Ms. No. JA993387I Neomycin–Acridine Conjugate: A Potent Inhibitor of Rev-RRE Binding Sarah R. Kirk, Nathan W. Luedtke and Yitzhak Tor* Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0358. Supporting Information: 1. Synthetic procedures and characterization of neo–acridine (2) and its synthetic intermediates •Figure S1. Synthetic scheme of neo–acridine 2. Synthetic procedures and characterization of Rev peptides and the RRE RNA 3. Experimental procedures and additional data for gel-mobility shift experiments •Figure S2a. Gel-mobility shift for neo–acridine/RRE binding •Figure S2b. Gel-mobility shift for Rev/RRE binding 4. Experimental procedures and conditions for fluorescence anisotropy measurements 5. Equations for calculating K d and K i values •Figure S3a. Binding curve of RRE to Rev-Fl •Figure S3b. Displacement of Rev off the RRE by neomycin B 6. Experimental procedures and additional data for enzymatic footprinting experiments •Figure S4. Enzymatic footprinting on the RRE by RNase V1, RNase T1, and RNase A •Figure S5 . Quantitative representation of RNase V1 cleavage of the RRE in the presence of Rev •Figure S6 . Quantitative representation of RNase V1 cleavage of the RRE in the presence of neo–acridine •Figure S7 . Quantitative representation of RNase T1 and RNase A cleavage of the RRE in the presence of Rev •Figure S8 . Quantitative representation of RNase T1 and RNase A cleavage of the RRE in the presence of neo–acridine
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Ms. No. JA993387I
Neomycin–Acridine Conjugate: A Potent Inhibitor of Rev-RRE Binding
Sarah R. Kirk, Nathan W. Luedtke and Yitzhak Tor*
Department of Chemistry and Biochemistry, University of California, San Diego,
La Jolla, CA 92093-0358.
Supporting Information:
1. Synthetic procedures and characterization of neo–acridine (2) and its synthetic intermediates
•Figure S1. Synthetic scheme of neo–acridine
2. Synthetic procedures and characterization of Rev peptides and the RRE RNA
3. Experimental procedures and additional data for gel-mobility shift experiments
•Figure S2a. Gel-mobility shift for neo–acridine/RRE binding
•Figure S2b. Gel-mobility shift for Rev/RRE binding
4. Experimental procedures and conditions for fluorescence anisotropy measurements
5. Equations for calculating Kd and Ki values
•Figure S3a. Binding curve of RRE to Rev-Fl
•Figure S3b. Displacement of Rev off the RRE by neomycin B
6. Experimental procedures and additional data for enzymatic footprinting experiments
•Figure S4. Enzymatic footprinting on the RRE by RNase V1, RNase T1, and RNase A
•Figure S5 . Quantitative representation of RNase V1 cleavage of the RRE in the presence
of Rev
•Figure S6 . Quantitative representation of RNase V1 cleavage of the RRE in the presence
of neo–acridine
•Figure S7. Quantitative representation of RNase T1 and RNase A cleavage of the RRE in
the presence of Rev
•Figure S8. Quantitative representation of RNase T1 and RNase A cleavage of the RRE in
the presence of neo–acridine
Neo–acridine (2) Synthesis - experimental
Compound 1a and 1b have previously been reported (Michael, K.; Wang, H.; Tor, Y. Bioorg.
Med. Chem. 1999, 7, 1361-1371)
Compound 1c. Freshly cut sodium metal (0.56 g, 24.4 mmol) was dissolved in ethanol (25 mL,
degassed under argon). Aminoethanethiol·HCl (1.337 g, 11.8 mmol) was added to the solution at
room temperature and stirred for 15 min under argon. Compound 1b (0.40 g, 0.27 mmol) was
dissolved in ethanol (10 mL) and canulated into the thiolate salt solution. The reaction mixture was
stirred at room temperature for 5 hours. The reaction was quenched with cold CH2Cl2 (400 mL)
and sodium phosphate (50 mL, pH 5-6) on ice. The CH2Cl2 solution was then washed with brine
(2 × 50 mL). The organic layer was neutralized with sodium bicarbonate (2 × 50 mL), dried over
Na2SO4, and concentrated in vacuo. Flash column chromatography (9% CH3OH in CH2Cl2)
afforded the desired product as a white solid (0.228 g, 66%). Rf 0.24 (10% CH3OH in CH2Cl2);
Upon binding the RRE, only minor changes in the emission spectrum of fluorescein were seen
(about 10% quenching of Rev-Fl). We have taken the change in anisotropy as being directly
proportional to the fraction of Rev-Fl bound by the RRE. At the starting point of each titration
there is 5 nM of unbound Rev-Fl, 5 nM Rev-Fl/RRE complex and 3.5 nM free RRE (see figure
S3a). Following complex formation, an inhibitor [I] is titrated into the thermocontrolled cuvette,
and a decrease in anisotropy is observed (see figure S3b for an example). At high concentrations
of inhibitor, the change in anisotropy saturates at 0.081 (the same value observed for the unbound
Rev-Fl peptide). Sufficient mixing time was always provided to allow for equilibrium to be
reached. At the concentration of inhibitor which disrupts half of the formed Rev-RRE complex,
there must be 2.5 nM of the Rev-Fl/RRE complex, 7.5 nM of free Rev-Fl, 5.23 nM of the
RRE/inhibitor complex, and 0.77 nM of free RRE (calculated from the Kd of Rev-Fl/RRE). From
a simple, three component, competitive binding equilibrium, the following equation can be derived:
The Kd of the Rev-RRE interaction is calculated by titration of the prefolded 67-nt RRE fragment
into a solution of 10 nM Rev-Fl.
[ RRE ] + [ RevFl ] [ RevFl - RRE ]
Kd = 2.3 nM+
[ I ]
Ki
[ I - RRE ]
[ RevFl ]
Kd [ RevFl-RRE ]
[ I - RRE ]Ki =
[ I ]
KD = 2.4 nM
0 50 100 150 200 250 300
0.080
0.085
0.090
0.095
0.100
0.105
0.110
0.115
0 2 4 6 8 10 12 14 160.080
0.082
0.084
0.086
0.088
0.090
0.092
0.094
0.096
0.098
Kd = 2.3 nMA
niso
trop
y
Concentration of Neomycin B (µM)
Ani
sotr
opy
Concentration of RRE (nM)
IC50 = 0.8 µM
a.
b.
A= anisotropy of the Rev-Fl A0= anisotropy of the Rev-Fl in the absence of RNA∆A=the total change in anisotropy at saturation of the Rev-Fl
Figure S3a. Both line shape and gel shift analysis indicate a 1:1 complex of the RRE to Rev-Fl. Given this, a Kd of 2.3 nM was calculated by nonlinear regression using the equation:
Figure S4. Enzymatic cleavage of RRE by ribonuclease T1, ribonuclease A, and ribonuclease V1. All lanes contained 25 nM RRE with trace 5’-32P labeled RRE. Lane 1, control; Lanes 2–10 contain 1000 units ribonuclease T1; lane 3, 0.5 µM Rev; lane 4, 2 µM Rev; lane 5, 5 µM Rev; lane 6, 10 µM Rev; lane 7, 0.5 µM neo−acridine; lane 8, 2 µM neo−acridine; lane 9, 5 µM neo−acridine; lane 10, 10 µM neo−acridine; lane 11, T1 sequencing; lane 12, U2 sequencing; lane 13, base-generated ladder. Lanes 14–22 contain 1 × 10-5 units ribonuclease A; lane 15, 0.5 µM Rev; lane 16, 2 µM Rev; lane 17, 5 µM Rev; lane 18, 10 µM Rev; lane 19, 0.5 µM neo −acridine; lane 20, 2 µM neo−acridine; lane 21, 5 µM neo−acridine; lane 22, 10 µM neo−acridine. Lanes 23–31 contain 7.2 units ribonuclease V1; lane 24, 0.5 µM Rev; lane 25, 2 µM Rev; lane 26, 5 µM Rev; lane 27, 10 µM Rev; lane 28, 0.5 µM neo−acridine; lane 29, 2 µM neo−acridine; lane 30, 5 µM neo−acridine; lane 31, 10 µM neo−acridine; lane 32, base-generated ladder.