Cinnamides Target Leishmania amazonensis Arginase Selectively

Caffeic acid and related natural compounds were previously described as Leishmania amazonensis arginase (L-ARG) inhibitors, and against the whole parasite in vitro. In this study, we tested cinnamides that were previously synthesized to target human arginase. The compound caffeic acid phenethyl amide (CAPA), a weak inhibitor of human arginase (IC50 = 60.3 ± 7.8 μM) was found to have 9-fold more potency against L-ARG (IC50 = 6.9 ± 0.7 μM). The other compounds that did not inhibit human arginase were characterized as L-ARG, showing an IC50 between 1.3–17.8 μM, and where the most active was compound 15 (IC50 = 1.3 ± 0.1 μM). All compounds were also tested against L. amazonensis promastigotes, and only the compound CAPA showed an inhibitory activity (IC50 = 80 μM). In addition, in an attempt to gain an insight into the mechanism of competitive L-ARG inhibitors, and their selectivity over mammalian enzymes, we performed an extensive computational investigation, to provide the basis for the selective inhibition of L-ARG for this series of compounds. In conclusion, our results indicated that the compounds based on cinnamoyl or 3,4-hydroxy cinnamoyl moiety could be a promising starting point for the design of potential antileishmanial drugs based on selective L-ARG inhibitors.

The structure of the RCAN1:CN complex explains the inhibition of and substrate recruitment by calcineurin

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the Ser/Thr phosphatase calcineurin (CN). It has been shown that excessive inhibition of CN is a critical factor for Down syndrome and Alzheimer’s disease. Here, we determined RCAN1’s mode of action. Using a combination of structural, biophysical, and biochemical studies, we show that RCAN1 inhibits CN via multiple routes: first, by blocking essential substrate recruitment sites and, second, by blocking the CN active site using two distinct mechanisms. We also show that phosphorylation either inhibits RCAN1-CN assembly or converts RCAN1 into a weak inhibitor, which can be reversed by CN via dephosphorylation. This highlights the interplay between posttranslational modifications in regulating CN activity. Last, this work advances our understanding of how active site inhibition of CN can be achieved in a highly specific manner. Together, these data provide the necessary road map for targeting multiple neurological disorders.

A phase I study of pazopanib with weekly paclitaxel and carboplatin in advanced solid tumors.

3021 Background: Pazopanib (pazo) is an oral tyrosine kinase inhibitor of VEGFR, PDGFR and c-Kit. It is a weak inhibitor of CYP3A4 and CYP2C8 and may decrease paclitaxel (P) clearance. Daily pazo with P and carboplatin (C) every 21 days was not feasible on a previous study. We hypothesized that pazo dosed intermittently and on a different day from P and C may be tolerable. We sought to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacokinetics (PK) of pazo with weekly P and C. Methods: Using a 3+3 standard design, a schedule of P 60-80 mg/m2 and C AUC2 on days 1, 8, and 15 with pazo 400-800 mg on days 2-5, 9-12, and 16-26 on a 28-day cycle was evaluated. Pazo alone could be continued if P and C were omitted due to maximal benefit or toxicity. PK was collected during cycles 1 and 2. Results: 34 patients (pts) were treated over 6 dose levels (Table). Mean age 57 (37-79). Tumor types: breast (22), lung (3) and other (9); 27 had prior platinum. Delay in starting cycle 2 due to grade 3 neutropenia was a DLT at dose level 2 and 5. Pts on 5A missed dosing during C1 and C2 due to neutropenia and required subsequent growth factor, and this was deemed unlikely to be sustainable long-term. All grade toxicities included anemia (62%), neutropenia (59%), and thrombocytopenia (56%). Protocol-defined MTD was not determined. PK analysis showed a dose proportional increase in pazo concentration, consistent with previous reports. Pazo did not alter the PK of C. Cmax of P was higher C2D1 vs C1D1; mean Cmax ratio between C2D1:C1D1 was 1.63 (95% CI:1.29-1.96). There were 11 objective responses (3 CRs, 8 PRs). Five breast pts were on pazo alone for a median of 9 cycles (2-52) with CR (2), PR (2) and SD (1); a squamous cell of unknown primary in CR received 22 cycles. Clinical trial information: NCT01407562. Conclusions: PK confirm that pazo is a weak inhibitor of CYP3A4 and CYP2C8. Myelosuppression was a major adverse event at all dose levels. MTD was not determined. Antitumor activity was achieved with this alternate combination schedule and sustained responses from sequential pazo monotherapy was observed.[Table: see text]

Anti-Parkinson Drug Biperiden Inhibits Enzyme Acetylcholinesterase

Biperiden is a drug used in Parkinson disease treatment and it serves also as an antiseizures compound in organophosphates poisoning. It acts as antagonist of muscarinic receptor activated by acetylcholine while the enzyme acetylcholinesterase (AChE) cleaves acetylcholine in synaptic junction into choline and acetic acid. This enzyme is inhibited by various compounds; however there has not been proposed evidence about interaction with biperiden molecule. We investigated this interaction using standard Ellman’s assay and experimental findings were critically completed with an in silico prediction by SwissDock docking software. Uncompetitive mechanism of action was revealed from Dixon plot and inhibition constant (Ki) was calculated to be 1.11 mmol/l. The lowest predicted binding energy was −7.84 kcal/mol corresponding to H-bond between biperiden molecule and Tyr 341 residuum in protein structure of AChE. This interaction seems to be further stabilized by π-π interaction with Tyr 72, Trp 286, and Tyr 341. In conclusion, biperiden appears as a very weak inhibitor but it can serve as a lead structure in a pharmacological research.

Determining the IC50 Values for Vorozole and Letrozole, on a Series of Human Liver Cytochrome P450s, to Help Determine the Binding Site of Vorozole in the Liver

Vorozole and letrozole are third-generation aromatase (cytochrome P450 19A1) inhibitors. [11C]-Vorozole can be used as a radiotracer for aromatase in living animals but when administered by IV, it collects in the liver. Pretreatment with letrozole does not affect the binding of vorozole in the liver. In search of finding the protein responsible for the accumulation of vorozole in the liver, fluorometric high-throughput screening assays were used to test the inhibitory capability of vorozole and letrozole on a series of liver cytochrome P450s (CYP1A1, CYP1A2, CYP2A6, and CYP3A4). It was determined that vorozole is a potent inhibitor of CYP1A1 (IC50 = 0.469 μM) and a moderate inhibitor of CYP2A6 and CYP3A4 (IC50 = 24.4 and 98.1 μM, resp.). Letrozole is only a moderate inhibitor of CYP1A1 and CYP2A6 (IC50 = 69.8 and 106 μM) and a very weak inhibitor of CYP3A4 (<10% inhibition at 1 mM). Since CYP3A4 makes up the majority of the CYP content found in the human liver, and vorozole inhibits it moderately well but letrozole does not, CYP3A4 is a good candidate for the protein that [11C]-vorozole is binding to in the liver.

Antiviral Activity of Broad-Spectrum and Enterovirus-Specific Inhibitors against Clinical Isolates of Enterovirus D68

ABSTRACTWe investigated the susceptibility of 10 enterovirus D68 (EV-D68) isolates (belonging to clusters A, B, and C) to (entero)virus inhibitors with different mechanisms of action. The 3C-protease inhibitors proved to be more efficient than enviroxime and pleconaril, which in turn were more effective than vapendavir and pirodavir. Favipiravir proved to be a weak inhibitor. Resistance to pleconaril maps to V69A in the VP1 protein, and resistance to rupintrivir maps to V104I in the 3C protease. A structural explanation of why both substitutions may cause resistance is provided.

Effect of Purine Alkaloids on the Proliferation of Lettuce Cells Derived from Protoplasts

To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 μM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with >250 μM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.