Dann, Charles E.

Deis, Siobhan M.

Elsevier - Publisher Connector 

Analysis of Antifolate Drugs with Disease Tissue Specificity 

Sunday, February 8, 2015 55astability, conformation and binding affinity by equilibrium unfolding using
steady state fluorescence and proteolytic digestion assay. These data show
that imatinib binds to hFGF-1 and enhances its thermal stability and solvent
accessibility. In addition, Biacore analysis was carried out to determine the
binding affinity of imatinib to hFGF-1. 1H-15N HSQC NMR was also per-
formed in order to determine exact binding sites and stoichiometry of binding
between imatinib and hFGF-1.
273-Pos Board B53
A Shared Binding Site for Propofol and Thiopental in ELIC
Monica N. Kinde1, Weiming Bu2, Edom Seyoum1, Qiang Chen1,
Marta M. Wells1, David D. Mowrey1, Roderic G. Eckenhoff2, Yan Xu1,
Pei Tang1.
1Department of Anesthesiology, University of Pittsburgh School of
Medicine, Pittsburgh, PA, USA, 2Department of Anesthesiology & Critical
Care, University of Pennsylvania Perelman School of Medicine,
Philadelphia, PA, USA.
The intravenous general anesthetics propofol and thiopental target pentameric
ligand-gated ion channels (pLGIC) and inhibit cation-conducting nAChRs.
These drugs also inhibit ELIC, a prokaryotic pLGIC. However, the binding
sites for these anesthetics are unknown for either nAChRs or ELIC. Here, using
photoaffinity labeling, two-electrode voltage clamp electrophysiology and mo-
lecular docking, we identified a functionally relevant binding site for thiopental
and propofol in ELIC. Molecular docking identified two binding pockets: an
intrasubunit site near M265 of TM3, partially overlapped with the previously
identified propofol binding site in GLIC; and an intersubunit site near W220
of TM1, which overlaps with the bromoform binding location identified previ-
ously. We generated two mutants, one targeted both predicted binding sites
(W220F/W224F/M265C) and another targeted only the intrasubunit site
(M265C). Functional measurements on Xenopus oocytes expressing the
W220F/W224F/M265C mutant show a significant decrease of anesthetic inhi-
bition, with a five-fold increase in the propofol IC50 and abolishment of thio-
pental inhibition. Interestingly, the M265C mutation alone could produce the
same effect as the W220F/W224F/M265C mutant. Photoaffinity labeling ex-
periments with a light-activated derivative of propofol (aziPm), in conjunction
with mass spectrometry, confirmed the binding site at M265 for aziPm. Alto-
gether, the results show that propofol and thiopental bind to a common func-
tionally relevant site. This intrasubunit action site may also be shared by
other intravenous anesthetics. Research supported by grants from the NIH.
274-Pos Board B54
Crystal View of Anesthetics and Alcohols Bound in the Pore of ELIC
Qiang Chen1, Monica N. Kinde1, Aina E. Cohen2, Pei Tang1, Yan Xu1.
1Department of Anesthesiology, University of Pittsburgh School of
Medicine, Pittsburgh, PA, USA, 2Stanford Synchrotron Radiation
Lightsource, Menlo Park, CA, USA.
Cys-loop receptors, including the acetylcholine, glycine, 5-HT3 and GABA re-
ceptors, are molecular targets of general anesthetics and alcohols. Molecular
mechanisms of anesthetics and alcohols interacting with Cys-loop receptors
are still unclear. ELIC is a prokaryotic homolog of Cys-loop receptors and
can be inhibited by general anesthetics and alcohols. Here, we report crystal
structures (~3.1 A˚) of ELIC bound with the volatile general anesthetic isoflur-
ane, and bound with 2-bromoethanol. The crystal structures were obtained in
the presence and absence of the agonist propylamine. Isoflurane was found in-
side the pore at two sites near T237(60) and A244(130), respectively, but 2-bro-
moethanol was only found near T237(60). In addition, 2-bromoethanol also
bound near Y102 and E150 in the extracellular domain. The presence of propyl-
amine had no obvious effect on the binding sites for both isoflurane and 2-bro-
moethanol. This is the first time that an anesthetic or alcohol has been observed
in the pore at an atomic resolution. The newly identified binding sites of isoflur-
ane and 2-bromoethanol in ELIC are significantly different from previously re-
ported anesthetic and alcohol binding sites. Neither isoflurane binding nor
2-bromoethanol binding introduced significant structural perturbation. The
binding of isoflurane and 2-bromoethanol inside the pore suggests the possibil-
ity of channel occlusion as a mechanism for channel inhibition of Cys-loop re-
ceptors by general anesthetics and alcohols. Supported by grants from NIH.
275-Pos Board B55
Analysis of Antifolate Drugs with Disease Tissue Specificity
Siobhan M. Deis1, Charles E. Dann III2.
1Biochemistry, Indiana University, Bloomington, IN, USA, 2Chemistry,
Indiana University, Bloomington, IN, USA.
Antifolates, analogues of the essential vitamin folic acid, are used in the clinic
to treat cancers and inflammatory diseases. Antifolates are primarily trans-
ported into cells via the endogenously expressed reduced folate carrier
(RFC). Conversely, our collaborators in Aleem Gangjee’s group at DuquesneUniversity have synthesized antifolates (AG antifolates) that transport poorly
by the RFC, but are efficiently transported by the folate receptor (hFR). The
GPI-anchored hFR is lowly expressed on the apical surface in a subset of
normal epithelial lineages, but is highly expressed in many cancers of epithelial
origin and on activated macrophages in inflammatory disease. Therefore, AG
antifolate molecules have specificity for transport into disease cells over
healthy cells. These newly developed AG antifolates cause cell death via inhi-
bition of an enzyme involved in de novo purine synthesis, glycinamide ribonu-
cleotide (GAR) transformylase.
We analyzed a series of AG antifolates using biophysical and biochemical tech-
niques to understand both the specificity for transport by the folate receptor as
well as the inhibition of the GAR transformylase in order to drive informed,
hypothesis-based drug design. Our data, including pH-dependent binding pro-
files, enzyme inhibition data, and crystallographic models of protein in com-
plex with AG molecules will be presented in the context of drug design and
development.
276-Pos Board B56
PKA-Dependent Potentiation Mechanisms of Human CFTR Activity
Guangyu Wang.
University of California Davis, Davis, CA, USA.
Both curcumin and VX-770 potentiate the channel activity of human CFTR
(hCFTR) and two most common cystic fibrosis (CF) mutants G551D and
F508delta in an ATP-independent but PKA-dependent manner. The underlying
molecular mechanisms are unclear. Herein, HEK-293T cells cultured in a Fe3þ-
containingmediumwere transiently transfectedwith hCFTR constructs and cur-
cumin with well-known chemical nature was employed as a template to explore
PKA-dependent potentiation mechanisms of hCFTR activity. The results
showed that curcumin potentiation of Fe3þ-sensitive hCFTR activity was
partially weakened by Fe3þ-insensitive mutations at the interface of the R
domain and intracellular loop (ICL) 3 and completely suppressed by sufficient
Fe3þ. Thus, release of the inhibitory Fe3þ-bound R domain from ICL3 by cur-
cumin may be critical for curcumin potentiation. Further study indicated that
curcumin potentiation was significantly prohibited by a missense alanine muta-
tion of F157,Y161 orK166 from ICL1, or R1066, F1074 or F1078 from ICL4, or
S795 or S813 from the R domain with or without the involvement of nucleotide-
binding domain 2 (NBD2). More importantly, curcumin potentiation was also
suppressed by the R811A/S813D or Y808A/S813D mutation and disulfide
crosslinking of K162C to S795C enhanced channel opening. Therefore, the
phosphorylated R domain may function as a length- and gating-regulatory
cross-linker between two transmembrane domains (TMD1 and TMD2). Curcu-
min may potentiate hCFTR activity by stabilizing the stimulatory ICL1/ICL4-R
interactions that promote channel opening by pulling all ICLs together and thus
triggering a gating inward-to-outward reorientation of TMDs. Possible chemical
interactions may involve cation-p interactions, p-p interactions and hydrogen
bonding. Taken together, both release of the R domain from ICL3 and the stim-
ulatory R-ICL1/ICL4 interactionsmay be necessary for PKA-dependent hCFTR
activation and potentiation. These findings may help optimize the potentiators
for treating those CF mutants with an ATP-dependent gating defect.
277-Pos Board B57
Kinase Structural Dynamics Enables Tight and Selective Binding of
Inhibitors
Roman V. Agafonov, Chris Wilson, Vanessa Buosi, Renee Otten,
Dorothee Kern.
Biochemistry, HHMI / Brandeis University, Waltham, MA, USA.
Protein kinases are obvious drug targets against cancer due to their central role
in cellular regulation. With oncologic diseases being the second leading cause
of death in the US kinases rapidly gain attention and are likely to become the
number one drug target. Using NMR and fast kinetics, we establish a novel
model that solves a longstanding question of high selectivity of clinically rele-
vant drug Gleevec that effectively inhibits Abl tyrosine kinase while closely
related Src family of kinases is affected much less. Our study of an entirely
different family of Ser/Thr Aurora kinases and its specific inhibitors suggests
that an energy landscape that provides tight affinity via an induced-fit and bind-
ing plasticity via a conformational selection mechanism is likely to be general
for many inhibitors.
278-Pos Board B58
Glutathione Reductase of Plasmodium Falciparum as an Antimalarial
Drug Target of Methylene Blue
Socheata Lim, Judith H. Prieto.
Chemistry Department, Western Connecticut State University, Danbury,
CT, USA.
Plasmodium falciparum is the cause of human malaria and is one of two ma-
laria parasites known to have drug resistance. Since there are no preventative


file:///data/core-remote/dit/data/elsevier/pdf/e89/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUxNDAxNTQzNA==.pdf

Analysis of Antifolate Drugs with Disease Tissue Specificity

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector