Tanja Schirmeister

Novel Peptidomimetics Containing a Vinyl Ester Moiety as Highly Potent and Selective Falcipain-2 Inhibitors

This paper describes the synthesis and biological evaluation of a new class of peptidomimetic falcipain-2 inhibitors based on a 1,4-benzodiazepine scaffold combined with various alpha,beta-unsaturated electrophilic functions such as vinyl-ketone, -amide, -ester, and -nitrile. The profile of reactivity of this class of derivatives has been evaluated and 4c, containing a vinyl ester warhead, proved to be a highly potent and selective falcipain-2 inhibitor.

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.

Allicin and derivates are cysteine protease inhibitors with antiparasitic activity.

Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure-activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei.

Aziridine-2,3-Dicarboxylates, Peptidomimetic Cysteine Protease Inhibitors with Antileishmanial Activity

ABSTRACT Chemotherapy of leishmaniasis is mainly based on antimonials. However, they are extremely toxic and cause serious side effects, and there is a worldwide increasing frequency of chemoresistance to antimonials. These issues emphasize the urgent need for affordable alternative drugs against leishmaniasis. Leishmania cysteine proteases are essential for parasite growth, differentiation, pathogenicity, and virulence and are thus attractive targets for combating leishmaniasis. Herein we demonstrate that the cysteine protease inhibitors aziridine-2,3-dicarboxylates 13b and 13e impaired promastigote growth at mid-micromolar concentrations and decreased the infection rate of peritoneal macrophages at concentrations 8- to 13-fold lower than those needed to inhibit parasite replication. Simultaneous treatment of infected cells with compound 13b and gamma interferon resulted in an even further reduction of the concentration needed for a significant decrease in macrophage infection rate. Notably, treatment with the compounds alone modulated the cytokine secretion of infected macrophages, with increased levels of interleukin-12 and tumor necrosis factor alpha. Furthermore, the decreased infection rate in the presence of compound 13b correlated with increased nitric oxide production by macrophages. Importantly, at the concentrations used herein, compounds 13b and 13e were not toxic against fibroblasts, macrophages, or dendritic cells. Together, these results suggest that the aziridine-2,3-dicarboxylates 13b and 13e are potential antileishmanial lead compounds with low toxicity against host cells and selective antiparasitic effects.

Antioxidant and Anti-Protease Activities of Diazepinomicin from the Sponge-Associated Micromonospora Strain RV115

Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP) assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2) and human promyelocytic (HL-60) cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC50 of 70–90 µM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC50 of 13.5 µM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate.

Michael Acceptor Based Antiplasmodial and Antitrypanosomal Cysteine Protease Inhibitors with Unusual Amino Acids

New peptidic Michael acceptor based cysteine protease inhibitors displaying antiparasitic activity were identified by testing a broad series of 45 compounds in total, containing Asn, Gln, or Phe. As target enzymes, falcipain-2 and -3 from P. falciparum and rhodesain from T. b. rhodesiense were used. In the case of the Asn/Gln containing compounds, the trityl-protected, diastereomeric E-configured vinylogous dipeptide esters 16 (Boc-(S)-Phg-(R/S)-vGln(Trt)-OEt) were discovered as most active inhibitors concerning both protease inhibition and antiparasitic acitivity, with inhibition constants in the submicromolar range. The compounds were shown to display time-dependent and competitive inhibition. In the case of the Phe containing compounds, the maleic acid derivatives 42 and 43 (BnO-Phe<--Mal-Phe-OBn, BnO-Phe<--Mal-Phe-Ala-OBn, Mal = maleic acid) displayed good inhibition of rhodesain as well as good antitrypanosomal activity, while the fumaric acid derived E-analogue 14 (BnO-Phe<--Fum-Phe-OBn) only displayed inhibition of the target enzymes but no antiparasitic activity. Inhibition by these Phe derivatives was shown to be time-independent and competitive.

On the origin of the stabilization of the zwitterionic resting state of cysteine proteases: A theoretical study

Papain-like cysteine proteases are ubiquitous proteolytic enzymes. The protonated His199/deprotonated Cys29 ion pair (cathepsin B numbering) in the active site is essential for their proper functioning. The presence of this ion pair stands in contrast to the corresponding intrinsic residue p K a values, indicating a strong influence of the enzyme environment. In the present work we show by molecular dynamics simulations on quantum mechanical/molecular mechanical (QM/MM) potentials that the ion pair is stabilized by a complex hydrogen bond network which comprises several amino acids situated in the active site of the enzyme and 2-4 water molecules. QM/MM reaction path computations for the proton transfer from His199 to the thiolate of the Cys29 moiety indicate that the ion pair is about 32-36 kJ mol (-1) more stable than the neutral form if the whole hydrogen bonding network is active. Without any hydrogen bonding network the ion pair is predicted to be significantly less stable than the neutral form. QM/MM charge deletion analysis and QM model calculations are used to quantify the stabilizing effect of the active-site residues and the L1 helix in favor of the zwitterionic form. The active-site water molecules contribute about 30 kJ mol (-1) to the overall stabilization. Disruption of the hydrogen bonding network upon substrate binding is expected to enhance the nucleophilic reactivity of the thiolate.

Tetracycline-Inducible Expression of Individual Secreted Aspartic Proteases in Candida albicans Allows Isoenzyme-Specific Inhibitor Screening

ABSTRACT The yeast Candida albicans possesses a gene family that encodes secreted aspartic proteases (Saps), which are important for the virulence of this human fungal pathogen. Inhibitors of the Saps could therefore be used as novel antimycotic agents for the treatment of C. albicans infections. In the present study, we established a bioassay which allows testing of the activity of potential protease inhibitors against specific Sap isoenzymes by their ability to inhibit protease-dependent growth of C. albicans. In a medium containing bovine serum albumin (BSA) as the sole source of nitrogen, C. albicans specifically expresses the Sap2p isoenzyme, which degrades the BSA and thereby enables the fungus to grow. As the other SAP genes are not significantly expressed under these conditions, mutants lacking SAP2 are unable to utilize BSA as a nitrogen source and cannot grow in such a medium. To investigate whether forced expression of SAP genes other than SAP2 would also allow growth on BSA, we constructed a set of strains expressing each of the 10 SAP genes from a tetracycline-inducible promoter in a sap2Δ mutant background. Expression of Sap1p, Sap2p, Sap3p, Sap4p, Sap5p, Sap6p, Sap8p, and a C-terminally truncated, secreted Sap9p restored the growth of the sap2Δ mutant with different efficiencies. This set of strains was then used to test the activities of various aspartic protease inhibitors against specific Sap isoenzymes by monitoring growth on BSA in the presence of the inhibitors. While pepstatin blocked the activity of all of the Saps tested, the human immunodeficiency virus protease inhibitors ritonavir and saquinavir inhibited growth of the strains expressing Sap1p to Sap3p and Sap1p, respectively, but not that of strains expressing other Saps. Therefore, the strain set can be used to test the activity of new protease inhibitors against individual C. albicans Sap isoenzymes by their ability to block the growth of the pathogen.

Novel Dengue Virus NS2B/NS3 Protease Inhibitors

ABSTRACT Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.

Anti-protease and Immunomodulatory Activities of Bacteria Associated with Caribbean Sponges

Marine sponges and their associated bacteria have been proven to be a rich source of novel secondary metabolites with therapeutic usefulness in cancer, infection, and autoimmunity. In this study, 79 strains belonging to 20 genera of the order Actinomycetales and seven strains belonging to two genera of the order Sphingomonadales were cultivated from 18 different Caribbean sponges and identified by 16S rRNA gene sequencing. Seven of these strains are likely to represent novel species. Crude extracts from selected strains were found to exhibit protease inhibition against cathepsins B and L, rhodesain, and falcipain-2 as well as immunomodulatory activities such as induction of cytokine release by human peripheral blood mononuclear cells. These results highlight the significance of marine sponge-associated bacteria to produce bioactive secondary metabolites with therapeutic potential in the treatment of infectious diseases and disorders of the immune system.