Lecithin:cholesterol acyltransferase activation by sulfhydryl-reactive small molecules: Role of cysteine-31s

Article abstract of DOI:10.1124/jpet.117.240457

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin: cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, an

Full text of DOI:10.1124/jpet.117.240457

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TITLE PAGE
Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small
Molecules: Role of Cysteine-31
Lita A. Freeman, Stephen J. Demosky Jr., Monika Konaklieva, Rostislav Kuskovsky,
Angel Aponte, Alice F. Ossoli, Scott M. Gordon, Ross F. Koby, Kelly A. Manthei, Min
Shen, Boris L. Vaisman, Robert D. Shamburek, Ajit Jadhav, Laura Calabresi, Marjan
Gucek, John J.G. Tesmer, Rodney L. Levine, Alan T. Remaley
Primary laboratory of origin: Lipid Metabolism Section, Cardiovascular and
Pulmonary Branch, NHLBI, NIH
Affiliations:
Lipid Metabolism Section, Cardiovascular and Pulmonary Branch, NHLBI, NIH (LAF,
SJD, SMG, BLV, RDS, ATR)
Department of Chemistry, American University (MK, RK)
Systems Biology Center, NHLBI, NIH (AA, MG)
University of Milano, Milano, Italy (AFO, LC)
Department of Chemistry, Vanderbilt University (RFK)
Life Sciences Institute, Departments of Pharmacology and Biological Chemistry,
University of Michigan, Ann Arbor (KAM, JJGT)
NCATS (MS, AJ)
Laboratory of Biochemistry, NHLBI, NIH (RLL)
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RUNNING TITLE PAGE
Running Title: LCAT Activation by Sulfhydryl-Reactive Small Molecules
Corresponding Author:
Alan T. Remaley
Building 10, Room 2C433
1
0 Center Dr
Bethesda, MD 20814
Telephone: (301) 402-9796
Email: aremaley1@mail.nih.gov
Number of text pages: 41
Number of tables: 2
Number of figures: 10
Number of references: 50
Number of words in abstract: 250
Number of words in introduction: 845
Number of words in discussion: 1,496
Abbreviations: CE, cholesteryl ester; CER, cholesterol esterification rate; DTNB,
dithiobis-nitrobenzoic acid; FC, free cholesterol; FED; fish-eye disease; FLD, familial
LCAT deficiency; HDL, high-density lipoprotein; LCAT, lecithin cholesterol
acyltransferase; msrA, methionine sulfoxide reductase A; MUP, methylumbelliferyl
palmitate; POPC, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; rhLCAT,
recombinant human LCAT; RMSF, root-mean-square fluctuation.
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ABSTRACT
BACKGROUND: Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma
cholesteryl ester formation and is defective in Familial LCAT Deficiency (FLD), an
autosomal recessive disorder characterized by low HDL, anemia and renal disease.
OBJECTIVE: To investigate the mechanism by which Compound A, a small
heterocyclic amine, activates LCAT. METHODS: Effect of Compound A on LCAT was
tested in human plasma and with recombinant LCAT. Mass spectrometry and NMR was
used to determine Compound A adduct formation with LCAT. Molecular modeling was
performed to gain insight into Compound A effects on LCAT structure and activity.
RESULTS: Compound A increased LCAT activity in a subset (3 out of 9) of LCAT
mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-
Cys31Gly prevented activation by Compound A. Substitution of Cys31 with charged
residues (Glu, Arg and Lys) decreased LCAT activity, whereas bulky hydrophobic
groups (Trp, Leu, Phe and Met) increased activity up to 3-fold (p<0.005). Mass
spectrometry of a tryptic digest of LCAT incubated with Compound A revealed a
+103.017 m/z adduct on Cys31, consistent with addition of a single hydrophobic
cyanopyrazine ring. Molecular modeling identified potential interactions of Compound A
near Cys31 and structural changes correlating with enhanced activity. Functional
groups important for LCAT activation by Compound A were identified by testing
Compound A derivatives. Finally, sulfhydryl-reactive β-lactams were developed as a
new class of LCAT activators. CONCLUSIONS: Compound A activates LCAT,
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including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus
providing a mechanistic rationale for the design of future LCAT activators.
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INTRODUCTION
High-density lipoprotein-cholesterol (HDL-C) levels in plasma are inversely
related to cardiovascular disease (CVD) risk, but most therapeutic approaches for
raising HDL-C in clinical trials have not shown benefit(Rader, 2016). It may be the
function rather than the cholesterol content of HDL that accounts for its
atheroprotection(Karathanasis et al., 2017). High-density lipoproteins (HDL) are a
complex mixture of particles with many different protein and lipid components.
Protective functions ascribed to HDL include reverse cholesterol transport from aortic
foam cells to the liver, as well as anti-inflammatory, antioxidative, and antiprotease
effects(Gordon, 2016; Rosenson et al., 2016; Rye and Barter, 2014). Many of these
functions of HDL are performed by larger, spherical particles, so maturation of HDL from
small discoidal particles to large spherical particles may be important for CVD
protection(Asztalos et al., 2000; Asztalos et al., 2011; Karathanasis et al., 2017).
Lecithin:cholesterol acyltransferase (LCAT), a plasma protein secreted by liver, is
an enzyme critical for HDL particle maturation(Ahsan et al., 2014). LCAT catalyzes
cholesteryl ester (CE) production from free cholesterol (FC) and phosphatidylcholine
(lecithin). Because CEs are more hydrophobic than FC, LCAT activity promotes
partitioning of cholesterol from the surface of HDL into its core, converting small
discoidal HDL to larger, spherical HDL particles. Moreover, esterification promotes the
net egress of cholesterol from cells because unlike FC, CE does not freely equilibrate
between cells and lipoproteins. Esterification thereby makes cholesterol efflux to HDL
irreversible(Czarnecka and Yokoyama, 1996). Once formed, CE is preferentially
delivered to the liver(Schwartz et al., 2004).
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CE production by LCAT takes place in two steps. First, LCAT cleaves fatty acids
from the sn-2 position of phospholipids, using its phospholipase A-2 activity. Ser181,
part of the catalytic triad of LCAT (Ser181, Asp345, His377), acts as the nucleophile
and is acylated during this step(Ahsan et al., 2014). Next, LCAT transfers the cleaved
fatty acid from Ser181 to the A-ring hydroxyl group of cholesterol to produce CE.
Phosphatidylcholine is the preferred phospholipid substrate, particularly those with
unsaturated fatty acids in the sn-2 position(Ahsan et al., 2014).
Recent X-ray crystallographic studies of LCAT confirm the presence of a flexible
lid (residues 226-246) that may alter accessibility of the catalytic site to its lipid
substrates(Glukhova et al., 2015; Gunawardane et al., 2016; Piper et al., 2015). Certain
lid residues (227-DNQGIPVMSS-236) may also contribute to substrate
specificity(Glukhova et al., 2015). Residues 36-101 comprise the membrane binding
domain of LCAT(Glukhova et al., 2015), with residues 50-74 forming part of the
HDL/LDL interfacial binding domain(Adimoolam and Jonas, 1997). LCAT has six
cysteines, four in disulfide bonds (Cys50-Cys74 and Cys313-Cys356) that are critical for
LCAT activity(Glukhova et al., 2015). The two free cysteines (Cys31 and Cys184) are
near the active site, with the Cys31 backbone amide forming part of the oxyanion
hole(Glukhova et al., 2015; Holleboom et al., 2011).
Many different LCAT mutations have been described in human patients(Ahsan et
al., 2014). The most severe result in familial LCAT deficiency (FLD), characterized by
low HDL-C, corneal opacities, anemia, and proteinuria that typically progresses by age
4
0-50 to end-stage renal disease, the primary cause of morbidity and mortality. LpX, an
abnormal multivesicular-like lipoprotein that accumulates in FLD patients, contributes to
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their renal disease(Ossoli et al., 2016). Less severe LCAT mutations lead to fish-eye
disease (FED), characterized by low HDL-C and corneal opacities but none of the other
clinical features of FLD. LCAT is also secondarily decreased in an IL-6-dependent
pathway under some conditions(Feister et al., 2002), likely contributing to decreased
HDL during inflammation(Khovidhunkit et al., 2004). Decreased LCAT activity has also
been observed in “Disappearing HDL Syndrome” in several disorders associated with
extremely high levels of IL-10(Moraitis et al., 2015).
We recently developed recombinant human LCAT (rhLCAT) as a potential
therapy and showed that it safely increased HDL-C levels in both CAD and FLD
patients(Shamburek et al., 2016a; Shamburek et al., 2016b). Because rhLCAT raises
HDL and enhances cholesterol efflux, it might protect against CVD, particularly acute
coronary syndrome, similar to findings with reconstituted HDL in early-stage clinical
trials(Krause and Remaley, 2013; Remaley et al., 2008; Remaley et al., 2014). rhLCAT
therapy, however, will likely be relatively expensive and require intravenous infusion. An
oral small-molecule activator that increases activity in FLD patients with some residual
activity is a possible alternative to rhLCAT therapy and would also be a preferable
treatment for CVD. Compound A (3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-
carbonitrile), the only known small-molecule LCAT activator, was found by Amgen in a
high-throughput screen (Kayser et al., 2008) and was shown in hamsters to increase
plasma CE and HDL-C levels, hepatic abcg5/g8 and cyp7a1 gene expression, and
sterol biliary excretion (Chen et al., 2012).
Our objective was to investigate the mechanism for LCAT activation by Compound A
and to determine if it can also increase activity of some natural LCAT mutations. We
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found that Compound A can indeed activate a subset of FLD mutations by forming a
hydrophobic adduct with Cys31 near the active site. We also identified functional groups
in Compound A responsible for LCAT activation and describe a new class of LCAT
activators based on sulfhydryl-reactive β-lactams. Our findings provide important new
mechanistic insights into LCAT activation that can be used to design novel LCAT
activators.
METHODS
Cholesterol Esterification Rate - Human plasma for these studies was obtained
under informed consent from protocols approved by the NHLBI IRB and the Milano Area
C Ethic Committee. Cholesterol esterification rate (CER) was determined by the rate of
3
esterification of [ H] cholesterol, using 125 µl of plasma samples before and after
incubation of plasma at 37°C for 1 h. Before starting the reaction, plasma from subjects
with LCAT deficiency carrying different mutations were incubated with Compound A at a
final concentration of 10 µg/ml. The reaction was stopped with cold ethanol, cholesterol
was extracted, and CE was separated from FC by TLC(Dobiasova, 1983; Dobiasova and
Schutzova, 1986; Vaisman and Remaley, 2013).
Alternatively, CER was quantitated in some plasma from Italian carriers of LCAT
deficiency by measuring FC content before and after incubation of plasma at 37°C for 1
h by an enzymatic colorimetric method(Kempen et al., 2016). A conversion factor
between the radioactive and absorbance CER methods was determined by measuring
several samples with both methods.
LCAT Plasmid constructs – Human LCAT cDNA was optimized for expression
and cloned into pcDNA™4 containing a C-terminal 6-His tag(Glukhova et al., 2015).
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Mutants were made using the QuikChange II Site-Directed Mutagenesis kit
(Stratagene). Primer sequences are in Supplemental Table S1.
Expression of rhLCAT in tissue culture cells - WT or mutant LCAT plasmid (with
C-terminal 6-His tag) was transfected into freshly thawed HEK293 cells, using the
TrueFect-Lipo Transfection Reagent (United Biosystems, Herndon, VA) in Opti-MEM
media. 24 h later, cells were checked for efficiency of transfection by GFP expression.
Media was then changed to serum-free DMEM containing antibiotics, glutamine and
3
.75 mM valproic acid sodium salt (Sigma, St. Louis, MO). The conditioned media was
o
removed 48 - 72 h later, centrifuged for 5 min at 500 x g at 4 C and stored frozen at -
8
0oC until use.
Continuous Fluorescence Assay for LCAT - A novel, sensitive fluorescent 96-well
plate assay for LCAT, using methylumbelliferyl palmitate (MUP) as a substrate, was
developed to measure phospholipase activity of LCAT. The activity was measured by
following the increase in fluorescence due to methylumbelliferone formation.
Methylumbelliferone (Sigma M-1381), prepared at 100 mM in DMSO and diluted further
in PBS, was used to generate standard curves. For apparent K
m
and Vmax calculations,
1
ml of rhLCAT(Rousset et al., 2010) at 50 µg/ml in Assay Buffer (0.1 M sodium
phosphate, pH 7.4) was incubated with 2.8 µl of Compound A (10 mg/ml in DMSO) or
o
just DMSO (vehicle) at 37 C for 1 h. 20 µl of each reaction (LCAT minus Compound A,
LCAT plus Compound A, or assay buffer) were then added to black 96-well plates
(Greiner) in triplicate. During the incubation of LCAT with Compound A, MUP stock (1
mg/ml) was prepared by adding 30 µl Triton X-100 to 1 mg MUP in a glass vial, and
then adding DMSO to 1.0 ml. Immediately before use, the MUP stock solution was
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diluted in assay buffer to 0, 0.01, 0.02, 0.04, 0.06, 0.1 and 0.15 mM MUP and then 180
µl of each dilution was immediately added to the wells to start the reaction of LCAT with
MUP. LCAT activity (methylumbelliferone formation) was measured by fluorescence at
an excitation wavelength of 340 nm and an emission wavelength of 460 nm.
Background due to MUP self-hydrolysis (“assay buffer only” wells) was subtracted from
each result. Specific activity (mol MUP hydrolyzed/g LCAT) in conditioned media was
determined by dividing mol MUP hydrolyzed by LCAT mass, which was determined by
an ELISA (BioVendor, Asheville, NC). Apparent K
m
and Vmax values were calculated
using GraphPad Prism software.
Reaction of Compound A with methionine sulfoxide reductase A and glutathione
and analysis by mass spectrometry - Recombinant mouse methionine sulfoxide
reductase A (msrA) was produced and purified as described(Kim et al., 2010), with the
three cysteine residues not at the active site mutated to serine (C107S/C218S/C227S) to
avoid complications from oxidation or disulfide formation(Lim et al., 2011). Ten µl of the
modified msrA, at 0.5 mg/ml in 12.5 mM Tris, pH 7.4, was adjusted to 0.1 mg/ml in
Compound A by the addition of 0.1 µl 10 mg/ml Compound A stock solution prepared in
dimethylformamide. After 30 min incubation at 37ºC, 2 µl of the reaction was injected onto
a reverse phase column with an autosampler set to 4°C (Agilent 1100 series HPLC,
Agilent Technologies, Santa Clara, CA). The system contained a Zorbax 300Å
StableBond C18 MicroBore column (865630-902, 1.0 x 50mm, 3.5 µm). The initial solvent
was 0.1% formic acid with gradient elution by acetonitrile/0.1% formic acid, increasing at
2
%/min with a flow rate of 20 µL/min. Positive ion electrospray ionization (ESI) mass
spectra were obtained with an Agilent 6520 mass spectrometer equipped with a time-of-
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flight detector. The capillary voltage was 3500 V, fragmentor voltage was 235 V, nitrogen
gas temperature was 350ºC, and data was collected in the mass range of 500-2500 m/z.
Mass spectra were analyzed and deconvoluted using Agilent software, MassHunter
version B.05.
For the reaction of Compound A with glutathione, a solution of 100 µM
glutathione was prepared in 50 mM Tris, 1 mM diethylenetriamine-pentaacetic acid, pH
7
.5. One µl of 10 mg/ml Compound A in dimethylformamide was added to 99 µl
glutathione and incubated at 37ºC for 30 min, after which 1 µl was injected into the
same HPLC-mass spectrometry system described above. For this analysis, the
instrument was run as a tandem mass spectrometer in order to sequence the modified
glutathione. The capillary voltage was 3500 V, fragmentor voltage was 175 V, nitrogen
gas temperature was 300ºC, and the collision cell energy was ramped linearly from 9 to
6
9 V for the m/z range from 300 to 1500.
NMR analysis of the reaction of Compound 5 (3-((5-(methylthio)-1,3,4-thiadiazol-
2
-yl)thio)pyrazine-2-carbonitrile) with glutathione (GSH) - To a solution of GSH (Aldrich)
(
3.3 mg, 10.7 mmol) in 1 ml of D
ml of dDMF, followed by the addition of (2.86 mg,10.7 mmol) of Compound 5 (Table 1).
The resulting 2:1 dDMF:D O solution was left at room temperature for 48 h with
2
O in an NMR tube at room temperature were added 2
2
occasional shaking. The reaction proceeded cleanly, with no unreacted Compound 5
rd
detected. The reaction resulted in attachment of GSH to the 3 position of the pyrazine
ring of Compound A and departure of the 5-(methylthio)-1,3,4-thiadiazole-2-thiol
(thiadiazole ring) as the leaving group.
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Reaction of Compound A with LCAT and analysis by mass spectrometry -
Compound A was dissolved in DMSO to a concentration of 10 mg/ml and then diluted to
1
1
.0 mM in PBS, pH 7.4. Next, 1.5 µl of 1.0 mM Compound A or vehicle was added to
o
5.0 µl rhLCAT at 1 mg/ml and incubated at 37 C for 30 min. LCAT, incubated with
either vehicle or Compound A, was deglycosylated under denaturing conditions using
Protein Deglycosylation Mix (New England Biolabs, Ipswich, MA). To each tube
2
containing 15 µl rhLCAT, 1.5 µl H O and 2.0 µl 10X Glycoprotein Denaturing Buffer was
o
added and the reaction was heated at 100 C for 15 min. The reaction was chilled on ice
and centrifuged for 10 s. To each tube, 5.0 µl 10X Glyco2 buffer, 5 µl 10% NP-40 and
1
2
5 µl H O was added. After gentle mixing, 5.0 µl Deglycosylation Enzyme Cocktail was
o
added. The solution was mixed again and incubated at 37 C for 4 h and then subjected
to SDS-PAGE on a 4-12% Bis-Tris NuPAGE gel (Thermo-Fisher Scientific/Invitrogen,
Carlsbad, CA) and stained with SimplyBlue (Invitrogen). Gel pieces were destained with
2
00 mM sodium bicarbonate/40% ACN (1 x 400, 1 x 200 µl) with frequent vortexing,
dried in a Speedvac treated with tributylphosphine and iodoacetamide using the
ProteoPrep Reduction and Alkylation Kit (Sigma), washed 2X with 25 mM NH CO
00 µl NH CO in 50% ACN 15 minutes, and then desiccated in a Speedvac.
4
3
then
2
4
3
For trypsin digestion, 20 µg trypsin (sequencing grade; Promega, Madison, WI)
was dissolved in 1.6 ml 25 mM NH CO (in H O) and enough of the trypsin solution was
added to just cover the gel pieces. Gel pieces were rehydrated on ice for 30 min and
4
3
2
excess solution was removed. 25 mM NH
4
CO
3
was then added to cover the gel pieces.
o
After a brief spin, the samples were placed in a 37 C incubator overnight. After briefly
spinning down the gel pieces, the supernatant (containing peptides) was transferred to
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a clean tube. Peptides remaining in the gel slices were extracted by incubating the gel
pieces, with shaking, in Extraction Buffer (50% acetonitrile, 5% formic acid) at room
temperature for 30 min and collecting the supernatant (twice). All three supernatants
containing tryptic peptides were then combined, concentrated in a Speedvac, desalted
using Ziptips (Millipore, Billerica, MA) and adjusted to 0.1% formic acid. The solution
was subjected to analysis by LC/MS on an Orbitrap Fusion coupled with an Ultimate
3
000-nLC (Thermo Fisher Scientific). Peptides were separated on an EASY-Spray C18
column (Thermo Scientific; 75 μm x 25 cm inner diameter, 2 μm particle size and 100 Å
pore size). Separation was achieved by applying a 5–35% linear gradient of acetonitrile
-1
plus 0.1% formic acid over 40 min at 300 nl·min . An electrospray voltage of 1.9 kV
was applied to the eluent via the EASY-Spray column electrode. The Orbitrap Fusion
1
was operated in positive ion data-dependent mode. Full scan MS was performed in the
Orbitrap with a normal precursor mass range of 350–1,500 m/z at a resolution of 120k.
The automatic gain control (AGC) target and maximum accumulation time settings
5
2
were set to 4
x
10 and 150 ms, respectively. MS was triggered by selecting the
3
most intense precursor ions above an intensity threshold of 1
x
10 for collision-induced
2
dissociation (CID)-MS fragmentation with an AGC target and maximum accumulation
4
time settings of 5
x
10 and 250 ms, respectively. Mass filtering was performed by the
quadrupole with 1.2 m/z transmission window, followed by CID fragmentation in the
Orbitrap at a resolution of 15k and a normalized collision energy (NCE) of 37%. To
improve the spectral acquisition rate parallelizable time was activated. The number of MS2
spectra acquired between full scans was restricted to a duty cycle of 3 s.
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Raw data files were processed with Proteome Discoverer (v1.4, Thermo Fisher
Scientific), using Sequest HT search node. Sequest HT searches were performed
against SwissProt human database (February 1, 2015). The mass tolerances for parent
ions and fragment ions were set as 10 ppm and 0.06 Da, respectively. Trypsin was set
as the enzyme and a maximum of two missed cleavages was allowed.
Carbamidomethylation of cysteine, methionine oxidation, deamidation (N,Q) and
Compound A (103.0269 m/z) adduct on cysteine were selected as variable
modifications. Peptide spectral matches (PSM) were performed against a target decoy
database. PSM’s were filtered with a false discovery rate (FDR) cutoff of 1%.
Synthesis of Compound A and derivatives - Methods for synthesis of Compound
A (3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile) and its derivatives,
along with NMR spectra of synthesized compounds are described in Supplemental
Materials.
Molecular Modeling – Cholesterol, phospholipid [1-Palmitoyl-2-oleoyl-sn-glycero-
3
-phosphocholine (POPC)] and Compound A binding to LCAT were modeled using
Maestro software (Release 2015-3; Schrodinger). LCAT coordinates from a recent X-
ray structure analysis (PDB: 4X96)(Glukhova et al., 2015) were used for molecular
docking, using the Glide application. Ligands were docked into the active site of LCAT,
using flexible ligand sampling.
To evaluate the influence of Cys31 mutations or Compound A and DNTB
adducts on LCAT structure, a high resolution LCAT structure was used (PDB:
4
XWG)(Piper et al., 2015). To prepare this structure, the Prime application was used to
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replace missing residues 231-237, the Tyr31 mutation was modified back to cysteine,
and energy minimization was performed to relax the modified structure. This starting
structure was then mutated in silico at Cys31 to simulate the various amino acid
substitutions or adduct formation by Compound A and DNTB. All-atom molecular
dynamics simulations were then performed, using the Desmond application in SPC
solvent with 0.15 M NaCl and at a temperature of 310 K. Simulations were run for 20 ns
on the NIH High Performance Computing Biowulf Cluster.
All computations for the electrostatic potential of Compound A were run using
Gaussian 09 software. Visualizations were made using Gaussview. A 6-31G* basis set
was used to optimize the structure and calculate the electrostatic potential of Compound
A using the Hartree-Fock method.
Statistical Analysis- Unless otherwise indicated, all results are expressed as the
(mean +/- SD) of at least triplicates. Values were compared by a 2-tailed t-test and P-
values <0.05 were considered to be statistically significant.
RESULTS
Naturally Occurring LCAT Mutations Activated By Compound A
Compound A and radiolabelled cholesterol were added to plasma from patients
bearing different LCAT mutations to measure the CER. Figure 1 shows that Compound
A increased LCAT activity in healthy control plasma (WT) by 5 to 6-fold. The M293R
LCAT homozygote and the compound LCAT heterozygotes Y312C/E354K and
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S91P/A141T had the highest response to Compound A, and reached activity levels that
were at least 50% of WT untreated plasma control samples. Two other LCAT mutations,
namely K218N and V46E, also showed considerable activation with Compound A, but
absolute activity levels of LCAT were still relatively low after Compound A treatment.
Mutations near the active site, which had no detectable baseline LCAT activity (R147W
and F278S), showed no response to Compound A.
Compound A increases apparent K and Vmax of LCAT by interacting with Cysteine 31
m
Using methylumbelliferyl palmitate as a substrate to monitor the phospholipase
step of the LCAT reaction, we tested the effect of Compound A on the apparent K and
m
Vmax of recombinant LCAT (Fig. 2). Compound A had only a limited effect on the
apparent K of LCAT but increased the apparent Vmax by almost 2-fold (control 7.47 +/-
m
0
.26 µM MU/30 min; Compound A treatment 14.9 +/- 0.6 µM MU/30 min).
It has previously been reported that Cys31 plays a role in modulating LCAT
activity(Francone and Fielding, 1991; Gunawardane et al., 2016; Kayser et al., 2008;
Piper et al., 2015; Qu et al., 1993). Dithiobis-nitrobenzoic acid (DTNB), a well-known
inhibitor of LCAT (Supplemental Fig. 1), forms a mixed disulfide bond with Cys31,
resulting in the addition of a nitrobenzoic acid group to Cys31(Francone and Fielding,
1
991). To test whether Compound A, which has three sulfur groups (Table I), may also
modulate LCAT activity by covalently reacting with Cys31, we used a codon-optimized
full-length human LCAT cDNA tagged with 6-His(Glukhova et al., 2015) and changed
Cys31 to glycine (C31G) by site-directed mutagenesis. We also mutated Cys184 to
glycine (C184G), because it is the only other free cysteine in LCAT. After transfection
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into cells, conditioned media from the cells were tested for LCAT activity, using MUP as
the substrate in the presence and absence of Compound A. As can be seen in Figure 3,
LCAT-C31G was not activated by Compound A, whereas LCAT-C184G was activated
to a similar extent as WT-LCAT.
In Figure 4, we show that replacement of Cys31 with either positively (Arg and
Lys) or negatively (Glu) charged amino acids inhibits LCAT activity (Fig. 4), consistent
with the proposed mechanism for DTNB inhibition. In contrast, replacement of Cys31
with bulky hydrophobic amino acids (Trp, Tyr, Phe, Leu and Met) resulted in a 2-3 fold
activation of LCAT. By comparison, replacement of Cys31 with small polar amino acids
(Ser and Thr) or His had a more limited effect (Fig. 4). These combined data are
consistent with a model wherein Compound A could activate LCAT by forming a
hydrophobic adduct with Cys31.
Compound A alkylates thiols by adding a cyanopyrazine moiety
To explore the reaction of Compound A with proteins containing a free cysteine,
we first investigated its effect on a mutated form of mouse msrA, which is 26 kDa in size
and contains a single, highly reactive low-pK cysteine in its active site(Lim et al., 2012).
a
Compound A was incubated with the mutated msrA and the reaction mixture was
analyzed for a mass shift directly without any trypsinization on a QTOF mass
spectrometer. The major modified form of msrA found after 30 min exposure to
Compound A gained +103.0 Da in mass and was stable to treatment with 25 mM DTT
for 90 min. Based on the structure of Compound A, this result is consistent with an
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alkylation of the free cysteine of msrA with the cyanopyrazine ring from Compound A
(Figure 5A), which is calculated to increase the mass by 103.02 Da.
Figure 5B shows a similar experiment after incubating Compound A with the
tripeptide glutathione, which also contains a single highly reactive cysteine. A molar
excess of glutathione readily reacted with Compound A to form a derivative with a
monoisotopic mass of 410.1021 and another product of mass 177.9710, which is
Compound A less the cyanopyrazine moiety. MS/MS of the derivatized glutathione
showed a difference of 103.0269 mass units, matching the mass difference in the y2 ion
plus/minus the cyanopyrazine Compound A adduct (Figure 5B).
The reaction of glutathione with Compound 5 (Table 1), a derivative of
Compound A that also contains a cyanopyrazine ring and activates LCAT, was
monitored by proton NMR (Fig. 6A). The resonance for one of two hydrogens (H ) on
a
the cyanopyrazine ring, shifted downfield from 8.79 to 8.59 ppm, indicative of a more
electron-withdrawing group (S-5-membered thiadiazole ring) in the para position being
replaced with a less electron-withdrawing group (Glutathione S). The entire NMR
spectrum for this reaction is shown in Supplemental Figure 2 and is consistent with the
proposed alkylation reaction of glutathione with the cyanopyrazine ring of Compound A
and its derivatives, as shown in the scheme in Figure 6B.
To gain further insight into how Compound A reacts with cysteines, we calculated
the relative electrostatic potential of Compound A by the Hartree-Fock method (Fig.
7
A,B). The carbon residue (atom C3, Fig. 7A) in the cyanopyrazine ring that alkylates
Cys31 of LCAT was predicted to be the most electropositive part of the molecule
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+0.433) (see also Table 2). This carbon, which is connected to an electron-withdrawing
(
S-thiadiazole group, is also at the end of an alpha-beta unsaturated conjugated π-
system due to the presence of the cyano group on the cyanopyrazine ring of Compound
A. This likely explains the electropositivity of this site and its susceptibility to nucleophilic
attack by reactive cysteine residues in msrA and glutathione. The charges on the
analogous carbons in Compound 5 and Compound 10 are similar (+0.414) (Fig. 7C,D;
Table 2).
In Figure 8, rhLCAT was incubated with Compound A or vehicle and then
deglycosylated, gel-purified and trypsinized. Tryptic peptides were analyzed by LC/MS
2
on an Orbitrap Fusion. In the MS mode, the monoisotopic m/z of the z=4 LCAT peptide
fragments containing Cys31 increased from 640.8445 to 666.5986 after incubation with
Compound A (Figures 8A and 8B). This corresponds to a mass difference of 103.0164
Da, nearly identical to the size shift seen with msrA and glutathione after incubation with
Compound A. The adduct was present only on LCAT incubated with Compound A and
3
+2
was not detected in LCAT incubated with vehicle. In the MS mode, the b16 fragment
m/z of the peptide containing Cys31 increased after incubation with Compound A from
8
44.94153 to 896.45148, or a mass difference of 103.01990 (Figure 8C, D). Consistent
with the formation of a cyanopyrazine adduct with an S-C bond rather than a disulfide
bond, DTT did not reverse LCAT activation by Compound A (Supplemental Figure 3).
Overall, the combined mass spectrometry and NMR data indicate that the
sulfhydryl group of Cys31 of LCAT acts as a nucleophile to attack Compound A,
resulting in its alkylation by the attachment of a cyanopyrazine group to the side chain of
Cys31.
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Molecular Modeling of WT-LCAT Treated with Compound A and LCAT Mutants
Recent X-ray structural analyses of LCAT(Glukhova et al., 2015; Gunawardane
et al., 2016; Piper et al., 2015) reveal that Cys31 lies near the catalytic triad active site,
with its backbone nitrogen contributing to the oxyanion hole. Therefore, to gain more
insight on how WT LCAT and its variants are altered by Compound A, we performed
molecular modeling studies.
First, we showed that both LCAT substrates, cholesterol and
phosphatidylcholine, were able to dock in the active site of WT LCAT in an orientation
that would be expected to promote their catalysis (Supplemental Figures 4A and 4B).
Compound A docked in the active site as well, with its cyanopyrazine ring positioned
close to the sulfhydryl group of Cys31, which would facilitate their reaction
(Supplemental Figure 4C; Fig. 9A). A close-up view of the WT active site docked with
phosphatidylcholine also shows how adding a hydrophobic adduct on Cys31 or
replacing Cys31 with an amino acid with a hydrophobic side chain could potentially
enhance phospholipid binding by increasing hydrophobic interactions with the glycerol
backbone of phosphatidylcholine (Fig. 9B).
For LCAT-C31E (Supplemental Figure 4D), which is catalytically inactive, the
positively charged choline head group of phosphatidylcholine was predicted to be
located near the negative side chain of glutamic acid in position 31 (Supplemental Fig.
4
E), thus preventing the proper orientation of the phospholipid substrate for catalysis
(compare Supplemental Figure 4D to Supplemental Figure 4B).
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Next, we did all-atom molecular dynamics simulations on WT-LCAT and several
site-directed mutants, as well as WT-LCAT modified by Compound A and DTNB. The
various changes in LCAT structure were made in silico, using WT-LCAT as the starting
o
structure, and were then allowed to relax in water at 37 C for 20 ns to reach a stable
state (Supplemental Fig. 5). As can be seen in Fig. 10A, the simulation predicted that
WT-LCAT undergoes considerable motion in the backbone of the membrane binding
domain (residues 36-101), which is adjacent to the active site(Glukhova et al., 2015).
Another dynamic region was predicted to be the lid region (residues 225-248) (Fig.
1
0A), which is near residues in the cap domain that help shape the substrate binding
pocket(Glukhova et al., 2015; Piper et al., 2015). When the root-mean-square
fluctuation (RMSF) difference in the backbone motion of LCAT modified with Compound
A or DTNB versus unmodified LCAT was calculated, several positions within the
membrane-binding domain and the lid and substrate binding pocket region showed
differential responses to the modifications (Fig. 10B). A similar analysis was performed
for various Cys31 mutants (C31M, C31E, C31F, C31L) that either activated or inhibited
LCAT (Fig. 4) and a plot correlating the backbone motion with LCAT activity was made
(Fig. 10C). This plot showed that areas of increased motion in the membrane binding
domain, especially residues 83-90, positively correlate with increased activity. In
contrast, increased motion around the lid region, particularly amino acids 238-242,
negatively correlated with LCAT activity. Overall, these results suggest that adduct
formation by Compound A on LCAT modifies its dynamic structure in particular regions
of LCAT that lead to enhanced activity.
Effect of Compound A Derivatives on LCAT activation
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To determine the structural requirements of Compound A for LCAT activation, we
made and tested several derivatives of Compound A (Table 1).
First, the alkyl substituent of the alkylthio group on the thiadiazole ring of
Compound A was changed from an ethyl to a methyl group, forming Compound 5
(Supplemental Figure 6A, Scheme 1). Compound 5 retained LCAT activation but its
EC50 was increased and its ECmax was reduced compared to Compound A (Table 1).
Replacement of the thiadiazole ring of Compound A with other groups, such as
thiophene (Supplemental Figure 6A, Scheme 1, Compound 6), as well as substituted
benzenes (Supplemental Figure 6A, Scheme 1, Compounds 7, 8), negated the ability of
these derivatives to activate LCAT. In fact, Compounds 6, 7, and 8 were inhibitory at
high concentrations (IC50 > 0.06). This is most likely due to the lack of Lewis bases,
such as nitrogen atoms, on the group attached to position 3 of the pyrazine ring. The
presence of atoms that are Lewis bases might be necessary for an appropriate
positioning of Compound A in close proximity to the Cys31 (Supplemental Figure 7),
which is a more likely reason for lack of activity of Compounds 6-8 (Supplemental
Figure 6A, Scheme 1) rather than their ability to become good leaving groups.
Compounds substituting the electron-withdrawing CN group on the pyrazine ring
of Compound A with CF
3
or NO
2
groups and changing the pyrazine group to pyridine
(Supplemental Figure 6B, Scheme 2, Compounds 12-14), or situating the CN group on
the opposite side of the ring as compared to Compound A (Compound 15) also did not
activate LCAT. In fact, Compound 14 was also an inhibitor at high concentrations (IC50
=
6.8 µM for 14). Furthermore, alteration of 13 (Supplemental Figure 6B, Scheme 2) by
substituting the methylsulfanyl-[1,3,4]thiadiazol-2-ylsulfanyl group with a 4-nitro-
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phenylsulfanyl group (14, Supplemental Figure 6B, Scheme 2) did not rescue its ability
to activate LCAT. The lack of Lewis bases (such as electron-rich nitrogen atoms) as in
Compounds 17 and 18 (Supplemental Figure 6C, Scheme 3) led to compounds that
were weak inhibitors of LCAT.
Next, we docked Compound A with LCAT to investigate potential interactions
with amino acid residues near the active site (Supplemental Fig. 7A, B). The S-ethyl
group attached to the thiadiazole ring was predicted to fit into and almost fully occupy a
pocket within the substrate binding pocket that contains both hydrophobic and
hydrophilic elements (Supplemental Fig. 7A, B). We, therefore, redesigned Compound
A by replacing the S-ethyl tail with a propionamide group (Compound 10, Supplemental
Figure 6A, Scheme 1), so that it could potentially form hydrogen bonds with amino acid
side chains in the active site (Supplemental Fig. 7C, D). Compound 10, however, was
only a relatively weak activator of LCAT compared to Compound A (Table 1). Upon
further inspection, the modeling revealed a potential clash between the propionamide
group of Compound 10 with Thr246 (Supplemental Fig. 7C,D), possibly explaining its
decreased potency (Table I). Finally, a comparison of the docking of Compound A
(
Supplemental Figure 7A, B) and Compound 5 (Supplemental Figure 7E, F) with the
LCAT active site revealed that Compound A, with its S-CH -CH tail, fit into the pocket
better than Compound 5, with its shorter S–CH tail, which likely accounts for the
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3
3
superior LCAT activation by Compound A. For all three compounds, Cys31 and Thr248
were shown to interact with electron-rich nitrogen atoms (Lewis bases) in Compound A
and its derivatives.
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Finally, we synthesized a novel class of thiol-reactive compounds based on
monocyclic β-lactams (Compounds 19 and 20, Table 1; Supplemental Figure 6D,
Scheme 4). Both of these compounds contain a methylthio group, which could form a
disulfide bond with LCAT by S-methylating Cys31. Because the C31M mutation in LCAT
had increased activity (Fig. 4), we predicted that these compounds would activate
LCAT. Both of these compounds were nearly good as Compound A in activating LCAT
(Table 1). In the case of Compound 19, LCAT activation could be reversed with DTT,
unlike Compound A, consistent with Compound 19 forming an adduct with LCAT by
forming of a disulfide bond.
DISCUSSION
A potential clinical indication for small-molecule LCAT activators, such as
Compound A, is for the treatment of FLD. We previously investigated the use of rhLCAT
as a potential therapy for FLD and found that it was safe and well-tolerated(Shamburek
et al., 2016a; Shamburek et al., 2016b). Moreover, rhLCAT treatment almost completely
normalized HDL-C levels, improved anemia, and stabilized renal function in a FLD
patient with advanced kidney disease(Shamburek et al., 2016a). rhLCAT, however, is
an expensive biologic that requires repeated intravenous infusions. A small-molecule
treatment approach would be a preferable therapy, if such a drug would activate the
LCAT mutations that cause FLD. Compound A has already been shown to increase
LCAT activity and raise HDL-C in plasma of mice and hamsters (see (Chen et al.,
2
012)). We observed, in this study, that three out of nine FLD mutations were activated
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by Compound A to a level comparable to FLD heterozygotes. Because FLD
heterozygotes are asymptomatic except for their low normal HDL-C levels, these results
suggest that small molecule LCAT activators could be a feasible therapeutic approach
for a subset of FLD patients, especially since the primary site of action of LCAT is in the
plasma.
A second possible clinical indication for small-molecule LCAT activators is for the
treatment of CVD. The strong negative correlation between plasma HDL-C levels and
CVD risk and the ability of HDL to promote cholesterol efflux from cholesterol-loaded
macrophages suggest that HDL-raising therapies might offer protection against CVD.
Although some HDL-C raising treatments, such as CETP inhibitors(Karathanasis et al.,
2
017), have not so far met with success in protecting against CVD, the possible efficacy
of LCAT as a CVD therapeutic has not been tested. Studies of the role of LCAT,
however, in the pathogenesis of atherosclerosis in both human and animal studies have
yielded conflicting results(Ahsan et al., 2014; Rousset et al., 2011). A Mendelian
randomization study of subjects with low HDL-C due to LCAT SNPs concluded that low
levels of LCAT activity were not associated with an increased risk of CVD(Haase et al.,
2
012). In contrast, increased levels of pre-β HDL and low LCAT activity have been
found in a cohort of patients with CVD(Sethi et al., 2010). In addition,
some(Duivenvoorden et al., 2011; Hovingh et al., 2005; van den Bogaard et al., 2012)
but not all(Calabresi et al., 2009) FLD heterozygotes appear to have increased carotid
atherosclerosis(Ahsan et al., 2014). Moreover, intravenous rhLCAT infusion reduced
atherosclerosis in rabbits(Zhou, 2009) and also improved plasma cholesterol efflux and
raised plasma HDL-C levels in CVD patients(Shamburek et al., 2016b). If ongoing
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clinical trials of rhLCAT demonstrate benefit in reducing atherosclerosis and/or in
reducing clinical events in CVD patients, this will likely greatly stimulate efforts to
develop small molecule activators of LCAT.
Another major finding from this study is that Compound A forms a hydrophobic
adduct with Cys31 of LCAT and increases the apparent Vmax of LCAT for at least the
first step of the reaction. Site-directed mutagenesis also showed that amino acids with
hydrophobic side chains at residue 31 increased LCAT activity, whereas charged amino
acid residues, both positive and negative, at the same position, strongly inhibited LCAT.
These results are consistent with the qualitative findings for LCAT mutants first reported
in the patent for Compound A (Kayser et al., 2008). Replacing Cys31 with Tyr has also
been previously described to enhance LCAT activity without significantly altering its
structure(Piper et al., 2015). Interestingly, in chickens, the amino acid corresponding to
Cys31 in human LCAT is the hydrophobic amino acid phenylalanine(Hengstschlager-
Ottnad et al., 1995), and chickens have relatively high plasma levels of HDL-C.
Consistent with our observation of decreased activity of the C31E mutation, DTNB,
which adds a negatively charged adduct to Cys, is also known to inhibit LCAT(Francone
and Fielding, 1991) (Supplemental Figure 1). Mass spectrometry and NMR analysis of
msrA and glutathione incubated with Compound A or its derivatives were all consistent
with the presence of a hydrophobic cyanopyrazine adduct on the single cysteine in
these two model compounds. Finally, mass spectrometry of LCAT incubated with
Compound A revealed a +103.016 m/z adduct on the tryptic peptide containing Cys31,
thus confirming the addition of a cyanopyrazine ring to Cys31 of LCAT.
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Cys31 is located within the active site of LCAT(Glukhova et al., 2015), and
therefore it is not unexpected that modification of this residue can affect LCAT activity.
Like other interfacial enzymes, LCAT is unable to synthesize CE until it binds to a
lipoprotein surface(Ahsan et al., 2014). The interaction with lipoproteins provides the
substrates for LCAT but is also predicted to cause the displacement of the lid region
that blocks the substrate binding pocket(Ahsan et al., 2014). By our modeling studies,
adducts to Cys31 that activate LCAT or site directed mutations in Cys31 that activate
LCAT all show increased movement of the membrane binding domain, which may
account for the increased LCAT activity after Compound A treatment. Changes in the
movement of other regions of LCAT, such as in the lid and substrate binding region,
also correlated with changes in activity. From the crystal structures of WT-
LCAT(Glukhova et al., 2015) and LCAT-C31Y(Piper et al., 2015), the cavity formed by
the substrate binding pocket is relatively large and should be able to readily
accommodate adducts at Cys31. As opposed to charge adducts at this position that
inhibit LCAT, such as occurs with DTNB, hydrophobic adducts appear to activate LCAT
by either promoting the binding of its hydrophobic lipid substrates and/or by promoting a
more favorable orientation of the lipid substrate for catalysis. Ongoing efforts to obtain
the crystal structure of LCAT modified by Compound A may provide further insight into
the mechanism of LCAT activation.
None of the Compound A derivatives that we synthesized showed improvements
over the original compound first produced by Amgen in activating LCAT. In fact, many of
them turned out to be inhibitors of LCAT at higher concentrations. This may have
occurred because Cys31 is in close proximity to the catalytic triad (Ser181, Asp345,
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His377), particularly Ser181, which acts as a nucleophile in the first step of the LCAT
reaction(Ahsan et al., 2014). This makes preparation of LCAT activators acting at Cys31
challenging, because they will have to be electrophiles that selectively react only with
Cys31, and not with the active site serine residue. Alkylating reagents under future
consideration should, therefore, be relatively inactive toward oxygen nucleophiles like
Ser. The fragment-based synthetic studies that we did on Compound A revealed the
importance of the presence of an α, β – unsaturated system (cyanopyrazine ring), as
well as the presence of Lewis bases (e.g. nitrogen atoms in the cyanopyrazine ring).
The S-ethyl group tail of Compound A also appears to be necessary for appropriately
situating the molecule via hydrogen-bonding toward the Cys31 for nucleophilic attack.
A potential limitation of Compound A is that its reaction with glutathione may
indicate a lack of specificity. Many drugs that target sulfhydryl groups are in fact non-
specific and may react with reactive cysteines on other proteins(Zuniga et al., 2012).
There are, however, many examples of sulfhydryl-targeting drugs in current use.
Reaction of compound A with LCAT may be somewhat specific, since the reaction with
LCAT was complete at 30-60 minutes, whereas reaction of Compound A with
glutathione took 48 hours to complete. The Cys31 sulfhydryl in LCAT may be more
reactive, possibly due to a lower pKa induced by the effect of neighboring residues in
the three-dimensional structure. In general, the rate of reaction of a cysteine is known to
be directly proportional to the fraction that is ionized to the thiolate – and that is
controlled by the pKa. The excellent fit of Compound A inside the LCAT binding pocket
may also add to its specificity for LCAT.
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We did succeed in producing a new class of small molecule activators of LCAT
based on thiol-reactive β-lactams. Because of their reversibility after DTT treatment,
these new compounds likely activate LCAT by forming a mixed disulfide with LCAT by
S-methylating Cys31. Again, a limitation of this new class of compounds is that many
drugs that target sulfhydryl groups are non-specific and may react with reactive
cysteines on other proteins(Zuniga et al., 2012). Previous studies of the N-methylthio
class of lactams have shown that the N–S bond is relatively resistant to cleavage by
most types of nucleophiles but is susceptible to thiophiles(Shah and Cama, 1987;
Woulfe et al., 1985) Moreover, N-thiolated lactams are also known to be non-toxic to
mammalian cells(Revell et al., 2007). If the lactam ring of these compounds can be
appropriately decorated in order to improve selectivity, perhaps by making it structurally
resemble a lipid substrate, it may be possible to make specific LCAT activators based
on this class of molecules.
In summary, we describe the mechanism for Compound A activation of LCAT
and show that some natural LCAT mutations can be considerably activated by
Compound A, thus revealing a possible new treatment strategy for FLD. We also
describe a new class of small molecule activators of LCAT based on sulfhydryl-reactive
β-lactams. These results provide important new insights into the design of future LCAT
activators.
ACKNOWLEDGMENTS
We thank Dr. Tim Hanusa for advice with the Hartree-Fock modeling studies and Dr.
Alisa Glukhova for construction of the WT-LCAT-6-His pcDNA4 plasmid.
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AUTHORSHIP CONTRIBUTIONS
Participated in research design: Freeman, Demosky, Konaklieva, Kuskovsky,
Aponte, Ossoli, Gordon, Koby, Shen, Vaisman, Jadhav, Calabresi, Gucek, Levine,
Remaley.
Conducted experiments: Freeman, Demosky, Konaklieva, Kuskovsky, Aponte,
Ossoli, Koby, Levine.
Contributed new reagents or analytic tools: Konaklieva, Kuskovsky, Shamburek,
Calabresi, Levine.
Performed data analysis: Freeman, Demosky, Konaklieva, Kuskovsky, Aponte,
Ossoli, Gordon, Koby, Shen, Jadhav, Levine, Remaley.
Wrote or contributed to the writing of the manuscript: Freeman, Konaklieva,
Aponte, Ossoli, Manthei, Tesmer, Levine, Remaley.
Supporting Information: Additional material is online in Supplementary Materials.
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