11 research outputs found
Transcriptional Activity of the Islet β Cell Factor Pdx1 is Augmented by Lysine Methylation Catalyzed by the Methyltransferase Set7/9
The transcription factor Pdx1 is crucial to islet β cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass spectrometric analysis using β cells verified Lys methylation of endogenous Pdx1. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed a requirement of Pdx1 residue Lys-131, but not Lys-123, for transcriptional augmentation by Set7/9. Lys-131 was not required for high-affinity interactions with DNA in vitro, suggesting that its methylation likely enhances post-DNA binding events. To define the role of Set7/9 in β cell function, we generated mutant mice in which the gene encoding Set7/9 was conditionally deleted in β cells (SetΔβ). SetΔβ mice exhibited glucose intolerance similar to Pdx1-deficient mice, and their isolated islets showed impaired glucose-stimulated insulin secretion with reductions in expression of Pdx1 target genes. Our results suggest a previously unappreciated role for Set7/9-mediated methylation in the maintenance of Pdx1 activity and β cell function
Interrogating alkyl and arylalkylpolyamino (bis)urea and (bis)thiourea isosteres as potent antimalarial chemotypes against multiple lifecycle forms of Plasmodium falciparum parasites
A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized
and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal
substituents increased the potency of analogues in the compound library 3-fold, with the most active
compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM,
respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro
evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria
parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary
biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds
target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have
the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology
against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these
results and postulate a refined structure–activity relationship (SAR) model for antiplasmodial polyamine
analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea–polyamine analogues featuring a
3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials
with activities in the low nanomolar range, and high selectivity against various lifecycle forms of
P. falciparum parasites.South African National Research Foundation (FA2007050300003 & UID: 84627), the University of Pretoria and the South African Medical Research Council Strategic
Health Initiatives Partnerships with the Medicines for Malaria Venture.http://www.elsevier.com/locate/bmc2016-08-31hb201
Structure- and Ligand-Based Modeling of Beta-Secretase 1 (BACE1) Inhibitors
There exists a broad consensus among the Alzheimer's disease research community that the key to successful treatment lies in the specific inhibition of beta-amyloid converting enzyme 1 (BACE1). A series of transition-state analogues of BACE1 inhibitors containing fused aryl or biaryl moieties were designed computationally to probe the S2 pocket of BACE1, synthesized, and tested for inhibitory activity. The structure-activity relationship of these inhibitors will be discussed. It has been shown that unlike the bi-aryl, the fused-ring moiety is successfully accommodated in the binding site resulting in ligands with excellent inhibitory activity. When mouse neuroblastoma cells (N2a) are treated with active BACE1 inhibitors 5b and 5c, a reduction in Aβ40 production, c.a. 65% and 35% respectively, compared to the control, was observed. To get additional insights into BACE1 ligand design, we have developed a quantitative structure activity relationship (QSAR) for ‘non-peptidomimetic' BACE1 inhibitors using comparative molecular field analysis (CoMFA). The reported 3D-QSAR model is statistically significant thereby demonstrating a sound SAR for inhibitors that bind to the catalytic site of BACE1. For CoMFA analysis, the statistical parameters are: R2NV = 0.98, R2CV = 0.64, R2LOO = 0.67, SEE = 0.154, F = 287.219 and R2PRED = 0.74. This model should be useful for the identification, design and development of potential BACE1 inhibitors
Low Molecular Weight Amidoximes that Act as Potent Inhibitors of Lysine-Specific Demethylase 1
The recently discovered enzyme lysine-specific demethylase
1 (LSD1)
plays an important role in the epigenetic control of gene expression,
and aberrant gene silencing secondary to LSD1 dysregulation is thought
to contribute to the development of cancer. We reported that (bis)Âguanidines,
(bis)Âbiguanides, and their urea- and thiourea isosteres are potent
inhibitors of LSD1 and induce the re-expression of aberrantly silenced
tumor suppressor genes in tumor cells in vitro. We now report a series
of small molecule amidoximes that are moderate inhibitors of recombinant
LSD1 but that produce dramatic changes in methylation at the histone
3 lysine 4 (H3K4) chromatin mark, a specific target of LSD1, in Calu-6
lung carcinoma cells. In addition, these analogues increase cellular
levels of secreted frizzle-related protein (SFRP) 2, H-cadherin (HCAD),
and the transcription factor GATA4. These compounds represent leads
for an important new series of drug-like epigenetic modulators with
the potential for use as antitumor agents
Mechanism of Inactivation of GABA Aminotransferase by (<i>E</i>)- and (<i>Z</i>)‑(1<i>S</i>,3<i>S</i>)‑3-Amino-4-fluoromethylenyl-1-cyclopentanoic Acid
When
γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter
in the mammalian central nervous system, falls below a threshold level,
seizures occur. One approach to raise GABA concentrations is to inhibit
GABA aminotransferase (GABA-AT), a pyridoxal 5′-phosphate-dependent
enzyme that degrades GABA. We have previously developed (1<i>S</i>,3<i>S</i>)-3-amino-4-difluoromethylene-1-cyclopentanoic
acid (CPP-115), which is 186 times more efficient in inactivating
GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT.
We also developed (<i>E</i>)- and (<i>Z</i>)-(1<i>S</i>,3<i>S</i>)-3-amino-4-fluoromethylenyl-1-cyclopentanoic
acid (<b>1</b> and <b>2</b>, respectively), monofluorinated
analogs of CPP-115, which are comparable to vigabatrin in inactivating
GABA-AT. Here, we report the mechanism of inactivation of GABA-AT
by <b>1</b> and <b>2</b>. Both produce a metabolite that
induces disruption of the Glu270–Arg445 salt bridge to accommodate
interaction between the metabolite formyl group and Arg445. This is
the second time that Arg445 has interacted with a ligand and is involved
in GABA-AT inactivation, thereby confirming the importance of Arg445
in future inactivator design
Interrogating alkyl and arylalkylpolyamino (bis)urea and (bis)thiourea isosteres as potent antimalarial chemotypes against multiple lifecycle forms of Plasmodium falciparum parasites
A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized
and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal
substituents increased the potency of analogues in the compound library 3-fold, with the most active
compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM,
respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro
evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria
parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary
biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds
target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have
the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology
against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these
results and postulate a refined structure–activity relationship (SAR) model for antiplasmodial polyamine
analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea–polyamine analogues featuring a
3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials
with activities in the low nanomolar range, and high selectivity against various lifecycle forms of
P. falciparum parasites.South African National Research Foundation (FA2007050300003 & UID: 84627), the University of Pretoria and the South African Medical Research Council Strategic
Health Initiatives Partnerships with the Medicines for Malaria Venture.http://www.elsevier.com/locate/bmc2016-08-31hb201