A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

Comparison of two techniques for a comprehensive gut histopathological analysis: Swiss Roll versus Intestine Strips.

Assessing the gut mucosa milieu is important to grade the inflammatory process in conditions such as food hypersensitivity, allergy, gut parasitosis, etc. However, the gastrointestinal tract comprises a challenging system to evaluate, due to its thin tubular structure and mucosa, which suffer fast autolysis after death. Irrespective of the preferred inflammatory score system, it is important to choose the technique that will render the best tissue analysis. Thus, our aim was to compare two of the most frequently used methods to collect, process and analyze gut segments, the Swiss Roll and the Intestinal Strips. Normal C57Bl/6 mice were randomly assigned to Rolls or Strips group. After an overdose of anesthetics, segments of the duodenum, jejunum and ileum were collected and prepared accordingly for histological processing and analysis. Our results show the villi in the Rolls tend to be shorter and wider than those in the Strips in the duodenum and jejunum but not the ileum. No significant differences were observed in intra-epithelial lymphocytes and goblet cells counts. Finally, we staged each segment using our histomorphometric classification system, which revealed that although all animals presented a normal intestinal mucosa, those assigned to the Rolls group had their mucosa staged in the Infiltrative Stage while the Strips group had their mucosa staged as Normal. In conclusion, Swiss Rolls might be desirable for a wider assessment of the intestine, as it allows large segments to be analyzed at once, while Strips are better suited when detailed evaluation of each villus is intended.

Marine Diterpenes: Molecular Modeling of Thrombin Inhibitors with Potential Biotechnological Application as an Antithrombotic.

Thrombosis related diseases are among the main causes of death and incapacity in the world. Despite the existence of antithrombotic agents available for therapy, they still present adverse effects like hemorrhagic risks which justify the search for new options. Recently, pachydictyol A, isopachydictyol A, and dichotomanol, three diterpenes isolated from Brazilian marine brown alga Dictyota menstrualis were identified as potent antithrombotic molecules through inhibition of thrombin, a key enzyme of coagulation cascade and a platelet agonist. Due to the biotechnological potential of these marine metabolites, in this work we evaluated their binding mode to thrombin in silico and identified structural features related to the activity in order to characterize their molecular mechanism. According to our theoretical studies including structure-activity relationship and molecular docking analysis, the highest dipole moment, polar surface area, and lowest electronic density of dichotomanol are probably involved in its higher inhibition percentage towards thrombin catalytic activity compared to pachydictyol A and isopachydictyol A. Interestingly, the molecular docking studies also revealed a good shape complementarity of pachydictyol A and isopachydictyol A and interactions with important residues and regions (e.g., H57, S195, W215, G216, and loop-60), which probably justify their thrombin inhibitor effects demonstrated in vitro. Finally, this study explored the structural features and binding mode of these three diterpenes in thrombin which reinforced their potential to be further explored and may help in the design of new antithrombotic agents.

New approaches in tail-bleeding assay in mice: improving an important method for designing new anti-thrombotic agents.

This report describes a modified, simple, low-cost and more sensitive method to determine bleeding patterns and haemoglobin concentration in a tail-bleeding assay using BALB/c mice and tail tip amputation. The cut tail was immersed in Drabkin's reagent to promote erythrocyte lysis and haemoglobin release, which was monitored over 30 min. The operator was blinded to individual conditions of the mice, which were treated with either saline (NaCl 0.15m), DMSO (0.5%) or clinical anti-thrombotic drugs. Our experimental protocols showed good reproducibility and repeatability of results when using Drabkin's reagent than water. Thus, the use of Drabkin's reagent offered a simple and low-cost method to observe and quantify the bleeding and rebleeding episodes. We also observed the bleeding pattern and total haemoglobin loss using untreated animals or those under anti-coagulant therapy in order to validate the new Drabkin method and thus confirm that it is a useful protocol to quantify haemoglobin concentrations in tail-bleeding assay. This modified method provided a more accurate results for bleeding patterns in mice and for identifying new anti-thrombotic drugs.

Antiplatelet pyrazolopyridines derivatives: pharmacological, biochemical and toxicological characterization.

Platelet aggregation is one of the main events involved in vascular thrombus formation. Recently, N'-substituted-phenylmethylene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazides were described as antiplatelet derivatives. In this work, we explore the properties of these antiplatelet agents through a series of pharmacological, biochemical and toxicological studies. The antiplatelet activity of each derivative was confirmed as 3a, 3b and 3 h significantly inhibited human platelet aggregation induced by arachidonic acid, with no detectable effect on clotting factors or healthy erythrocytes. Importantly, mice treated with derivative 3a showed a higher survival rate at an in vivo model of pulmonary thromboembolism with a lower bleeding risk in comparison to aspirin. The in silico studies pointed a series of structural parameters related to thromboxane synthase (TXS) inhibition by 3a, which was confirmed by tracking plasma levels of PGE2 and TXB2 through an in vitro enzyme immunoassay. Derivative 3a showed selective TXS inhibition allied with low bleeding risk and increased animal survival, revealing the derivative as a promising candidate for treatment of cardiovascular diseases.

Synthesis and antiplatelet activity of antithrombotic thiourea compounds: biological and structure-activity relationship studies.

The incidence of hematological disorders has increased steadily in Western countries despite the advances in drug development. The high expression of the multi-resistance protein 4 in patients with transitory aspirin resistance, points to the importance of finding new molecules, including those that are not affected by these proteins. In this work, we describe the synthesis and biological evaluation of a series of N,N'-disubstituted thioureas derivatives using in vitro and in silico approaches. New designed compounds inhibit the arachidonic acid pathway in human platelets. The most active thioureas (compounds 3d, 3i, 3m and 3p) displayed IC50 values ranging from 29 to 84 µM with direct influence over in vitro PGE2 and TXA2 formation. In silico evaluation of these compounds suggests that direct blockage of the tyrosyl-radical at the COX-1 active site is achieved by strong hydrophobic contacts as well as electrostatic interactions. A low toxicity profile of this series was observed through hemolytic, genotoxic and mutagenic assays. The most active thioureas were able to reduce both PGE2 and TXB2 production in human platelets, suggesting a direct inhibition of COX-1. These results reinforce their promising profile as lead antiplatelet agents for further in vivo experimental investigations.

Structural model of haptoglobin and its complex with the anticoagulant ecotin variants: structure-activity relationship study and analysis of interactions.

Recently the literature described the binding of Haptoglobin (HP) with ecotin, a fold-specific serine-proteases inhibitor with an anticoagulant profile and produced by Escherichia coli. In this work, we used some in silico and in vitro techniques to evaluate HP 3D-fold and its interaction with wild-type ecotin and two variants. Our data showed HP models conserved trypsin fold, in agreement to the in vitro immunological recognition of HP by trypsin antibodies. The analysis of the three ecotin-HP complexes using the mutants RR and TSRR/R besides the wild type revealed several hydrogen bonds between HP and ecotin secondary site. These data are in agreement with the in vitro PAGE assays that showed the HP-RR complex in native gel conditions. Interestingly, the ternary complex interactions varied depending on the inhibitor structure and site-directed mutation. The interaction of HP with TSRR/R involved new residues compared to wild type, which infers a binding energy increase caused by the mutation.

Human thromboxane synthase: comparative modeling and docking evaluation with the competitive inhibitors Dazoxiben and Ozagrel.

Thromboxane synthase (TXAS) is a P450 epoxygenase that synthesizes thromboxane A2 (TXA2), a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. This enzyme plays an important role in several human diseases, including myocardial infarction, stroke, septic shock, asthma and cancer. Despite of the increasing interest on developing TXAS inhibitors, the structure and activity of TXAS are still not totally elucidated. In this study, we used a comparative molecular modeling approach to construct a reliable model of TXAS and analyze its interactions with Dazoxiben and Ozagrel, two competitive inhibitors. Our results were compatible with experimental published data, showing feasible cation-π interaction between the iron atom of the heme group of TXAS and the basic nitrogen atom of the imidazolyl group of those inhibitors. In the absence of the experimental structure of thromboxane synthase, this freely available model may be useful for designing new antiplatelet drugs for diseases related with TXA2.

Platelets: still a therapeutical target for haemostatic disorders.

Platelets are cytoplasmatic fragments from bone marrow megakaryocytes present in blood. In this work, we review the basis of platelet mechanisms, their participation in syndromes and in arterial thrombosis, and their potential as a target for designing new antithrombotic agents. The option of new biotechnological sources is also explored.

High-throughput optofluidic screening of single B cells identifies novel cross-reactive antibodies as inhibitors of uPAR with antibody-dependent effector functions.

The urokinase-type plasminogen activator receptor (uPAR) is an essential regulator for cell signaling in tumor cell proliferation, adhesion, and metastasis. The ubiquitous nature of uPAR in many aggressive cancer types makes uPAR an attractive target for immunotherapy. Here, we present a rapid and successful workflow for developing cross-reactive anti-uPAR recombinant antibodies (rAbs) using high-throughput optofluidic screening of single B-cells from human uPAR-immunized mice. A total of 80 human and cynomolgus uPAR cross-reactive plasma cells were identified, and selected mouse VH/VL domains were linked to the trastuzumab (Herceptin®) constant domains for the expression of mouse-human chimeric antibodies. The resulting rAbs were characterized by their tumor-cell recognition, binding activity, and cell adhesion inhibition on triple-negative breast cancer cells. In addition, the rAbs were shown to enact antibody-dependent cellular cytotoxicity (ADCC) in the presence of either human natural killer cells or peripheral blood mononuclear cells, and were evaluated for the potential use of uPAR-targeting antibody-drug conjugates (ADCs). Three lead antibodies (11857, 8163, and 3159) were evaluated for their therapeutic efficacy in vivo and were shown to suppress tumor growth. Finally, the binding epitopes of the lead antibodies were characterized, providing information on their unique binding modes to uPAR. Altogether, the strategy identified unique cross-reactive antibodies with ADCC, ADC, and functional inhibitory effects by targeting cell-surface uPAR, that can be tested in safety studies and serve as potential immunotherapeutics.

Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2.

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC 50 ) values between 0.41 µM and 66 µM. In addition, eight compounds inhibited PLpro with IC 50 ranging from 1.7 µM to 46 µM. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

In Vivo Measurement of Granzyme Proteolysis from Activated Immune Cells with PET.

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects.

Identification of recombinant Fabs for structural and functional characterization of HIV-host factor complexes.

Viral infection and pathogenesis is mediated by host protein-viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-ß-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12-419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 ( 4 ) has an IC 50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC 50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC 50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC 50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

Antibody-Drug Conjugates Targeting the Urokinase Receptor (uPAR) as a Possible Treatment of Aggressive Breast Cancer.

A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily through endocytosis showed efficacy in a mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) of 2G10 were designed and produced bearing tubulin inhibitor payloads ligated through seven different linkers. Aldehyde tag technology was employed for linking, and either one or two tags were inserted into the antibody heavy chain, to produce site-specifically conjugated ADCs with drug-to-antibody ratios of either two or four. Both cleavable and non-cleavable linkers were combined with two different antimitotic toxins-MMAE (monomethylauristatin E) and maytansine. Nine different 2G10 ADCs were produced and tested for their ability to target uPAR in cell-based assays and a mouse model. The anti-uPAR ADC that resulted in tumor regression comprised an MMAE payload with a cathepsin B cleavable linker, 2G10-RED-244-MMAE. This work demonstrates in vitro activity of the 2G10-RED-244-MMAE in TNBC cell lines and validates uPAR as a therapeutic target for TNBC.

In vitro and in vivo analysis of the antithrombotic and toxicological profile of new antiplatelets N-acylhydrazone derivatives and development of nanosystems: determination of novel NAH derivatives antiplatelet and nanotechnological approach.

BACKGROUND: Cardiovascular diseases are the most frequent cause of morbidity and mortality worldwide. Among the most important cardiovascular diseases are atherothrombosis and venous thromboembolism that present platelet aggregation as a key event. Currently, the commercial antiplatelet agents display several undesirable effects, which prompt the search for new compounds with better therapeutic index, more efficient body distribution and mechanism. METHODS: In this work we characterized in vivo and in vitro the antithrombotic and toxicological profiles of novel antiplatelet N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides derivatives also comparing them with aspirin. In addition we also analyzed the stability of the more active compound after encapsulation in PLGA or PCL nanoparticles and the release profile of these new nanosystems. RESULTS: The biological results revealed not only the selective effect against arachidonic acid-induced platelet aggregation mainly for compounds 2c, 2e and 2h but also their in vivo active profile on thromboembolism pulmonary animal model with better survival rates (e.g. 82%) than aspirin (33%). The overall toxicological profile was determined by in vitro (MTT reduction tests, neutral red uptake in kidney VERO cells and hemolysis assays) and in vivo (pulmonary embolism) assays that pointed 2c as the most promising derivative with potential as a lead compound. By using the nanoprecipitation technique 2c was loaded into PLGA and PCL nanoparticles showing controlled release profile over 21days according to our drug release tests. CONCLUSION: According to our results compound 2c is the most interesting derivative for further studies as it showed the best activity and toxicological profile also allowing the nanoencapsulation process. Thus 2c may assist in determining a new potential therapy with favorable pharmacokinetics for treatment of thrombotic disorders.