In Vitro and In Silico Analysis of New n-Butyl and Isobutyl Quinoxaline-7-carboxylate 1,4-di-N-oxide Derivatives against Trypanosoma cruzi as Trypanothione Reductase Inhibitors.

American trypanosomiasis is a worldwide health problem that requires attention due to ineffective treatment options. We evaluated n-butyl and isobutyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives against trypomastigotes of the Trypanosoma cruzi strains NINOA and INC-5. An in silico analysis of the interactions of 1,4-di-N-oxide on the active site of trypanothione reductase (TR) and an enzyme inhibition study was carried out. The n-butyl series compound identified as T-150 had the best trypanocidal activity against T. cruzi trypomastigotes, with a 13% TR inhibition at 44 µM. The derivative T-147 behaved as a mixed inhibitor with Ki and Ki' inhibition constants of 11.4 and 60.8 µM, respectively. This finding is comparable to the TR inhibitor mepacrine (Ki = 19 µM).

An essential thioredoxin-type protein of Trypanosoma brucei acts as redox-regulated mitochondrial chaperone.

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.

Natural killer cell expression of Ki67 is associated with elevated serum IL-15, disease activity and nephritis in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder whose pathology involves multiple immune cell types, including B and T lymphocytes as well as myeloid cells. While it is clear that autoantibody-producing B cells, as well as CD4+ T cell help, are key contributors to disease, little is known regarding the role of innate lymphoid cells such as natural killer (NK) cells in the pathogenesis of SLE. We have characterized the phenotype of NK cells by multi-color flow cytometry in a large cohort of SLE patients. While the overall percentage of NK cells was similar or slightly decreased compared to healthy controls, a subset of patients displayed a high frequency of NK cells expressing the proliferation marker, Ki67, which was not found in healthy donors. Although expression of Ki67 on NK cells correlated with Ki67 on other immune cell subsets, the frequency of Ki67 on NK cells was considerably higher. Increased frequencies of Ki67+ NK cells correlated strongly with clinical severity and active nephritis and was also related to low NK cell numbers, but not overall leukopenia. Proteomic and functional data indicate that the cytokine interleukin-15 promotes the induction of Ki67 on NK cells. These results suggest a role for NK cells in regulating the immune-mediated pathology of SLE as well as reveal a possible target for therapeutic intervention.

Targeting a Large Active Site: Structure-Based Design of Nanomolar Inhibitors of Trypanosoma brucei Trypanothione Reductase.

Trypanothione reductase (TR) plays a key role in the unique redox metabolism of trypanosomatids, the causative agents of human African trypanosomiasis (HAT), Chagas' disease, and leishmaniases. Introduction of a new, lean propargylic vector to a known class of TR inhibitors resulted in the strongest reported competitive inhibitor of Trypanosoma (T.) brucei TR, with an inhibition constant Ki of 73 nm, which is fully selective against human glutathione reductase (hGR). The best ligands exhibited in vitro IC50 values (half-maximal inhibitory concentration) against the HAT pathogen, T. brucei rhodesiense, in the mid-nanomolar range, reaching down to 50 nm. X-Ray co-crystal structures confirmed the binding mode of the ligands and revealed the presence of a HEPES buffer molecule in the large active site. Extension of the propargylic vector, guided by structure-based design, to replace the HEPES buffer molecule should give inhibitors with low nanomolar Ki and IC50 values for in vivo studies.

Natural Sesquiterpene Lactones of the 4,15-iso-Atriplicolide Type are Inhibitors of Trypanothione Reductase.

In the course of our investigations on the antitrypanosomal potential of sesquiterpene lactones (STL), we have recently reported on the exceptionally strong activity of 4,15-iso-Atriplicolide tiglate, which demonstrated an IC50 value of 15 nM against Trypanosoma brucei rhodesiense, the etiologic agent responsible for East African human trypanosomiasis (HAT). Since STLs are known to often interact with their biological targets (e.g., in anti-inflammatory and anti-tumor activity) by means of the covalent modification of biological nucleophiles-most prominently free cysteine thiol groups in proteins-it was a straightforward assumption that such compounds might interfere with the trypanothione-associated detoxification system of trypanosomes. This system heavily relies on thiol groups in the form of the dithiol trypanothione (T(SH)2) and in the active centers of enzymes involved in trypanothione metabolism and homeostasis. Indeed, we found in the present study that 4,15-iso-atriplicolide tiglate, as well as its structural homologues, the corresponding methacrylate and isobutyrate, are inhibitors of trypanothione reductase (TR), the enzyme serving the parasites to keep T(SH)2 in the dithiol state. The TR inhibitory activity was demonstrated to be time-dependent and irreversible. Quite interestingly, of the several further STLs with different core structures that were also tested, none inhibited TR at a significant level. Thus, the TR inhibitory effect by the 4,15-iso-atriplicolide esters appears to be specific for this particular type of furanoheliangolide-type STL. Some structure-activity relationships can already be deduced on the basis of the data reported here, which may serve as the starting point for searching further, possibly more potent, TR inhibitors.

Inhibitor-Induced Dimerization of an Essential Oxidoreductase from African Trypanosomes.

Trypanosomal and leishmanial infections claim tens of thousands of lives each year. The metabolism of these unicellular eukaryotic parasites differs from the human host and their enzymes thus constitute promising drug targets. Tryparedoxin (Tpx) from Trypanosoma brucei is the essential oxidoreductase in the parasite's hydroperoxide-clearance cascade. In vitro and in vivo functional assays show that a small, selective inhibitor efficiently inhibits Tpx. With X-ray crystallography, SAXS, analytical SEC, SEC-MALS, MD simulations, ITC, and NMR spectroscopy, we show how covalent binding of this monofunctional inhibitor leads to Tpx dimerization. Intra- and intermolecular inhibitor-inhibitor, protein-protein, and inhibitor-protein interactions stabilize the dimer. The behavior of this efficient antitrypanosomal molecule thus constitutes an exquisite example of chemically induced dimerization with a small, monovalent ligand that can be exploited for future drug design.

Analysis of angiogenesis biomarkers for ramucirumab efficacy in patients with metastatic colorectal cancer from RAISE, a global, randomized, double-blind, phase III study.

Background: The phase III RAISE trial (NCT01183780) demonstrated that the vascular endothelial growth factor (VEGF) receptor (VEGFR)-2 binding monoclonal antibody ramucirumab plus 5-fluororuracil, leucovorin, and irinotecan (FOLFIRI) significantly improved overall survival (OS) and progression-free survival (PFS) compared with placebo + FOLFIRI as second-line metastatic colorectal cancer (mCRC) treatment. To identify patients who benefit the most from VEGFR-2 blockade, the RAISE trial design included a prospective and comprehensive biomarker program that assessed the association of biomarkers with ramucirumab efficacy outcomes. Patients and methods: Plasma and tumor tissue collection was mandatory. Overall, 1072 patients were randomized 1 : 1 to the addition of ramucirumab or placebo to FOLFIRI chemotherapy. Patients were then randomized 1 : 2, for the biomarker program, to marker exploratory (ME) and marker confirmatory (MC) groups. Analyses were carried out using exploratory assays to assess the correlations of baseline marker levels [VEGF-C, VEGF-D, sVEGFR-1, sVEGFR-2, sVEGFR-3 (plasma), and VEGFR-2 (tumor tissue)] with clinical outcomes. Cox regression analyses were carried out for each candidate biomarker with stratification factor adjustment. Results: Biomarker results were available from >80% (n = 894) of patients. Analysis of the ME subset determined a VEGF-D level of 115 pg/ml was appropriate for high/low subgroup analyses. Evaluation of the combined ME + MC populations found that the median OS in the ramucirumab + FOLFIRI arm compared with placebo + FOLFIRI showed an improvement of 2.4 months in the high VEGF-D subgroup [13.9 months (95% CI 12.5-15.6) versus 11.5 months (95% CI 10.1-12.4), respectively], and a decrease of 0.5 month in the low VEGF-D subgroup [12.6 months (95% CI 10.7-14.0) versus 13.1 months (95% CI 11.8-17.0), respectively]. PFS results were consistent with OS. No trends were evident with the other antiangiogenic candidate biomarkers. Conclusions: The RAISE biomarker program identified VEGF-D as a potential predictive biomarker for ramucirumab efficacy in second-line mCRC. Development of an assay appropriate for testing in clinical practice is currently ongoing. Clinical trials registration: NCT01183780.

Factors associated with long-term weight-loss maintenance following bariatric surgery in adolescents with severe obesity.

BACKGROUND/OBJECTIVES: Bariatric surgery produces robust weight loss, however, factors associated with long-term weight-loss maintenance among adolescents undergoing Roux-en-Y gastric bypass surgery are unknown. SUBJECTS/METHODS: Fifty adolescents (mean±s.d. age and body mass index (BMI)=17.1±1.7 years and 59±11 kg m-2) underwent Roux-en-Y gastric bypass surgery, had follow-up visits at 1 year and at a visit between 5 and 12 years following surgery (Follow-up of Adolescent Bariatric Surgery at 5 Plus years (FABS-5+) visit; mean±s.d. 8.1±1.6 years). A non-surgical comparison group (n=30; mean±s.d. age and BMI=15.3±1.7 years and BMI=52±8 kg m-2) was recruited to compare weight trajectories over time. Questionnaires (health-related and eating behaviors, health responsibility, impact of weight on quality of life (QOL), international physical activity questionnaire and dietary habits via surgery guidelines) were administered at the FABS-5+ visit. Post hoc, participants were split into two groups: long-term weight-loss maintainers (n=23; baseline BMI=58.2 kg m-2; 1-year BMI=35.8 kg m-2; FABS-5+ BMI=34.9 kg m-2) and re-gainers (n=27; baseline BMI=59.8 kg m-2; 1-year BMI=36.8 kg m-2; FABS-5+ BMI=48.0 kg m-2) to compare factors which might contribute to differences. Data were analyzed using generalized estimating equations adjusted for age, sex, baseline BMI, baseline diabetes status and length of follow-up. RESULTS: The BMI of the surgical group declined from baseline to 1 year (-38.5±6.9%), which, despite some regain, was largely maintained until FABS-5+ (-29.6±13.9% change). The BMI of the comparison group increased from baseline to the FABS-5+ visit (+10.3±20.6%). When the surgical group was split into maintainers and re-gainers, no differences in weight-related and eating behaviors, health responsibility, physical activity/inactivity, or dietary habits were observed between groups. However, at FABS-5+, maintainers had greater overall QOL scores than re-gainers (87.5±10.5 vs 65.4±20.2, P<0.001) and in each QOL sub-domain (P<0.01 all). CONCLUSIONS: Long-term weight outcomes for those who underwent weight-loss surgery were superior to those who did not undergo surgical treatment. While no behavioral factors were identified as predictors of success in long-term weight-loss maintenance, greater QOL was strongly associated with maintenance of weight loss among adolescents who underwent Roux-en-Y gastric bypass surgery surgery.

The cytosolic or the mitochondrial glutathione peroxidase-type tryparedoxin peroxidase is sufficient to protect procyclic Trypanosoma brucei from iron-mediated mitochondrial damage and lysis.

African trypanosomes express three virtually identical glutathione peroxidase (Px)-type enzymes that occur in the cytosol (Px I and II) and mitochondrion (Px III) and detoxify fatty acid-derived hydroperoxides. Selective deletion of the genes revealed that procyclic Trypanosoma brucei lacking either the cytosolic or mitochondrial enzyme proliferate nearly as wild-type parasites, whereas the knockout of the complete genomic locus is lethal. Flow cytometry and immunofluorescence analyses revealed that the Px I-III-deficient parasites lose their mitochondrial membrane potential, which is followed by a loss of the lysosomal signal but not the glycosomal one. Mitochondrial damage and cell lysis are prevented by Trolox, ubiquinone derivatives and the iron chelator deferoxamine, whereas starch-deferoxamine is inefficient. In glucose-rich medium, cell death is attenuated suggesting that oxidants generated by the respiratory chain contribute to the lethal phenotype. Thus, the Px-type peroxidases protect procyclic cells from an iron-mediated oxidative membrane damage that originates at the mitochondrion. This contrasts with the situation in bloodstream cells, where the lysosome is the primarily affected organelle. Strikingly, either the cytosolic or the mitochondrial form of the peroxidases is required and sufficient to protect the mitochondrion and prevent cell lysis.

Lipid changes 8 years post gastric bypass in adolescents with severe obesity (FABS-5+ study).

OBJECTIVE: Severe obesity in adolescents is increasing and few effective treatments exist. Bariatric surgery is one option, but the extent to which surgery influences cardiovascular risk factors over time in youth is not clear. We hypothesized that Roux-en Y gastric bypass (RYGB) would be associated with sustained improvements in lipids over time (>5 years). PARTICIPANTS/METHODS: Youth who underwent RYGB from 2001 to 2007 were recruited for the Follow-up of Adolescent Bariatric Surgery-5+ (FABS-5+) in 2011-2014. Baseline body mass index (BMI) and lipids were abstracted from medical records. Follow-up data were obtained at a research visit. Analyses included paired t-tests to assess changes in BMI and lipids over time. General linear models were used to evaluate predictors of high-density lipoprotein (HDL) and non-HDL-cholesterol at follow-up. A non-operative group was recruited for comparison. RESULTS: Surgical participants (n=58) were a mean±s.d. age of 17±2 years at baseline and 25±2 years at long-term follow-up. Eighty-six percent were Caucasian and 64% were female. At long-term follow-up BMI decreased by 29% and all lipids (except total cholesterol) significantly improved (P<0.01). Female sex was a significant predictor of non-HDL-cholesterol level at 1 year, while change in BMI from 1 year to long-term follow-up was a significant predictor of non-HDL-cholesterol and HDL-cholesterol during the same interval (P<0.05). In the non-operative group, BMI increased by 8% and lipid parameters were unchanged. CONCLUSIONS: This is the longest and most complete follow-up of youth following RYGB. Weight loss maintenance over time was significantly associated with improvements in lipid profile over 5 years.

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 compounds represent promising starting points for new rhodesain inhibitors.

Synthesis and in vitro antikinetoplastid activity of polyamine-hydroxybenzotriazole conjugates.

Thirteen new polyamine derivatives coupled to hydroxybenzotriazole have been synthesized and evaluated for their in vitro antikinetoplastid activity. Trypanosoma Trypanothione reductase (TryR) was envisioned as a potential target. Among all tested molecules, only one compound, a N3-spermidine-benzotriazole derivative, displayed relevant inhibitory activity on this enzyme but was not active on parasites. The corresponding Boc-protected spermidine-benzotriazole was however trypanocidal against Trypanosoma brucei gambiense with an IC50 value of 1µM and was completely devoid of cytotoxicity. On the intramacrophage amastigotes of Leishmania donovani, a N2-spermidine conjugate of this series, exhibited an interesting IC50 value of 3µM associated with both low cytotoxicity against axenic Leishmania donovani. These new compounds are promising leads for the development of antikinetoplastid agents and their targets have to be deciphered.

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 and likely target of the tacrine derivatives. In addition, the inhibition of trypanothione reductase by bistacrines was found. This flavoprotein oxidoreductase is the main defense against oxidative stress in the thiol redox system unique for protozoa.

Trypanocidal Activity of Quinoxaline 1,4 Di-N-oxide Derivatives as Trypanothione Reductase Inhibitors.

Chagas disease or American trypanosomiasis is a worldwide public health problem. In this work, we evaluated 26 new propyl and isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives as potential trypanocidal agents. Additionally, molecular docking and enzymatic assays on trypanothione reductase (TR) were performed to provide a basis for their potential mechanism of action. Seven compounds showed better trypanocidal activity on epimastigotes than the reference drugs, and only four displayed activity on trypomastigotes; T-085 was the lead compound with an IC50 = 59.9 and 73.02 µM on NINOA and INC-5 strain, respectively. An in silico analysis proposed compound T-085 as a potential TR inhibitor with better affinity than the natural substrate. Enzymatic analysis revealed that T-085 inhibits parasite TR non-competitively. Compound T-085 carries a carbonyl, a CF3, and an isopropyl carboxylate group at 2-, 3- and 7-position, respectively. These results suggest the chemical structure of this compound as a good starting point for the design and synthesis of novel trypanocidal derivatives with higher TR inhibitory potency and lower toxicity.

Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px)-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective stage of T. brucei. The respective knockout of the cytosolic px I-II in the procyclic insect form resulted in cells that were fully viable in Trolox-free medium.

In vitro and in vivo identification of tetradentated polyamine complexes as highly efficient metallodrugs against Trypanosoma cruzi.

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), a series of tetraamine-based compounds was prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by PCR and reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels by (1)H NMR and TEM studies. Finally, as tetraamines are potentially capable of casuing oxidative damage in the parasites, the study was completed by assessing their activity as potential iron superoxide dismutase (Fe-SOD) and trypanothione reductase (TR) inhibitors. High-selectivity indexes observed in vitro were the basis of promoting three of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Tetraamines 2 and 3 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression and curative rates of 33 and 50%, respectively. Tetraamine 3 turned out to be a great inhibitor of Fe-SOD and TR. The high anti-parasitic activity and low toxicity render these tetraamines appropriate molecules for the development of an affordable anti-Chagas agent.

Detection of thiol-based redox switch processes in parasites - facts and future.

Malaria and African trypanosomiasis are tropical diseases caused by the protozoa Plasmodium and Trypanosoma, respectively. The parasites undergo complex life cycles in the mammalian host and insect vector, during which they are exposed to oxidative and nitrosative challenges induced by the host immune system and endogenous processes. Attacking the parasite's redox metabolism is a target mechanism of several known antiparasitic drugs and a promising approach to novel drug development. Apart from this aspect, oxidation of cysteine residues plays a key role in protein-protein interaction, metabolic responses to redox events, and signaling. Understanding the role and dynamics of reactive oxygen species and thiol switches in regulating cellular redox homeostasis is crucial for both basic and applied biomedical approaches. Numerous techniques have therefore been established to detect redox changes in parasites including biochemical methods, fluorescent dyes, and genetically encoded probes. In this review, we aim to give an insight into the characteristics of redox networks in the pathogens Plasmodium and Trypanosoma, including a comprehensive overview of the consequences of specific deletions of redox-associated genes. Furthermore, we summarize mechanisms and detection methods of thiol switches in both parasites and discuss their specificity and sensitivity.

Dissecting the catalytic mechanism of Trypanosoma brucei trypanothione synthetase by kinetic analysis and computational modeling.

In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione (bis(glutathionyl)spermidine (T(SH)2)). Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays Km values for GSH, ATP, spermidine, and Gsp of 34, 18, 687, and 32 µm, respectively, as well as Ki values for GSH and T(SH)2 of 1 mm and 360 µm, respectively. As Gsp hydrolysis has a Km value of 5.6 mm, the in vivo amidase activity is probably negligible. To obtain deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This system's biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps, and (iii) T(SH)2 inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.

Antitrypanosomal isothiocyanate and thiocarbamate glycosides from Moringa peregrina.

O-Methyl (1), O-ethyl (2), and O-butyl (3) 4-[(α-L-rhamnosyloxy) benzyl] thiocarbamate (E), along with 4-(α-L-rhamnosyloxy) benzyl isothiocyanate (4) have been isolated from the aerial parts of Moringa peregrina. The compounds were tested for in vitro activity against Trypanosoma brucei rhodesiense and cytotoxicity in rat skeletal myoblasts (L6 cells). The most potent compound was 4 with an IC50 of 0.10 µM against T.b. rhodesiense and a selectivity index of 73, while the thiocarbamate glycosides 1, 2, and 3 showed only moderate activity. Intraperitoneal administration of 50 mg/kg body weight/day of 4 in the T.b. rhodesiense STIB 900 acute mouse model revealed significant in vivo toxicity. Administration of 10 mg/kg body weight/day resulted in a 95% reduction of parasitemia on day 7 postinfection, but did not cure the animals. Because of its high in vitro activity and its ability to irreversibly inhibit trypanothione reductase, an attractive parasite-specific target enzyme, 4-[(α-L-rhamnosyloxy) benzyl] isothiocyanate (4), can be considered as a lead structure for the development and characterization of novel antitrypanosomal drugs.

A multicenter, randomized phase 2 study to establish combinations of CBP501, cisplatin and nivolumab for ≥3rd-line treatment of patients with advanced pancreatic adenocarcinoma.

BACKGROUND: There is no standard of care for ≥ 3rd-line treatment of metastatic pancreatic adenocarcinoma (PDAC). CBP501 is a novel calmodulin-binding peptide that has been shown to enhance the influx of platinum agents into tumor cells and tumor immunogenicity. This study aimed to (1) confirm efficacy of CBP501/cisplatin/nivolumab for metastatic PDAC observed in a previous phase 1 study, (2) identify combinations that yield 35% 3-month progression-free survival rate (3MPFS) and (3) define the contribution of CBP501 to the effects of combination therapy. METHODS: CBP501 16 or 25 mg/m2 (CBP(16) or CBP(25)) was combined with 60 mg/m2 cisplatin (CDDP) and 240 mg nivolumab (nivo), administered at 3-week intervals. Patients were randomized 1:1:1:1 to (1) CBP(25)/CDDP/nivo, (2) CBP(16)/CDDP/nivo, (3) CBP(25)/CDDP and (4) CDDP/nivo, with randomization stratified by ECOG PS and liver metastases. A Fleming two-stage design was used, yielding a one-sided type I error rate of 2.5% and 80% power when the true 3MPFS is 35%. RESULTS: Among 36 patients, 3MPFS was 44.4% in arms 1 and 2, 11.1% in arm 3% and 33.3% in arm 4. Two patients achieved a partial response in arm 1 (ORR 22.2%; none in other arms). Median PFS and OS were 2.4, 2.1, 1.5 and 1.5 months and 6.3, 5.3, 3.7 and 4.9 months, respectively. Overall, all treatment combinations were well tolerated. Most treatment-related adverse events were grade 1-2. CONCLUSIONS: The combination CBP(25)/(16)/CDDP/nivo demonstrated promising signs of efficacy and a manageable safety profile for the treatment of advanced PDAC. CLINICAL TRIAL REGISTRATION: NCT04953962.