastasis, thus allowing a prognostic and therapeutic stratification of patients with palate SCCs.

Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus.

ABSTRACTOver the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro; also known as 3CLpro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents.IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2-Priming Protease TMPRSS2.

Background:Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry.Methods:We developed a cell-based assay to identify TMPRSS2 inhibitors. Inhibitory activity was established in SARS-CoV-2 viral load systems.Results:We identified the human extracellular serine protease inhibitor (serpin) alpha 1 anti-trypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity.Conclusions:Our data support the key role of extracellular serine proteases in SARS CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells.

Studies on Functional Molecules Based on Peptide Chemistry

Studies on functional molecules starting from syntheses of cysteine-containing peptides and protein are described. Starting from evaluation of a cysteine specific side-reaction, a specific reaction for disulfide-bond formation was developed. The reaction made it possible to independently construct a disulfide bridge without effecting the existing disulfide bonds, which resulted in a unique approach for the synthesis of human insulin by site-specific disulfide bond formation. In a series of studies on sulfur-containing amino acids, another cysteine related un-natural amino acid, α-methyl cysteine, was used for the total syntheses of natural products containing a unique thiazorine/thiazole ring system. Chloroimidazolidium coupling reagent developed by us was effective for the successive couplings of the α-methyl cysteine residues. Based on these synthetic studies, design and evaluation of protease inhibitors were then studied, since a stereo-specific synthesis of the key structure is crucial to make the inhibitor an effective functional molecule in the interactions with its target protease. As the target proteases, β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and chymotrypsin-like protease of severe acute respiratory syndrome (SARS 3CL protease) were selected: the former is a crucial enzyme for amyloid β production and the latter is an essential enzyme for the re-construction of SARS corona virus in host cells. Structure optimization procedure of the respective inhibitors are described based on X-ray crystal structure analyses of the inhibitor-protease complex.

DEELIG: A Deep Learning Approach to Predict Protein-Ligand Binding Affinity

Protein-ligand binding prediction has extensive biological significance. Binding affinity helps in understanding the degree of protein-ligand interactions and is a useful measure in drug design. Protein-ligand docking using virtual screening and molecular dynamic simulations are required to predict the binding affinity of a ligand to its cognate receptor. Performing such analyses to cover the entire chemical space of small molecules requires intense computational power. Recent developments using deep learning have enabled us to make sense of massive amounts of complex data sets where the ability of the model to “learn” intrinsic patterns in a complex plane of data is the strength of the approach. Here, we have incorporated convolutional neural networks to find spatial relationships among data to help us predict affinity of binding of proteins in whole superfamilies toward a diverse set of ligands without the need of a docked pose or complex as user input. The models were trained and validated using a stringent methodology for feature extraction. Our model performs better in comparison to some existing methods used widely and is suitable for predictions on high-resolution protein crystal (⩽2.5 Å) and nonpeptide ligand as individual inputs. Our approach to network construction and training on protein-ligand data set prepared in-house has yielded significant insights. We have also tested DEELIG on few COVID-19 main protease-inhibitor complexes relevant to the current public health scenario. DEELIG-based predictions can be incorporated in existing databases including RSCB PDB, PDBMoad, and PDBbind in filling missing binding affinity data for protein-ligand complexes.

Structure-Based Macrocyclization of Substrate Analogue NS2B-NS3 Protease Inhibitors of Zika, West Nile and Dengue viruses.

A series of cyclic active‐site‐directed inhibitors of the NS2B‐NS3 proteases from Zika (ZIKV), West Nile (WNV), and dengue‐4 (DENV4) viruses has been designed. The most potent compounds contain a reversely incorporated d‐lysine residue in the P1 position. Its side chain is connected to the P2 backbone, its α‐amino group is converted into a guanidine to interact with the conserved Asp129 side chain in the S1 pocket, and its C terminus is connected to the P3 residue via different linker segments. The most potent compounds inhibit the ZIKV protease with K i values <5 nM. Crystal structures of seven ZIKV protease inhibitor complexes were determined to support the inhibitor design. All the cyclic compounds possess high selectivity against trypsin‐like serine proteases and furin‐like proprotein convertases. Both WNV and DENV4 proteases are inhibited less efficiently. Nonetheless, similar structure‐activity relationships were observed for these enzymes, thus suggesting their potential application as pan‐flaviviral protease inhibitors.

Leucoefdin a potential inhibitor against SARS CoV-2 Mpro

Leucoefdin an important constituent of various fruits such as banana, raspberry, etc. was explored to target MPro protease of SARS Co-V 2. Ligand was found to bind at active site of MPro with large negative binding energies in molecular docking and simulation study. The docking results showed that Leucoefdin interacted with the MPro by forming hydrogen bonds, at Leu 141, His163, His 164, and Glu 166. Other non-bonded interactions were seen at Met49, Pro52, Tyr54, Phe140, Leu141, Cys145 and Met165. Results of Leucoefdin was in coherence with the recently reported MPro protease-inhibitor complex. It even displayed better binding energies (kcal/mol) in HTVS (-6.28), SP (-7.28), XP (-9.29) and MMGBSA (-44.71) as compared to the reference ligand [HTVS (-4.87), SP (-6.79), XP (-5.75) and MMGBSA (-47.76)]. Leucoefdin-MPro complex on molecular dynamic simulation showed initial fluctuations in RMSD plot for a certain period and attained equilibrium which remained stable during entire simulation for 150 ns. RMSF of protein showed less secondary structure fluctuations and a greater number of H-bond formation with Leucoefdin during 150 ns simulation. Post simulation MMGBSA analysis showed binding energy of -45.98 Kcal/mol. These findings indicated the potential of Leucoefdin as lead compound in R&D for drug discovery and development against SARS CoV-2. Communicated by Ramaswamy H. Sarma

Selective Modulation of the Protease Activated Protein C Using Exosite Inhibiting Aptamers.

Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities. Nonanticoagulant APC mutants show beneficial effects as cytoprotective agents. To study, if such biased APC signaling can be achieved by APC-binding ligands, the aptamer technology has been used. A G-quadruplex-containing aptamer, G-NB3, has been selected that binds to the basic exosite of APC with a KD of 0.2 nM and shows no binding to APC-related serine proteases or the zymogen protein C. G-NB3 inhibits the inactivation of activated cofactors V and VIII with IC50 values of 11.6 and 13.1 nM, respectively, without inhibiting the cytoprotective and anti-inflammatory functions of APC as tested using a staurosporine-induced apoptosis assay and a vascular barrier protection assay. In addition, G-NB3 prolongs the plasma half-life of APC through inhibition of APC-serine protease inhibitor complex formation. These physicochemical and functional characteristics qualify G-NB3 as a promising therapeutic agent usable to enhance the cytoprotective functions of APC without increasing the risk of APC-related hemorrhage.

Views on Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease by Dai et al. (2020).

The project for the report Dai et al. (2020) [1] seems to be well funded and the authors had access to many modern facilities and techniques. They were able to successfully design, synthesize and evaluate two lead inhibitors of SARS-CoV-2 main protease. Interestingly, they were able to crystallize protease-inhibitor complex to visualize real chemical interaction involved. According to x-ray diffraction images, they found that the aldehyde group of synthesized inhibitors is involved in covalent bond with Mpro active site. We can appreciate this finding by noting that computational modeling approaches like docking studies have difficulties in predicting covalent bonds. The design of these two novel inhibitors may be considered a continuation for several previous attempts of structure-based design of coronavirus main protease inhibitors, for example the well-known peptide inhibitor N3. I am only concerned about the interaction between lead inhibitors and water molecules in crystallization images. These water molecules may be active site reserved water or merely the result of crystallization process. It would be interesting to prepare several derivatives of these two lead inhibitors and then establish a structure-activity relationship study. This seems to be crucial for elucidating the structure of core scaffold.

Novel radial distribution function approach in the study of point mutations: the HIV-1 protease case study.

Background: Mutations are one of the engines of evolution. Under constant stress pressure, mutations can lead to the emergence of unwanted, drug-resistant entities. Methodology: The radial distribution function weighted by the number of valence shell electrons is used to design quantitative structure-activity relationship (QSAR)model relating descriptors with the inhibition constant for a series of wild-type HIV-1 protease inhibitor complexes. The residuals of complexes with mutant HIV-1 protease were correlated with the energy of the highest occupied molecular orbitals of the residues introduced to enzyme via point mutations. Conclusion: Successful identification of residues Ile3, Asp25, Val32 and Ile50 as the one whose substitution influences the inhibition constant the most, demonstrates the potential of the proposed methodology for the study of the effects of point mutations.

α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease–inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.

Global Fibrinolysis Analysed By Clofal Method Is Not Associated with Post-Thrombotic Syndrome or Presence of Residual Vein Thrombosis in Patients with DVT in the Chronic Phase

Introduction: Deep venous thrombosis (DVT) is a common disease and post-thrombotic syndrome (PTS) is a complication present in about 20-50% of patients after the thrombotic episode (Kahn, 2016 - Hematology Am Soc Hematol Educ Program). The presence of residual venous thrombosis (RVT) can contribute to DVT recurrence and its role in SPT is controversial (Pradoni et al., 2009 - Annals of Internal Medicine; Cosmi et al., 2010 - Eur J Vasc Endovasc Surg). Fibrinolysis is the mechanism responsible for blood clot dissolution and decreased plasma fibrinolytic potential (DPFP), leads to an increased risk of DVT and appears to be associated with PTS, but not with recurrence(Lisman, 2017 - Semin Thromb Hemost; Meltzer et al., 2008 - PLoS Med). Furthermore, hypercoagulability and hypofibrinolysis synergistically enhance the risk of a first venous event, driven probably by elevated plasma levels of TAFI and PAI-1(Bombardier et al., 2012 - Thromb Res; Lisman, 2017 - Semin Thromb Hemost). Relationship between fibrinolysis and DVT has been focused on plasma levels of proteins or protease-inhibitor complexes, and not on plasma fibrinolytic potential (PFP). CloFAL is a turbidimetric assay that calculates the coagulation index (CI) and fibrinolytic index (FI), and allows the evaluation of global fibrinolysis based on clot lysis, with good analytical sensitivity for hypercoagulability and hypofibrinolysis. Preliminary studies of our group demonstrated decreased median fibrinolytic index in patients with lower limb DVT when compared to controls (98% vs 146%, p = 0.003). With the objective to increase scientific knowledge, we evaluated PFP in DVT patients and its association with PTS, RVT, and PAI-1 levels. Methods: PFP was determined by CloFAL assay and PAI-1 by ELISA method. The reference values of CI and FI were calculated using the median values obtained in plasma samples of 20 healthy subjects, along with the percentiles 25th and 75th (for CI, median was 104%, interquartile range (IQR) = 70-140%; for F, median = 97%; IQR = 80-180%). PTS and RVT were evaluated by Villalta scale and doppler ultrasound (US), respectively. Outpatients with DVT of lower limbs up to two years after the acute episode attended at Hemostasis Clinic of Hemocentro (University of Campinas - UNICAMP, SP Brazil) from 2014 to 2019 were included. Healthy controls were workers from Hemocentro. Exclusion criteria were cancer, anticoagulation, renal or hepatic disease, and infection. Continuous variables among groups were compared with Mann-Whitney test. A multiple regression analysis was performed by to evaluate the effect of age, sex, type of thrombosis (spontaneous or provoked), PAI-1, RVT, time between DVT and blood collection and CI on the observed values of FI. Results: Seventy-eight patients and 42 controls were included. The samples were collected 16 months (range = 1-27) after acute DVT. Forty-three (55%) patients presented PTS and 41 (71%) showed RVT on US. Our results corroborate our previous study, as patients demonstrated a significant increase of CI (p = 0.0009) and decrease of FI (p = 0.0009) (Table 1). However, when patients were analyzed according to the presence of SPT or RVT, no difference was observed. Median PAI-1 levels corrected by BMI were higher in patients compared to controls (8.5 vs 4.8, p&lt; 0.0001). No difference was observed in PAI-1 levels when we compared patients according to SPT and RVT status. When multiple variables were analyzed with a multiple regression analysis, only CI statistically significantly predicted FI (R2= 0.278; p=0.003). Conclusions: Patients with DVT, even in the chronic phase, presented hypofibrinolysis, when compared to controls. We initially found higher PAI-1 levels in patients when compared to controls but with a multivariate analysis, CI was the only variable that predicted FI. Therefore, other factors not included in this study may contribute to the lower FI values observed in our cohort. The presence of RVT or PTS did not induce modifications of global fibrinolysis. Disclosures No relevant conflicts of interest to declare.

HIV-1 Protease Inhibitors Incorporating Stereochemically Defined P2' Ligands To Optimize Hydrogen Bonding in the Substrate Envelope.

A structure-guided design strategy was used to improve the resistance profile of HIV-1 protease inhibitors by optimizing hydrogen bonding and van der Waals interactions with the protease while staying within the substrate envelope. Stereoisomers of 4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties were explored as P2' ligands providing pairs of diastereoisomers epimeric at P2', which exhibited distinct potency profiles depending on the configuration of the hydroxyl group and size of the P1' group. While compounds with the 4-(1-hydroxyethyl)benzene P2' moiety maintained excellent antiviral potency against a panel of multidrug-resistant HIV-1 strains, analogues with the polar 4-(1,2-dihydroxyethyl)benzene moiety were less potent, and only the (R)-epimer incorporating a larger 2-ethylbutyl P1' group showed improved potency. Crystal structures of protease-inhibitor complexes revealed strong hydrogen bonding interactions of both (R)- and (S)-stereoisomers of the hydroxyethyl group with Asp30'. Notably, the (R)-dihydroxyethyl group was involved in a unique pattern of direct hydrogen bonding interactions with the backbone amides of Asp29' and Asp30'. The SAR data and analysis of crystal structures provide insights for optimizing these promising HIV-1 protease inhibitors.

Light scattering determination of the stoichiometry for protease-potato serine protease inhibitor complexes

The interaction between pancreatic proteases and a serine protease inhibitor purified from potato tubers was investigated by chromatography-coupled light scattering measurements. The molar mass distribution in the chromatogram was compared to theoretical values calculated for the different possible combinations of complexes and free components by three different approaches, namely section analyses of the chromatograms, full mass average determination and mass distribution analysis. This revealed that the inhibitor was able to bind trypsin in a 2:1 complex, whereas the data for chymotrypsin clearly showed a limitation to 1:1 complex regardless of the molar ratio in the injected samples. The same experiment carried out with elastase and the potato inhibitor gave only weak indications of complex formation under the conditions used.

Structures of Zika virus NS2B-NS3 protease in complex with peptidomimetic inhibitors

Zika virus NS2B-NS3 protease plays an essential role in viral replication by processing the viral polyprotein into individual proteins. The viral protease is therefore considered as an ideal antiviral drug target. To facilitate the development of protease inhibitors, we report three high-resolution co-crystal structures of bZiPro with peptidomimetic inhibitors composed of a P1-P4 segment and different P1′ residues. Compounds 1 and 2 possess small P1′ groups that are split off by bZiPro, which could be detected by mass spectrometry. On the other hand, the more potent compound 3 contains a bulky P1′ benzylamide structure that is resistant to cleavage by bZiPro, demonstrating that presence of an uncleavable C-terminal cap contributes to a slightly improved inhibitory potency. The N-terminal phenylacetyl residue occupies a position above the P1 side chain and therefore stabilizes a horseshoe-like backbone conformation of the bound inhibitors. The P4 moieties show unique intra- and intermolecular interactions. Our work reports the detailed binding mode interactions of substrate-analogue inhibitors within the S4-S1′ pockets and explains the preference of bZiPro for basic P1-P3 residues. These new structures of protease-inhibitor complexes will guide the design of more effective NS2B-NS3 protease inhibitors with improved potency and bioavailability.