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Inhibitory profile of a LEDGF/p75 peptide against HIV-1 integrase:
insight into integrase-DNA complex formation and catalysis
Laith Q. Al-Mawsawia, Frauke Christb, Raveendra Dayama, Zeger Debyserb, Nouri Neamatia,*
a
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of
Southern California, Los Angeles, California 90089, USA
b
The Laboratory for Molecular Virology and Gene Therapy, KULeuven and IRC KULAK,
Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
*Corresponding author: Nouri Neamati, Department of Pharmacology and Pharmaceutical
Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA.
Tel.: +1 323-442-2341; Fax: +1 323-442-1390; Email: [email protected]
2. Materials and methods (Supplemental)
2.1 Site Directed Mutagenesis
Site directed mutagenesis was conducted on the pET-15b-IN plasmid, a generous gift
from Dr. Robert Craigie, Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD. The
plasmid contains full length WT IN fused to a linker containing a 6-residue N-terminal histidine
tag downstream from a T7 promoter. A Quickchange site-directed mutagenesis kit (Stratagene)
was employed to make the point mutations according to the manufacturer’s instructions. The
primers used to introduce the amino acid substitutions at positions W132, M178, F181, and F185
were utilized previously in our laboratory and will be disclosed in an upcoming manuscript that
is currently under review.
Nucleotide replacements were confirmed by sequencing at the
USC/Norris Comprehensive Cancer Center Microchemical Core Facility (University of Southern
California).
2.2 Oligonucleotide Substrates and IN Inhibition Assays
Oligonucleotide substrates mimicking the HIV-1 U5 LTR DNA termini, 19-mer top
strand
(5’-GTGTGGAAAATCTCTAGCA-3’),
GTGTGGAAAATCTCTAGCAGT-3’)
and
21-mer
21-mer
top
strand
(5’-
bottom
strand
(5’-
ACTGCTAGAGATTTTCCACAC-3’), were purchased from USC/Norris Comprehensive
Cancer Center Microchemical Core Facility (University of Southern California) and purified by
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UV shadowing on polyacrylamide gel. In order to analyze the amount of both DNA reaction
events, the 21-mer top oligonucleotide was 5’-end-labeled using T4 polynucleotide kinase
(Epicenter, Madison, WI) and γ-[32P]ATP (MP Biomedicals, Irvine, CA). Afterwards the kinase
was heat inactivated, and 21-mer bottom oligonucleotide was added in 1.5 molar excess. The
sample was then heated to 95°C, cooled slowly, and subsequently passed through a G-25
Sephadex spin column (USA Scientific) to remove any unincorporated material from the
annealed double-stranded oligonucleotide.
IN inhibition assays for wild type (WT) and each mutant followed the same general
procedure. To determine the degree of inhibition on both 3’-processing and strand transfer,
purified IN enzyme (final concentration: 200 nM) was pre-incubated with peptide (dissolved in
water; stored at -20° C) in a reaction buffer [7.5 mM MnCl2, 0.1 mg/mL bovine serum albumin,
10 mM β-mercaptoethanol, and 25 mM MOPS, pH 7.2] for 30 min. at 30° C. For Mg2+ assay,
7.5 mM MgCl2 was substituted for MnCl2 in reaction buffer.
355
The LEDGF/p75 peptide
IHAEIKNSLKIDNLDVNRCIEAL377 was purchased from USC/Norris Comprehensive
Cancer Center Microchemical Core Facility (University of Southern California).
Mg2+
experiments were performed in the presence and absence of PEG in the reaction buffer to
determine optimal enzymatic conditions. This was followed by the addition of 20 nM of the 5’end
32
P-labeled linear double stranded oligonucleotide, and incubation was continued for an
additional one hour at the same temperature. For pre-assembled Ca2+ assay, 7.5 mM CaCl2 was
substituted for MnCl2 in above reaction buffer with the immediate addition of 20 nM 5’-end 32Plabeled linear double stranded oligonucleotide. The sample was then incubated for 30 min. at
30° C followed by the addition of inhibitor. After an additional incubation of 15 min. at 30° C,
MnCl2 (100mM) was added to the sample to initiate catalysis.
Upon completion the sample
reactions were quenched by adding half the total volume (8 µL) of loading dye (98% deionized
formamide, 10 mM EDTA, 0.025% xylene cyanol, and 0.025% bromophenol blue). A sample
aliquot was electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M Tris-borate, pH
8.3, 2 mM EDTA, 20% acrylamide, and 8 M urea).
Gels were vacuum dried, exposed in a PhosphorImager cassette, analyzed using a
Typhoon 8610 Variable Mode Imager (Amersham Biosciences), and quantified using
ImageQuant 5.2. Percent inhibition (% I) was calculated using the following equation:
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% I = 100 x [1 – (D – C)/(N – C)]
where C, N, and D are the fractions of 21-mer substrate converted to 19-mer (3’-processing
product) or strand transfer products for DNA alone, DNA plus IN, and IN plus drug,
respectively. IC50 values were determined by plotting the logarithm of drug concentration versus
the calculated percent inhibition to obtain the concentration that produced 50% inhibition.
Peptide Name: LEDGF IBD
Peptide Sequence: IHAEIKNSLKIDNLDVNRCIEAL
AU
1.0
0.5
0.0
-0.5
0
20
40
60
80
100
120
140
160
180
200
Minutes
2622.0
1.10e7
1.05e7
1.00e7
9.50e6
8.50e6
Intensity, cps
7.50e6
6.50e6
14PE-Pure
5.50e6
4.50e6
3.50e6
2.50e6
1.50e6
1.00e6
5.00e5
2200
2400
2600
2800
3000
3200
Mass, amu
Supplemental
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Figure
1.
The
synthesized
LEDGF/p75
peptide
IHAEIKNSLKIDNLDVNRCIEAL377 was determined to be 97.8% pure based on (A) C18
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reverse phase HPLC and corresponds to the correct mass of 2622 amu through (B) mass
spectrometric analysis.
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