Identification of Plakortide E from the Caribbean Sponge Plakortis halichondroides as a Trypanocidal Protease Inhibitor using Bioactivity-Guided Fractionation

In this paper, we report new protease inhibitory activity of plakortide E towards cathepsins and cathepsin-like parasitic proteases. We further report on its anti-parasitic activity against Trypanosoma brucei with an IC50 value of 5 mu M and without cytotoxic effects against J774.1 macrophages at 100 mu M concentration. Plakortide E was isolated from the sponge Plakortis halichondroides using enzyme assay-guided fractionation and identified by NMR spectroscopy and mass spectrometry. Furthermore, enzyme kinetic studies confirmed plakortide E as a non-competitive, slowly-binding, reversible inhibitor of rhodesain.

Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: Inhibition kinetics and docking studies

NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44 μM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7 μM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis…

Discovery of benzimidazole-based Leishmania mexicana cysteine protease CPB2.8ΔCTE inhibitors as potential therapeutics for leishmaniasis

Abstract: Chemotherapy is currently the only effective approach to treat all forms of leishmaniasis. However, its effectiveness is severely limited due to high toxicity, long treatment length, drug resistance, or inadequate mode of administration. As a consequence, there is a need to identify new molecular scaffolds and targets as potential therapeutics for the treatment of this disease. We report a small series of 1,2‐substituted‐1H‐benzo[d]imidazole derivatives (9ad) showing affinity in the submicromolar range (Ki = 0.150.69 μM) toward Leishmania mexicanaCPB2.8ΔCTE, one of the more promising targets for antileishmanial drug design. The compounds confirmed activity in vitro against intrace…

Antiproliferative Properties of a Few Auranofin-Related Gold(I) and Silver(I) Complexes in Leukemia Cells and their Interferences with the Ubiquitin Proteasome System

A group of triethylphosphine gold(I) and silver(I) complexes, structurally related to auranofin, were prepared and investigated as potential anticancer drug candidates. The antiproliferative properties of these metal compounds were assessed against two leukemia cell lines, i.e., CCRF-CEM and its multidrug-resistant counterpart, CEM/ADR5000. Interestingly, potent cytotoxic effects were disclosed for both series of compounds against leukemia cells, with IC50 values generally falling in the low-micromolar range, the gold derivatives being on the whole more effective than the silver analogues. Some initial structure-function relationships were drawn. Subsequently, the ability of the study compo…

Eliciting callus culture for production of hepatoprotective flavonoids and phenolics from Sequoia sempervirens (D. Don Endl)

The aim of our study is to estimate the hepatoprotective effects of the ethanolic extract of the leaves of Sequoia sempervirens by determination of liver biomarkers (ALT, AST, total bilirubin and albumin in serum) and by histopathological examinations using thioacetamide-induced (TAA) liver injury model. Concurrent administration of ethanolic extracts of S. sempervirens leaves improved the alterations in liver morphology where it was a potent protector of the liver. The potential of L-phenylalanine and silver nitrate as chemical elicitors as well as UV radiation as a physical elicitor on flavonoid production in callus culture of S. sempervirens were emphasized. Murashige and Skoog’s medium …

Dipeptidyl Enoates As Potent Rhodesain Inhibitors That Display a Dual Mode of Action

Dipeptidyl enoates were prepared through a high-yielding two-step synthetic route. They have a dipeptidic structure with a 4-oxoenoate moiety as a warhead with multiple reactive sites. Dipeptidyl enoates were screened against rhodesain and human cathepsins B and L, and were found to be potent and selective inhibitors of rhodesain. Among them (S,E)-ethyl 5-((S)-2-{[(benzyloxy)carbonyl]amino}-3-phenylpropanamido)-7-methyl-4-oxooct-2-enoate (6) was the most potent, with an IC50 value of 16.4 nm and kinact/Ki=1.6×106 m−1 s−1 against rhodesain. These dipeptidyl enoates display a reversible mode of inhibition at very low concentrations and an irreversible mode at higher concentrations. Inhibition…

Development of novel 1,4-benzodiazepine-based Michael acceptors as antitrypanosomal agents

Novel 1,4-benzodiazepines, endowed with a Michael acceptor moiety, were designed taking advantage of a computational prediction of their pharmacokinetic parameters. Among all the synthesized derivatives, we identified a new lead compound (i.e., 4a), bearing a vinyl ketone warhead and endowed with a promising antitrypanosomal activity against Trypanosoma brucei brucei (IC50 = 5.29 µM), coupled with a lack of cytotoxicity towards mammalian cells (TC50>100 µM).

Proline-Based Allosteric Inhibitors of Zika and Dengue Virus NS2B/NS3 Proteases

The NS2B/NS3 serine proteases of the Zika and Dengue flaviviruses are attractive targets for the development of antiviral drugs. We report the synthesis and evaluation of a new, proline-based compound class that displays allosteric inhibition of both proteases. The structural features relevant for protease binding and inhibition were determined to establish them as new lead compounds for flaviviral inhibitors. Based on our structure-activity relationship studies, the molecules were further optimized, leading to inhibitors with submicromolar IC50 values and improved lipophilic ligand efficiency. The allosteric binding site in the proteases was probed using mutagenesis and covalent modificati…

Identification of a new series of amides as non-covalent proteasome inhibitors

Proteasome inhibition has emerged as an important therapeutic strategy for the treatment of multiple myeloma (MM) and some forms of lymphoma, with potential application in other types of cancers. 20S proteasome consists of three different catalytic activities known as chymotrypsin-like (ChT-L), trypsin-like (T-L), and, post-glutamyl peptide hydrolyzing (PGPH) or caspase-like (C-L), which are located respectively on the β5, β2, and β1 subunits of each heptameric β rings. Currently a wide number of covalent proteasome inhibitors are reported in literature; however, the less widely investigated non-covalent inhibitors might be a promising alternative to employ in therapy, because of the lack o…

Taspase1: a 'misunderstood' protease with translational cancer relevance

Proteolysis is not only a critical requirement for life, but the executing enzymes also play important roles in numerous pathological conditions, including cancer. Therefore, targeting proteases is clearly relevant for improving cancer patient care. However, to effectively control proteases, a profound knowledge of their mechanistic function as well as their regulation and downstream signalling in health and disease is required. The highly conserved protease Threonine Aspartase1 (Taspase1) is overexpressed in numerous liquid and solid malignancies and was characterized as a 'non-oncogene addiction' protease. Although Taspase1 was shown to cleave various regulatory proteins in humans as well…

The putative PAINs nostotrebin 6 and derivatives from Nostoc sp. inhibit the trypanosomal cysteine protease rhodesain

New aziridine-based inhibitors of cathepsin L-like cysteine proteases with selectivity for the Leishmania cysteine protease LmCPB2.8

Abstract In the present work a series of aziridine-2,3-dicarboxylate inhibitors of papain-like cysteine proteases was designed, synthesized and tested. The compounds displayed selectivity for the parasitic protozoon Leishmania mexicana cathepsin L-like cysteine protease LmCPB2.8. The computational methods of homology modelling and molecular docking predicted some significant differences in the S2 pocket of LmCPB2.8 and cruzain, a related enzyme from Trypanosoma cruzi. Due to the presence of Tyr209 in LmCPB2.8 rather than Glu208 in cruzain sterically demanding, lipophilic ester groups (inhibitor 7d, 9d, 12d and 14d) are predicted to occupy the S2 pocket of the Leishmania protease, but do not…

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

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

Synthesis of C3/C1-Substituted Tetrahydroisoquinolines

A broad biological screening of the natural alkaloid N-methylisosalsoline (2) extracted from Hammada scoparia leaves against a panel of human and parasitic proteases revealed an interesting activity profile of 2 towards human 20S proteasome. This outcome suggests that the 1,2,3,4-tetrahydroisoquinoline skeleton may be exploited as a template for the development of novel anticancer agents. In this article, we report the synthesis and chemical characterization of a new series of isosalsoline-type alkaloids (10-11) with variations at N2 and C3 positions with respect to the natural Compound 2, obtained by a synthetic strategy that involves the Bischler-Napieralski cyclization. The substrate for…

Novel Opportunities for Cathepsin S Inhibitors in Cancer Immunotherapy by Nanocarrier-Mediated Delivery

Cathepsin S (CatS) is a secreted cysteine protease that cleaves certain extracellular matrix proteins, regulates antigen presentation in antigen-presenting cells (APC), and promotes M2-type macrophage and dendritic cell polarization. CatS is overexpressed in many solid cancers, and overall, it appears to promote an immune-suppressive and tumor-promoting microenvironment. While most data suggest that CatS inhibition or knockdown promotes anti-cancer immunity, cell-specific inhibition, especially in myeloid cells, appears to be important for therapeutic efficacy. This makes the design of CatS selective inhibitors and their targeting to tumor-associated M2-type macrophages (TAM) and DC an attr…

Fluorine Scan of Inhibitors of the Cysteine Protease Human Cathepsin L: Dipolar and Quadrupolar Effects in the π-Stacking of Fluorinated Phenyl Rings on Peptide Amide Bonds

The π-stacking of fluorinated benzene rings on protein backbone amide groups was investigated, using a dual approach comprising enzyme-ligand binding studies complemented by high-level quantum chemical calculations. In the experimental study, the phenyl substituent of triazine nitrile inhibitors of human cathepsin L (hCatL), which stacks onto the peptide amide bond Gly67-Gly68 at the entrance of the S3 pocket, was systematically fluorinated, and differences in inhibitory potency were measured in a fluorimetric assay. Binding affinity is influenced by lipophilicity (clog P), the dipole and quadrupole moments of the fluorinated rings, but also by additional interactions of the introduced fluo…

Quantum Chemical-Based Protocol for the Rational Design of Covalent Inhibitors.

We propose a structure-based protocol for the development of customized covalent inhibitors. Starting from a known inhibitor, in the first and second steps appropriate substituents of the warhead are selected on the basis of quantum mechanical (QM) computations and hybrid approaches combining QM with molecular mechanics (QM/MM). In the third step the recognition unit is optimized using docking approaches for the noncovalent complex. These predictions are finally verified by QM/MM or molecular dynamic simulations. The applicability of our approach is successfully demonstrated by the design of reversible covalent vinylsulfone-based inhibitors for rhodesain. The examples show that our approach…

Synthesis and biological evaluation of novel peptidomimetics as rhodesain inhibitors

Novel rhodesain inhibitors were developed by combining an enantiomerically pure 3-bromoisoxazoline warhead with a 1,4-benzodiazepine scaffold as specific recognition moiety. All compounds were proven to inhibit rhodesain with Ki values in the low-micromolar range. Their activity towards rhodesain was found to be coupled to an in vitro antitrypanosomal activity, with IC50 values ranging from the mid-micromolar to a low-micromolar value for the most active rhodesain inhibitor (R,S,S)-3. All compounds showed a good selectivity against the target enzyme since all of them were proven to be poor inhibitors of human cathepsin L. Novel rhodesain inhibitors were developed by combining an enantiomeri…

Modular Solid-Phase Synthesis of Antiprotozoal Barnesin Derivatives

Here, we applied and optimized a solid support (SP)-based Horner-Wadsworth-Emmons reagent to prepare SP-bound vinylogous amino acids. Subsequent SP-based peptide synthesis, global deprotection, and chemical modifications yielded 14 lipodipeptides carrying vinylogous amino acids, including the natural product barnesin A (1). Biological evaluation revealed that several synthesized derivatives show micromolar to nanomolar inhibitory activity against papain-like cysteine proteases, human cathepsin L, and rhodesain.

Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation.

Novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation were developed; some of them possess K(i) values in the micromolar range. We studied the structure-activity relationship of these derivatives and we performed docking studies, which allowed us to find out the key interactions established by the inhibitors with the target enzyme. Biological results indicate that the nature of the P2 and P3 substituents and their binding to the S2/S3 pockets is strictly interdependent.

Anticancer study of heterobimetallic platinum(II)-ruthenium(II) and platinum(II)-rhodium(III) complexes with bridging dithiooxamide ligand

Abstract Three heterobimetallic platinum (II)/ruthenium (II) and platinum (II)/rhodium (III) complexes, A: Pt{S–S2C2(NR)2H}{μ-S2C2(NR)2}-[Ru (p-cymene)Cl], R = isoamyl; B: Pt{S–S2C2(NR)2H}{μ-S2C2(NR)2}[Rh (phpy)2], R = isoamyl; C: [Pt{S–S2C2(NR)2H}{μ-S2C2(NR)2}-[Rh(C5Me5)Cl]], R = benzyl, were prepared from mononuclear complexes 1 and 2, 1: [Pt (H-isoamyl2DTO)2]; 2: [Pt (H-benzyl2DTO)2], DTO = dithiooxamide, by reaction of 1 or 2 with the corresponding chlorido-bridged dimers, [Rh(C5Me5)Cl (μ-Cl)]2, [Ru (p-cymene)Cl (μ-Cl)]2 or [Rh (phpy)2 (μ-Cl)]2, and then evaluated as anticancer agents for the inhibition of the three proteolytic activities of human 20S proteasome, one of the main target …

The discovery of novel antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense which seriously affects human health in Africa. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work herein describes the design and syntheses of novel antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines, with compound 13, the 4-(2-methoxyphenyl)-6-(pyridine-3-yl)pyrimidin-2-amine demonstrating an IC50 value of 0.38 μM and a promising off-target ADME-Tox profile in vitro. In silico molecular target investigations showed rhodesain to be a pu…

Antiprotozoal and cysteine proteases inhibitory activity of dipeptidyl enoates

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k2nd (M−1s−1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC50 values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and ev…

Inhibition of the Cysteine Protease Human Cathepsin L by Triazine Nitriles: Amide⋅⋅⋅Heteroarene π-Stacking Interactions and Chalcogen Bonding in the S3 Pocket.

We report an extensive "heteroarene scan" of triazine nitrile ligands of the cysteine protease human cathepsin L (hCatL) to investigate π-stacking on the peptide amide bond Gly67-Gly68 at the entrance of the S3 pocket. This heteroarene⋅⋅⋅peptide bond stacking was supported by a co-crystal structure of an imidazopyridine ligand with hCatL. Inhibitory constants (Ki ) are strongly influenced by the diverse nature of the heterocycles and specific interactions with the local environment of the S3 pocket. Binding affinities vary by three orders of magnitude. All heteroaromatic ligands feature enhanced binding by comparison with hydrocarbon analogues. Predicted energetic contributions from the ori…

Front Cover: Structure‐Activity Relationships of Benzamides and Isoindolines Designed as SARS‐CoV Protease Inhibitors Effective against SARS‐CoV‐2 (2/2021)

Synthesis and biological evaluation of papain-family cathepsin L-like cysteine protease inhibitors containing a 1,4-benzodiazepine scaffold as antiprotozoal agents.

Novel papain-family cathepsin L-like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure-activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzod…

sj-pdf-1-jla-10.1177_2472630319877374 – Supplemental material for FINDUS: An Open-Source 3D Printable Liquid-Handling Workstation for Laboratory Automation in Life Sciences

Supplemental material, sj-pdf-1-jla-10.1177_2472630319877374 for FINDUS: An Open-Source 3D Printable Liquid-Handling Workstation for Laboratory Automation in Life Sciences by Fabian Barthels, Ulrich Barthels, Marvin Schwickert and Tanja Schirmeister in SLAS Technology

Graphical Workflow System for Modification Calling by Machine Learning of Reverse Transcription Signatures

Modification mapping from cDNA data has become a tremendously important approach in epitranscriptomics. So-called reverse transcription signatures in cDNA contain information on the position and nature of their causative RNA modifications. Data mining of, e.g. Illumina-based high-throughput sequencing data, is therefore fast growing in importance, and the field is still lacking effective tools. Here we present a versatile user-friendly graphical workflow system for modification calling based on machine learning. The workflow commences with a principal module for trimming, mapping, and postprocessing. The latter includes a quantification of mismatch and arrest rates with single-nucleotide re…

New Tetromycin Derivatives with Anti-Trypanosomal and Protease Inhibitory Activities

Four new tetromycin derivatives, tetromycins 1-4 and a previously known one, tetromycin B (5) were isolated from Streptomyces axinellae Pol001(T) cultivated from the Mediterranean sponge Axinella polypoides. Structures were assigned using extensive 1D and 2D NMR spectroscopy as well as HRESIMS analysis. The compounds were tested for antiparasitic activities against Leishmania major and Trypanosoma brucei, and for protease inhibition against several cysteine proteases such as falcipain, rhodesain, cathepsin L, cathepsin B, and viral proteases SARS-CoV M(pro), and PL(pro). The compounds showed antiparasitic activities against T. brucei and time-dependent inhibition of cathepsin L-like proteas…

Peptidyl Vinyl Ketone Irreversible Inhibitors of Rhodesain: Modifications of the P2 Fragment.

In this paper, we report the design, synthesis and biological investigation of a series of peptidyl vinyl ketones obtained by modifying the P2 fragment of previously reported highly potent inhibitors of rhodesain, the main cysteine protease of Trypanosoma brucei rhodesiense. Investigation of the structure-activity relationship led us to identify new rhodesain inhibitors endowed with an improved selectivity profile (a selectivity index of up to 22 000 towards the target enzyme), and/or an improved antitrypanosomal activity in the sub-micromolar range.

Identification of noncovalent proteasome inhibitors with high selectivity for chymotrypsin-like activity by a multistep structure-based virtual screening.

Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors, e.g. carfilzomib, used in cancer therapy. A multistep hierarchical structure-based virtual screening (SBVS) of the 65,375 NCI lead-like compound library led to the identification of two compounds (9 and 28) which noncovalently inhibited the chymotrypsin-like (ChT-L) activity (Ki = 2.18 and 2.12 μM, respectively) with little or no effects on the other two major proteasome proteolytic activities, trypsin-like (T-L) and post-glutamyl peptide hydrolase (PGPH) activities. A subsequent hierarchical similarity search over the full NCI database with the most active tripeptide-based inh…

Dipeptidyl Nitroalkenes as Potent Reversible Inhibitors of Cysteine Proteases Rhodesain and Cruzain.

Dipeptidyl nitroalkenes are potent reversible inhibitors of cysteine proteases. Inhibitor 11 resulted to be the most potent one with Ki values of 0.49 and 0.44 nM against rhodesain and cruzain, respectively. According to enzymatic dilution and dialysis experiments, as well as computational and NMR studies, dipeptidyl nitroalkenes are tightly binding covalent reversible inhibitors. We thank Fundacion Española para la Ciencia y la Tecnología (Fecyt) and Generalitat Valenciana (AICO/2016/32) for financial support. T S. and B.E. thank the DFG (Deutsche Forschungsgemeinschaft) in the framework of the SFB630 for financial support. We thank Universitat Jaume I for technical suppport and funding. U…

Cytotoxicity of Endoperoxides from the Caribbean Sponge Plakortis halichondrioides towards Sensitive and Multidrug-Resistant Leukemia Cells: Acids vs. Esters Activity Evaluation.

The 6-epimer of the plakortide H acid (1), along with the endoperoxides plakortide E (2), plakortin (3), and dihydroplakortin (4) have been isolated from a sample of the Caribbean sponge Plakortis halichondrioides. To perform a comparative study on the cytotoxicity towards the drug-sensitive leukemia CCRF-CEM cell line and its multi-drug resistant subline CEM/ADR5000, the acid of plakortin, namely plakortic acid (5), as well as the esters plakortide E methyl ester (6) and 6-epi-plakortide H (7) were synthesized by hydrolysis and Steglich esterification, respectively. The data obtained showed that the acids (1, 2, 5) exhibited potent cytotoxicity towards both cell lines, whereas the esters s…

Tuning and Predicting Biological Affinity: Aryl Nitriles as Cysteine Protease Inhibitors

A series of aryl nitrile-based ligands were prepared to investigate the effect of their electrophilicity on the affinity against the cysteine proteases rhodesain and human cathepsin L. Density functional theory calculations provided relative reactivities of the nitriles, enabling prediction of their biological affinity and cytotoxicity and a clear structure-activity relationship.

Synthesis, solution behaviour and potential anticancer activity of new trinuclear organometallic palladium(II) complex of S-1-phenylethyl dithiooxamide: Comparison with the trinuclear heterobimetallic platinum(II) analogue

Abstract Addition of H2R2DTO (R = {S}-1-phenylethyl and DTO = dithiooxamide) to the bis(benzonitrile)palladium(II) chloride complex in chloroform afforded the mononuclear Pd(DTO)2·2HCl complex. The complex treated with NaHCO3 for removing of HCl and then reacted with [Pd(ƞ3-allyl)(µ-Cl)]2 for preparation of a new trimetallic organopalladium(II) complex. The molecular structure of the trimetallic complex was determined by X-ray diffraction indicating a planar geometry around each palladium center. Also, variable temperature spectroscopy for this complex was performed in CDCl3 in the range 298–390 K, and simulations of the dynamic spectra were performed using the gNMR program. A comparison be…

Ensemble-based ADME-Tox profiling and virtual screening for the discovery of new inhibitors of the Leishmania mexicana cysteine protease CPB2.8ΔCTE

Abstract: In an effort to identify novel molecular warheads able to inhibit Leishmania mexicana cysteine protease CPB2.8CTE, fused benzo[b]thiophenes and ,'-triketones emerged as covalent inhibitors binding the active site cysteine residue. Enzymatic screening showed a moderate-to-excellent activity (12%-90% inhibition of the target enzyme at 20m). The most promising compounds were selected for further profiling including in vitro cell-based assays and docking studies. Computational data suggest that benzo[b]thiophenes act immediately as non-covalent inhibitors and then as irreversible covalent inhibitors, whereas a reversible covalent mechanism emerged for the 1,3,3'-triketones with a Y-to…

Identification, Characterization and Synthesis of Natural Parasitic Cysteine Protease Inhibitors – More Potent Falcitidin Analogs

ABSTRACTProtease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogs of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C-terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization and absolute st…

The Medicinal Chemistry of Zika Virus

Arthropod-borne viruses, also known as arboviruses, are transmitted by bites of infected mosquito or tick vectors. In this context, the Flavivirus genus is mainly transmitted by mosquitoes from the Aedes genus, being the Ae. africanus, Ae. aegypti, and Ae. Albopictus species are responsible for transmitting the Zika virus (ZIKV). It is a lipid-enveloped virus constitute of an RNA genome, which is translated into a polyprotein encoding three structural proteins {(capsid (C), membrane (M), and envelope (E)} and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Several biological targets have been identified for developing antiviral agents against ZIKV, which could pre…

Drug Synergism: Studies of Combination of RK-52 and Curcumin against Rhodesain of Trypanosoma brucei rhodesiense

Rhodesain is an enzyme essential for the life of Trypanosoma brucei rhodesiense, a parasite causing a rapid-onset form of Human African Trypanosomiasis. RK-52 is a synthetic inhibitor of rhodesain,...

Approaching an experimental electron density model of the biologically active trans ‐epoxysuccinyl amide group—Substituent effects vs. crystal packing

The trans-epoxysuccinyl amide group as a biologically active moiety in cysteine protease inhibitors such as loxistatin acid E64c has been used as a benchmark system for theoretical studies of environmental effects on the electron density of small active ingredients in relation to their biological activity. Here, the synthesis and the electronic properties of the smallest possible active site model compound are reported to close the gap between the unknown experimental electron density of trans-epoxysuccinyl amides and the well-known function of related drugs. Intramolecular substituent effects are separated from intermolecular crystal packing effects on the electron density, which allows us…

Synthesis, X-ray Structure Determination, and Comprehensive Photochemical Characterization of (Trifluoromethyl)diazirine-Containing TRPML1 Ligands

Potential (trifluoromethyl)diazirine-based TRPML1 ion channel ligands were designed and synthesized, and their structures were determined by single-crystal X-ray diffraction analysis. Photoactivation studies via 19F NMR spectroscopy and HPLC-MS analysis revealed distinct kinetical characteristics in selected solvents and favorable photochemical properties in an aqueous buffer. These photoactivatable TRPML activators represent useful and valuable tools for TRPML photoaffinity labeling combined with mass spectrometry.

Evaluation of dipeptide nitriles as inhibitors of rhodesain, a major cysteine protease of Trypanosoma brucei

A series of dipeptide nitriles known as inhibitors of mammalian cathepsins were evaluated for inhibition of rhodesain, the cathepsin L-like protease of Trypanosoma brucei. Compound 35 consisting of a Leu residue fitting into the S2 pocket and a triarylic moiety consisting of thiophene, a 1,2,4-oxadiazole and a phenyl ring fitting into the S3 pocket, and compound 33 with a 3-bromo-Phe residue (S2) and a biphenyl fragment (S3) were found to inhibit rhodesain in the single-digit nanomolar range. The observed steep structure-activity relationship could be explained by covalent docking simulations. With their high selectivity indices (ca. 200) and the good antitrypanosomal activity (8μM) the com…

Structure‐Activity Relationships of Benzamides and Isoindolines Designed as SARS‐CoV Protease Inhibitors Effective against SARS‐CoV‐2

Abstract Inhibition of coronavirus (CoV)‐encoded papain‐like cysteine proteases (PLpro) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure‐activity relationships (SAR) of the noncovalent active‐site directed inhibitor (R)‐5‐amino‐2‐methyl‐N‐(1‐(naphthalen‐1‐yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS‐CoV PLpro. Moreover, we report the discovery of isoindolines as a new class of potent PLpro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS‐CoV‐2 replication in …

Nostotrebin 6 Related Cyclopentenediones and δ-Lactones with Broad Activity Spectrum Isolated from the Cultivation Medium of the Cyanobacterium Nostoc sp. CBT1153

Cyanobacteria are an interesting source of biologically active natural products, especially chemically diverse and potent protease inhibitors. On our search for inhibitors of the trypanosomal cysteine protease rhodesain, we identified the homodimeric cyclopentenedione (CPD) nostotrebin 6 (1) and new related monomeric, dimeric, and higher oligomeric compounds as the active substances in the medium extract of Nostoc sp. CBT1153. The oligomeric compounds are composed of two core monomeric structures, a trisubstituted CPD or a trisubstituted unsaturated δ-lactone. Nostotrebin 6 thus far has been the only known cyanobacterial CPD. It has been found to be active in a broad variety of assays, indi…

Selected cytotoxic gold compounds cause significant inhibition of 20S proteasome catalytic activities

Abstract Six structurally diverse cytotoxic gold compounds are reported to cause profound and differential inhibition of the three main catalytic activities of purified 20S proteasome whilst auranofin , an established gold(I) drug in clinical use, is nearly ineffective. In particular, the gold(I) complex [( pbiH ) Au ( PPh 3 )] PF 6 , turns out to be the most potent inhibitor of all three enzyme activities with sub-micromolar IC 50 values. The present results further support the view that proteasome inhibition may play a major – yet not exclusive – role in the cytotoxic actions of gold based anticancer agents.

Benchmark Study for the Cysteine-Histidine Proton Transfer Reaction in a Protein Environment: Gas Phase, COSMO, QM/MM Approaches.

Proton transfer reactions are of crucial interest for the investigation of proteins. We have investigated the accuracy of commonly used quantum chemical methods for the description of proton transfer reactions in different environments (gas phase, COSMO, QM/MM) using the proton transfer between the catalytic dyad residues cysteine 145 and histidine 41 of SARS coronavirus main protease as a case study. The test includes thermodynamic, kinetic, and structural properties. The study comprises computationally demanding ab initio approaches (HF, CC2, MP2, SCS-CC2, SCS-MP2, CCSD(T)), popular density functional theories (BLYP, B3LYP, M06-2X), and semiempirical methods (MNDO/d, AM1, RM1, PM3, PM6). …

BANΔIT: B’‐factor Analysis for Drug Design and Structural Biology

The analysis of B‐factor profiles from X‐ray protein structures can be utilized for structure‐based drug design since protein mobility changes have been associated with the quality of protein‐ligand interactions. With the BANΔIT (B’‐factor analysis and ΔB’ interpretation toolkit), we have developed a JavaScript‐based browser application that provides a graphical user interface for the normalization and analysis of B’‐factor profiles. To emphasize the usability for rational drug design applications, we have analyzed a selection of crystallographic protein‐ligand complexes and have given exemplary conclusions for further drug optimization including the development of a B’‐factor‐supported pha…

FINDUS: An Open-Source 3D Printable Liquid-Handling Workstation for Laboratory Automation in Life Sciences

3D-printed laboratory devices can enable ambitious research purposes even at a low-budget level. To follow this trend, here we describe the construction, calibration, and usage of the FINDUS (Fully Integrable Noncommercial Dispensing Utility System). We report the successful 3D printing and assembly of a liquid-handling workstation for less than $400. Using this setup, we achieve reliable and flexible liquid-dispensing automation with relative pipetting errors of less than 0.3%. We show our system is well suited for several showcase applications from both the biology and chemistry fields. In support of the open-source spirit, we make all 3D models, assembly instructions, and source code ava…

Metabolomics analysis and biological investigation of three Malvaceae plants

Introduction: Metabolomics is a fast growing technology that has effectively contributed to many plant-related sciences and drug discovery. Objective: To use the non-targeted metabolomics approach to investigate the chemical profiles of three Malvaceae plants, namely Hibiscus mutabilis L. (Changing rose), H. schizopetalus (Dyer) Hook.f. (Coral Hibiscus), and Malvaviscus arboreus Cav. (Sleeping Hibiscus), along with evaluating their antioxidant and anti-infective potential. Methodology: Metabolic profiling was carried out using liquid chromatography coupled with high-resolution electrospray ionisation mass spectrometry (LC-HR-ESI-MS) for dereplication purposes. The chemical composition of th…

Can Experimental Electron-Density Studies be Used as a Tool to Predict Biologically Relevant Properties of Low-Molecular Weight Enzyme Ligands?

The case of protease inhibitor model compounds incorporating an aziridine or epoxide ring is used to exemplify how application of experimental electron-density techniques can be used to explain the biological properties of low-molecular weight enzyme ligands. This is furthermore seen in the light of a comparison of crystal and enzyme environments employing QM/MM computations to elucidate to which extent the properties in the crystal can be used to predict behavior in the biological surrounding.

Cis autocatalytic cleavage of glycine-linked Zika virus NS2B-NS3 protease constructs.

The flaviviral heterodimeric serine protease NS2B-NS3, consisting of the NS3 protease domain and the NS2B co-factor, is essential for ZIKA virus maturation and replication in cells. For in vitro studies a 'linked' construct, where a polyglycine linker connects NS2BCF and NS3pro , is often used. This construct undergoes autocatalytic cleavage. Here, we show that linked ZIKV NS2BCF -NS3pro is cleaved in cis in the NS2BCF exclusively at position R95 and not at the previously proposed alternate cleavage site at residue R29 in the NS3pro . Cleavage neither affects protease stability nor activity, despite some observed differences in spectroscopic behavior. This minimally modified construct may t…

Evaluation of curcumin irreversibility

Dear Editor,We would like to reply to the letter to the Editor of Steverding (2018) on our research article “Drug combination studies of curcumin and genistein against rhodesain of Trypanosoma bruc...

Optimization of peptidomimetic boronates bearing a P3 bicyclic scaffold as proteasome inhibitors

Abstract A new series of pseudopeptide boronate proteasome inhibitors (2–3) was developed, through optimization of our previously described analogs of bortezomib, bearing a bicyclic 1,6-naphthyridin-5(6H)-one scaffold as P3 fragment (1). The biological evaluation on human 20S proteasome displayed a promising inhibition profile, especially for compounds bearing a P2 ethylene fragment, which exhibited Ki values in the nanomolar range for the ChT-L activity (e.g. 2a, Ki = 0.057 μM) and considerable selectivity for proteasome over bovine pancreatic α-chymotrypsin. Docking experiments into the yeast 20S proteasome revealed that the ligands are accommodated predominantly into the ChT-L site and t…

Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors.

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei (T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors (Ki < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC50 < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys–SOH) during crystallization. The P-glycoprotein efflux ratio was mea…

Electrostatic complementarity in pseudoreceptor modeling based on drug molecule crystal structures: the case of loxistatin acid (E64c)

After a long history of use as a prototype cysteine protease inhibitor, the crystal structure of loxistatin acid (E64c) is finally determined experimentally using intense synchrotron radiation, providing insight into how the inherent electronic nature of this protease inhibitor molecule determines its biochemical activity. Based on the striking similarity of its intermolecular interactions with those observed in a biological environment, the electrostatic potential of crystalline E64c is used to map the characteristics of a pseudo-enzyme pocket.

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

Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than …

Structural Determinants for the Mode of Action of Imidazopyridine DS2 at δ-Containing γ-Aminobutyric Acid Type A Receptors

Despite the therapeutic relevance of δ-containing γ-aminobutyric acid type A receptors (GABAARs) and the need for δ-selective compounds, the structural determinants for the mode and molecular site of action of δ-selective positive allosteric modulator imidazo[1,2-a]pyridine DS2 remain elusive. To guide the quest for insight, we synthesized a series of DS2 analogues guided by a structural receptor model. Using a fluorescence-based fluorometric imaging plate reader membrane potential assay, we found that the δ-selectivity and the pharmacological profile are severely affected by substituents in the 5-position of the imidazopyridine core scaffold. Interestingly, the 5-methyl, 5-bromo, and 5-chl…

Bistacrines as potential antitrypanosomal agents

Human African Trypanosomiasis (HAT) is caused by two subspecies of the genus Trypanosoma, namely Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. The disease is fatal if left untreated and therapy is limited due to only five non-adequate drugs currently available. In preliminary studies, dimeric tacrine derivatives were found to inhibit parasite growth with IC50-values in the nanomolar concentration range. This prompted the synthesis of a small, but smart library of monomeric and dimeric tacrine-type compounds and their evaluation of antiprotozoal activity. Rhodesain, a lysosomal cathepsin-L like cysteine protease of T. brucei rhodesiense is essential for parasite survival a…

Optimization of Triazine Nitriles as Rhodesain Inhibitors: Structure-Activity Relationships, Bioisosteric Imidazopyridine Nitriles, and X-ray Crystal Structure Analysis with Human Cathepsin L

The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure-based design and resulted in inhibitors with inhibition constants in the single-digit nanomolar range. Comprehensive structure-activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 subs…

Bistacrine derivatives as new potent antimalarials

Linking two tacrine molecules results in a tremendous increase of activity against Plasmodia in comparison to the monomer. This finding prompted the synthesis of a library of monomeric and dimeric tacrine derivatives in order to derive structure-activity relationships. The most active compounds towards chloroquine sensitive Plasmodium strain 3D7 and chloroquine resistant strain Dd2 show IC50 values in the nanomolar range of concentration, low cytotoxicity and target the cysteine protease falcipain-2, which is essential for parasite growth.

Synthesis and Pharmacological Evaluation of [11C]4-Methoxy-N-[2-(thiophen-2-yl)imidazo[1,2-a]pyridin-3-yl]benzamide as a Brain Penetrant PET Ligand Selective for the δ-Subunit-Containing γ-Aminobutyric Acid Type A Receptors

The α4/6βδ-containing GABAA receptors are involved in a number of brain diseases. Despite the potential of a δ-selective imaging agent, no PET radioligand is currently available for in vivo imaging...

Fluorovinylsulfones and -Sulfonates as Potent Covalent Reversible Inhibitors of the Trypanosomal Cysteine Protease Rhodesain: Structure–Activity Relationship, Inhibition Mechanism, Metabolism, and In Vivo Studies

Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, α-fluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (Ki = 3 nM). Furthermore, the nonfluorinated 2d-(H) shows favorabl…

Development of rhodesain inhibitors with a 3-bromoisoxazoline warhead

Novel rhodesain inhibitors were obtained by combining an enantiomerically pure 3-bromoisoxazoline warhead with a specific peptidomimetic recognition moiety. All derivatives behaved as inhibitors of rhodesain, with low micromolar Ki values. Their activity against the enzyme was found to be paralleled by an in vitro antitrypanosomal activity, with IC50 values in the mid-micromolar range. Notably, a preference for parasitic over human proteases, specifically cathepsins B and L, was observed.

Development of Novel Benzodiazepine-Based Peptidomimetics as Inhibitors of Rhodesain from Trypanosoma brucei rhodesiense.

Starting from the reversible rhodesain inhibitors 1 a-c, which have Ki values towards the target protease in the low-micromolar range, we have designed a series of peptidomimetics, 2 a-g, that contain a benzodiazepine scaffold as a β-turn mimetic; they are characterized by a specific peptide sequence for the inhibition of rhodesain. Considering that irreversible inhibition is strongly desirable in the case of a parasitic target, a vinyl ester moiety acting as Michael-acceptor was introduced as the warhead; this portion was functionalized in order to evaluate the size of corresponding enzyme pocket that could accommodate this substituent. With this investigation, we identified an irreversibl…

Protocol for rational design of covalently interacting inhibitors.

The inhibition potencies of covalent inhibitors mainly result from the formation of a covalent bond to the enzyme during the inhibition mechanism. This class of inhibitors has essentially been ignored in previous target-directed drug discovery projects because of concerns about possible side effects. However, their advantages, such as higher binding energies and longer drug-target residence times moved them into the focus of recent investigations. While the rational design of non-covalent inhibitors became standard the corresponding design of covalent inhibitors is still in its early stages. Potent covalent inhibitors can be retrieved from large compound libraries by covalent docking approa…

Applicability of a single‐use bioreactor compared to a glass bioreactor for the fermentation of filamentous fungi and evaluation of the reproducibility of growth in pellet form

Abstract The implementation of single‐use technologies offers several major advantages, e.g. prevention of cross‐contamination, especially when spore‐forming microorganisms are present. This study investigated the application of a single‐use bioreactor in batch fermentation of filamentous fungus Penicillium sp. (IBWF 040‐09) from the Institute of Biotechnology and Drug Research (IBWF), which is capable of intracellular production of a protease inhibitor against parasitic proteases as a secondary metabolite. Several modifications to the SU bioreactor were suggested in this study to allow the fermentation in which the fungus forms pellets. Simultaneously, fermentations in conventional glass b…

Optimization Strategy of Novel Peptide-Based Michael Acceptors for the Treatment of Human African Trypanosomiasis

This paper describes an optimization strategy of the highly active vinyl ketone 3 which was recognized as a strong inhibitor of rhodesain of Trypanosoma brucei rhodesiense, endowed with a ksecond v...

Vinyl sulfone building blocks in covalently reversible reactions with thiols

In the present study we use quantum-chemical calculations to investigate how the reactivity of vinyl sulfone-based compounds can be modified from an irreversible to a reversible reaction with thiols. Based on the predictions from theory, an array of nine different vinyl sulfones with systematically varying substitution pattern was synthesized and their crystal structures were determined. Subsequent Hirshfeld surface analyses employing the principle of electrostatic complementarity aid the understanding of the crystal packing of the synthesized compounds. Reactivity studies against the nucleophile 2-phenylethanethiol mirror the properties predicted by the quantum-chemical computations in sol…

Screening of cyanobacteria extracts for inhibitory activity against the cysteine protease rhodesain of Trypanosoma brucei

SAR of novel benzothiazoles targeting an allosteric pocket of DENV and ZIKV NS2B/NS3 proteases

In recent years, dengue virus (DENV) and Zika virus (ZIKV), both mosquito-borne members of the Flaviviridae family, have emerged as intercontinental health issues since their vectors have spread from their tropical origins to temperate climate zones due to climate change and increasing globalization. DENV and ZIKV are positive-sense, single-stranded RNA viruses, whose genomes consist of three structural (capsid, membrane precursor, envelope) and seven non-structural (NS) proteins, all of which are initially expressed as a single precursor polyprotein. For virus maturation, the polyprotein processing is accomplished by host proteases and the viral NS2B/NS3 protease complex, whose inhibitors …

sj-pdf-1-jla-10.1177_2472630319877374 – Supplemental material for FINDUS: An Open-Source 3D Printable Liquid-Handling Workstation for Laboratory Automation in Life Sciences

Supplemental material, sj-pdf-1-jla-10.1177_2472630319877374 for FINDUS: An Open-Source 3D Printable Liquid-Handling Workstation for Laboratory Automation in Life Sciences by Fabian Barthels, Ulrich Barthels, Marvin Schwickert and Tanja Schirmeister in SLAS Technology

Evidence for substrate binding-induced zwitterion formation in the catalytic Cys-His dyad of the SARS-CoV main protease.

The coronavirus main protease (M(pro)) represents an attractive drug target for antiviral therapy of coronavirus (CoV) infections, including severe acute respiratory syndrome (SARS). The SARS-CoV M(pro) and related CoV proteases have several distinct features, such as an uncharged Cys-His catalytic dyad embedded in a chymotrypsin-like protease fold, that clearly separate these enzymes from archetypical cysteine proteases. To further characterize the catalytic system of CoV main proteases and to obtain information about improved inhibitors, we performed comprehensive simulations of the proton-transfer reactions in the SARS-CoV M(pro) active site that lead to the Cys(-)/His(+) zwitterionic st…

Targeting of the Leishmania Mexicana cysteine protease CPB2.8 ΔCTE by decorated fused benzo[b] thiophene scaffold.

A potent and highly selective anhydride-based inhibitor of Leishmania mexicana cysteine protease CPB2.8ΔCTE (IC50 = 3.7 μM) was identified. The details of the interaction of the ligand with the enzyme active site were investigated by NMR biomimetic experiments and docking studies. Results of inhibition assays, NMR and theoretical studies indicate that the ligand acts initially as a non-covalent inhibitor and later as an irreversible covalent inhibitor by chemoselective attack of CYS 25 thiolate to an anhydride carbonyl.

Development of a New Antileishmanial Aziridine-2,3-Dicarboxylate-Based Inhibitor with High Selectivity for Parasite Cysteine Proteases

ABSTRACT Leishmaniasis is one of the major neglected tropical diseases of the world. Druggable targets are the parasite cysteine proteases (CPs) of clan CA, family C1 (CAC1). In previous studies, we identified two peptidomimetic compounds, the aziridine-2,3-dicarboxylate compounds 13b and 13e, in a series of inhibitors of the cathepsin L (CL) subfamily of the papain clan CAC1. Both displayed antileishmanial activity in vitro while not showing cytotoxicity against host cells. In further investigations, the mode of action was characterized in Leishmania major . It was demonstrated that aziridines 13b and 13e mainly inhibited the parasitic cathepsin B (CB)-like CPC enzyme and, additionally, ma…

Biochemistry and medicinal chemistry of the dengue virus protease.

The Significance of Ionic Bonding in Sulfur Dioxide: Bond Orders from X-ray Diffraction Data

A novel refinement technique for X‐ray diffraction data has been employed to derive S-O bond orders in sulfur dioxide experimentally. The results show that ionic S-O bonding dominates over hypervalency.

Cathepsin B in Antigen-Presenting Cells Controls Mediators of the Th1 Immune Response during Leishmania major Infection

Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb) and L (Ctsl) play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrop…

Interfering with Host Proteases in SARS-CoV-2 Entry as a Promising Therapeutic Strategy

Abstract: Due to its fast international spread and substantial mortality, the coronavirus disease COVID-19 evolved to a global threat. Since there is currently no causative drug against this viral infection available, science is striving for new drugs and other approaches to treat the new disease. Studies have shown that the cell entry of coronaviruses into host cells takes place through the binding of the viral spike (S) protein to cell receptors. Priming of the S protein occurs via hydrolysis by different host proteases. The inhibition of these proteases could impair the processing of the S protein, thereby affecting the interaction with the host-cell receptors and preventing virus cell …

Synthesis and Molecular Modeling Studies of Derivatives of a Highly Potent Peptidomimetic Vinyl Ester as Falcipain-2 Inhibitors

Herein we report the synthesis of a set of constrained peptidomimetics endowed with an electrophilic vinyl ester warhead and structurally related to a previously identified lead compound, a potent and irreversible inhibitor of falcipain-2 (FP-2). FP-2 is the main hemoglobinase of the malaria parasite P. falciparum. The new compounds were evaluated for their inhibition against FP-2, and the results were rationalized on the basis of docking experiments. These studies underscore the pivotal role of both the ester function at the P1' site and the trifluoromethyl group of the P3 side chain in determining the correct orientation of the Michael acceptor warhead in the catalytic site, and as a cons…

Development of Novel Peptide-Based Michael Acceptors Targeting Rhodesain and Falcipain-2 for the Treatment of Neglected Tropical Diseases (NTDs)

This paper describes the development of a class of peptide-based inhibitors as novel antitrypanosomal and antimalarial agents. The inhibitors are based on a characteristic peptide sequence for the inhibition of the cysteine proteases rhodesain of Trypanosoma brucei rhodesiense and falcipain-2 of Plasmodium falciparum. We exploited the reactivity of novel unsaturated electrophilic functions such as vinyl-sulfones, -ketones, -esters, and -nitriles. The Michael acceptors inhibited both rhodesain and falcipain-2, at nanomolar and micromolar levels, respectively. In particular, the vinyl ketone 3b has emerged as a potent rhodesain inhibitor (k2nd = 67 × 106 M-1 min-1), endowed with a picomolar b…

Structure of the Human TRPML2 Ion Channel Extracytosolic/Lumenal Domain.

Summary TRPML2 is the least structurally characterized mammalian transient receptor potential mucolipin ion channel. The TRPML family hallmark is a large extracytosolic/lumenal domain (ELD) between transmembrane helices S1 and S2. We present crystal structures of the tetrameric human TRPML2 ELD at pH 6.5 (2.0 A) and 4.5 (2.95 A), corresponding to the pH values in recycling endosomes and lysosomes. Isothermal titration calorimetry shows Ca2+ binding to the highly acidic central pre-pore loop which is abrogated at low pH, in line with a pH-dependent channel regulation model. Small angle X-ray scattering confirms the ELD dimensions in solution. Changes in pH or Ca2+ concentration do not affect…

Lead Discovery of SARS-CoV-2 Main Protease Inhibitors through Covalent Docking-Based Virtual Screening

During almost all 2020, coronavirus disease 2019 (COVID-19) pandemic has constituted the major risk for the worldwide health and economy, propelling unprecedented efforts to discover drugs for its prevention and cure. At the end of the year, these efforts have culminated with the approval of vaccines by the American Food and Drug Administration (FDA) and the European Medicines Agency (EMA) giving new hope for the future. On the other hand, clinical data underscore the urgent need for effective drugs to treat COVID-19 patients. In this work, we embarked on a virtual screening campaign against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro chymotrypsin-like cysteine pro…

GNPS-guided discovery of xylacremolide C and D, evaluation of their putative biosynthetic origin and bioactivity studies of xylacremolide A and B

Targeted HRMS2-GNPS-based metabolomic analysis of Pseudoxylaria sp. X187, a fungal antagonist of the fungus-growing termite symbiosis, resulted in the identification of two lipopeptidic congeners of xylacremolides, named xylacremolide C and D, which are built from d-phenylalanine, l-proline and an acetyl-CoA starter unit elongated by four malonyl-CoA derived ketide units. The putative xya gene cluster was identified from a draft genome generated by Illumina and PacBio sequencing and RNAseq studies. Biological activities of xylacremolide A and B were evaluated and revealed weak histone deacetylase inhibitory (HDACi) and antifungal activities, as well as moderate protease inhibition activity …

A Hyaluronic acid-pentamidine bioconjugate as macrophage mediated drug targeting delivery system for the treatment of Leishmaniasis

Leishmaniasis is still a serious public health problem worldwide, especially in tropical areas where this infectious disease is endemic. The most severe form of the disease (i.e. visceral) can claim victims if left untreated and the few accessible drugs have several drawbacks including major side effects and parenteral administration. In this context, the investigation of new delivery modalities which might reduce the toxicity and increase the bioavailability of the drugs currently on the market represents a valid strategy to counter these problems. Herein we present the development of a macrophage mediated drug targeting delivery system by conjugating the anti-leishmanial drug pentamidine …

Conformational Dynamics of the Dengue Virus Protease Revealed by Fluorescence Correlation and Single-Molecule FRET Studies.

The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET …

Similarities and differences between crystal and enzyme environmental effects on the electron density of drug molecules

Abstract The crystal interaction density is generally assumed to be a suitable measure of the polarization of a low‐molecular weight ligand inside an enzyme, but this approximation has seldomly been tested and has never been quantified before. In this study, we compare the crystal interaction density and the interaction electrostatic potential for a model compound of loxistatin acid (E64c) with those inside cathepsin B, in solution, and in vacuum. We apply QM/MM calculations and experimental quantum crystallography to show that the crystal interaction density is indeed very similar to the enzyme interaction density. Less than 0.1 e are shifted between these two environments in total. Howeve…

Die Bedeutung ionischer Bindungsanteile in Schwefeldioxid - Bindungsordnungen aus Röntgenbeugungsdaten

Development of Novel Selective Peptidomimetics Containing a Boronic Acid Moiety, Targeting the 20S Proteasome as Anticancer Agents

This paper describes the design, synthesis, and biological evaluation of peptidomimetic boronates as inhibitors of the 20S proteasome, a validated target in the treatment of multiple myeloma. The synthesized compounds showed a good inhibitory profile against the ChT-L activity of 20S proteasome. Compounds bearing a β-alanine residue at the P2 position were the most active, that is, 3-ethylphenylamino and 4-methoxyphenylamino (R)-1-{3-[4-(substituted)-2-oxopyridin-1(2H)-yl]propanamido}-3-methylbutylboronic acids (3 c and 3 d, respectively), and these derivatives showed inhibition constants (Ki ) of 17 and 20 nM, respectively. In addition, they co-inhibited post glutamyl peptide hydrolase act…

Natural products as inhibitors of recombinant cathepsin L of Leishmania mexicana.

Cysteine proteinases (cathepsins) from Leishmania spp. are promising molecular targets against leishmaniasis. Leishmania mexicana cathepsin L is essential in the parasite life cycle and a pivotal in virulence factor in mammals. Natural products that have been shown to display antileishmanial activity were screened as part of our ongoing efforts to design inhibitors against the L. mexicana cathepsin L-like rCPB2.8. Among them, agathisflavone (1), tetrahydrorobustaflavone (2), 3-oxo-urs-12-en-28-oic acid (3), and quercetin (4) showed significant inhibitory activity on rCPB2.8 with IC50 values ranging from 0.43 to 18.03 µM. The mechanisms of inhibition for compounds 1–3, which showed Ki values…

Ruthenium(II) and platinum(II) homo- and heterobimetallic complexes: Synthesis, crystal structures, theoretical calculations and biological studies

Four Ru-Pd heterobimetallic complexes, each one in two different coordination modes (NNSS and NS) having metals connected by a binucleating dialkyldithiooxamidate [N(R)SC-CS(R)N] [R = methyl, ethyl, n-butyl and isopropyl], were prepared by reacting the monochelate [(trinpropyl-phosphine)ClPd(HR2C2N2S2κ-S,S-Pd)] with [(η6-p-cymene)RuCl2]2. Furthermore, two palladium homobimetallic complexes having two (trinpropyl-phosphine)ClPd moieties joined by a diethyldithiooxamidate in both κ-N,S Pd, κ-N',S' Pd' and κ-N,N' Pd, κ-S,S' Pd' coordination modes were synthesized. For both kinds of complexes, homo- and heterobimetallic, at room temperature and in chloroform solution, the NNSS coordination mode…

Development of peptidomimetic boronates as proteasome inhibitors.

Abstract Proteasome inhibition has emerged over the past decade as an effective therapeutic approach for the treatment of hematologic malignancies. It is a multicatalytic complex, whose proteolytic activity relies in three types of subunits: chymotrypsin-like (β5), trypsin-like (β2) and caspase-like (β1). Most important for the development of effective antitumor agents is the inhibition of the β5 subunits. In this context, the dipeptide boronate bortezomib (Velcade ® ) represents the first proteasome inhibitor approved by the FDA and the lead compound in drug discovery. This paper describes the synthesis and biological evaluation of a series of conformationally constrained pseudopeptide bor…

Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A

Abstract Staphylococcus aureus is one of the most frequent causes of nosocomial and community‐acquired infections, with drug‐resistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active‐site cysteine. A broad series of derivatives were synthesized to derive structure‐activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were f…

Prospective Evaluation of Free Energy Calculations for the Prioritization of Cathepsin L Inhibitors.

Improving the binding affinity of a chemical series by systematically probing one of its exit vectors is a medicinal chemistry activity that can benefit from molecular modeling input. Herein, we compare the effectiveness of four approaches in prioritizing building blocks with better potency: selection by a medicinal chemist, manual modeling, docking followed by manual filtering, and free energy calculations (FEP). Our study focused on identifying novel substituents for the apolar S2 pocket of cathepsin L and was conducted entirely in a prospective manner with synthesis and activity determination of 36 novel compounds. We found that FEP selected compounds with improved affinity for 8 out of …

Promising trypanocidal heterocyclic compounds of natural origin and their synthetic analogs

Abstract Diseases caused by members of the order Trypanosomatidae include human African trypanosomiasis (HAT) and Chagas disease, caused by species of Trypanosoma brucei and Trypanosoma cruzi, respectively, as well as leishmaniasis, caused by various species of Leishmania spp. These infections belong to the so-called neglected tropical diseases group, which are a diverse group of communicable diseases that prevail in tropical and subtropical conditions in 149 countries and affect more than one billion people in addition to costing developing economies billions of dollars every year. The available pharmacotherapies for combatting these diseases are limited and associated with strong side eff…

Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes

AbstractRhodesain is the lysosomal cathepsin L-like cysteine protease ofT. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression ofT. brucei rhodesiensepro-rhodesain inE. coliand determined its crystal structure. The trypanosomal pr…

2 H-1,2,3-Triazole-Based Dipeptidyl Nitriles: Potent, Selective, and Trypanocidal Rhodesain Inhibitors by Structure-Based Design.

Macrocyclic inhibitors of rhodesain (RD), a parasitic cysteine protease and drug target for the treatment of human African trypanosomiasis, have shown low metabolic stability at the macrocyclic ether bridge. A series of acyclic dipeptidyl nitriles was developed using structure-based design (PDB ID: 6EX8). The selectivity against the closely related cysteine protease human cathepsin L (hCatL) was substantially improved, up to 507-fold. In the S2 pocket, 3,4-dichlorophenylalanine residues provided high trypanocidal activities. In the S3 pocket, aromatic residues provided enhanced selectivity against hCatL. RD inhibition (Ki values) and in vitro cell-growth of Trypanosoma brucei rhodesiense (I…

CCDC 1498221: Experimental Crystal Structure Determination

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CCDC 916055: Experimental Crystal Structure Determination

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CCDC 897063: Experimental Crystal Structure Determination

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