1300
A. R. Kore et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1296–1300
10. Data for MeOSuc-APAF-CH2Cl 11: 1H NMR (D2O, 400 MHz) d 7.27 to 7.07 (m,
7H), 6.52 (d, J = 6.8 Hz, 1H), 4.81 (m, 1H), 4.61 (m, 1H), 4.35 (m, 2H), 4.28 (d,
J = 16.4 Hz, 1H), 4.04 (d, J = 16.4 Hz, 1H), 3.72 (m, 1H), 3.64 (s, 3H), 3.54 (m, 1H),
3.16 (m, 1H), 2.96 (m, 1H), 2.61 (m, 2H), 2.50 (m, 2H), 2.11–1.81 (m, 44), 1.32
(d, J = 6.8 Hz, 3H), 1.21 (d, J = 7.2 Hz, 3H); MS (m/z): 551 [M+H]+.
4 provides data from a protein gel in which the marker ladder
ranges in size from 10 to 250 kDa, intact Ultrapure BSA substrate
(Applied Biosystems) is at about 70 kDa, and proteinase K is at
about 35 kDa. Figure 4 shows that MeOSuc-AAPF-CH2Cl 7 was
11. Preparation of stop solution with inhibitor: Typically, stop solution was prepared
capable of inhibiting proteinase K (100
lg/ml) at concentrations
in 100-
EGTA pH 9.0, 88 mM; compound 1, 7, or 11, at 11 mM; placental RNase
inhibitor protein, 2.2 U/ l; and DTT, 0.11 mM. The solution pH was adjusted to
ll volumes, containing the following components: Tris pH 8.0, 11 mM;
as low as 0.125 mM, while MeOSuc-AAPV-CH2Cl 1 was only capa-
l
ble of inhibiting proteinase K (100
low as 0.25 mM. (Data not shown)
lg/ml) at a concentrations as
8.0, if required, by dilute HCl. A dilution solution was prepared identically,
except that inhibitor analog was omitted. A series of solutions was made for
each inhibitor were made by diluting the initial solutions with the dilution
solution so that the final concentrations were 11, 8.25, 5.5, 2.75, and 1.10 mM
(0, 25%, 50%, and 75% dilutions).
In conclusion, we have synthesized and evaluated the new po-
tent proteinase K inhibitor MeOSuc-AAPF-CH2Cl 7, which inhibits
proteinase K activity at 10-fold lower concentrations than the cur-
rently-known inhibitor, MeOSuc-AAPV-CH2Cl 1. Computer model-
ing studies indicated the presence of an additional small binding
pocket in the vicinity of the proteinase K active site, which we pos-
tulate is filled by the aromatic phenylalanine moiety. The phenyl-
alanine residue aromatic ring, which fits into this binding pocket
and potentially offers additional van der Waals, hydrogen bonding,
and/or hydrophobic interactions, enhances the inhibitor potency.
We believe that the new inhibitor would be especially useful in
molecular biology applications, especially sample preparation
areas, where proteinase K used to digest away cellular components
including nucleases can be inhibited in situ without its removal,
and as a result, it will allow RT-qPCR to be subsequently performed
in the same reaction mixture. In addition, due to the runaway pro-
tease activity occurring in some pathogenic situations, this mole-
cule may have applications in the medical field in the future as
well.
12. Proteinase K treatment of reverse transcription reactions. All reactions were
carried out in triplicate. Fifty microliters of proteinase K solution (without
DNase mixed in) from the TaqManÒ gene expression Cells-to-CTTM kit (Applied
Biosystems) were aliquoted into 18 tubes. To each of these (these are
equivalent to lysis mixtures in the Cells-to-CTTM workflow),
5 ll of the
various stop solutions was added, resulting in 1, 0.75, 0.5, 0.25, and 0.11 mM
concentrations of the inhibitor analogs in the proteinase K solution. These were
incubated 2 min at room temperature. For the control reaction, 50
proteinase K solution was mixed with 5 l of the dilution solution and heated
at 95 °C for 10 min to thermally inactivate the proteinase K reaction. Next, the
reverse transcription reagents were prepared, that is, 15
l 2ꢁ RT buffer, and
1.5
l of 20ꢁ RT enzyme mix, were aliquoted and mixedin the wells of a 96-
well plate. To this solution, 13.5 l of each proteinase K + stop mixture was
ll of
l
l
l
l
added and the solution was mixed well. The resulting mixtures, containing
0.45, 0.3375, 0.224, 0.1125, and 0.045 mM of the appropriate analog was
incubated at room temperature for 1 h for residual PK activity to degrade the
RT enzyme. After 1 h incubation at room temperature, 1
ll of Xeno RNA
(diluted to 25,000 copies/ l in 10 ng/ l poly A) was added to all wells except
l
l
for the no template control wells. Then the reverse transcription (RT) reaction
was incubated at 37 °C for 60 min, 95 °C for 5 min, and cooled to 4 °C, using the
GeneAmpÒ 9700 PCR system (Applied Biosystems).
13. Real-time PCR: Real-time PCR was performed by using the Xeno AOD (Assay on
Demand) TaqManÒ Cells-to-CTTM control kit, on a 7500 Fast Real-Time PCR
Acknowledgments
system (Applied Biosystems). In each reaction, the equivalent of 12.5
TaqMan GEX master mix mixed with 1.25
l of 20ꢁ Xeno AOD was dispensed
in each well of a 96-well plate. 11.25 l of each RT reaction was then added to
each well. For three control reactions, 11.25 l of water was added to the three
ll of
l
l
We are thankful to Rick Conrad (Life Technologies, Inc.) for
commenting and critical reading of this manuscript. We also thank
to Brett C. Petrie, Robert Setterquist, and Brian S. Kim for encour-
agement and Richard Fekete for stimulating discussion.
l
wells. Then the 96-well plate was run by using the gene expression parameters
as per manufacturer’s instruction.
14. Wolf, W. M.; Bajorath, J.; Muller, A.; Raghunathan, S.; Singh, T. P.; Hinrichs, W.;
Saenger, W. J. Biol. Chem. 1991, 266, 17695.
15. Navia, M. A.; McKeever, B. M.; Springer, J. P.; Lin, T. Y.; Williams, H. R.; Fluder, E.
M.; Dorn, C. P.; Hoogsteen, K. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 7.
16. Emsley, P.; Cowtan, K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004, 60, 2126.
17. DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific, Palo
References and notes
1. Kettner, C.; Shaw, E. Methods Enzymol. 1981, 80, 820.
2. Schoellmann, G.; Shaw, E. Biochemistry 1963, 2, 252.
3. Shaw, E.; Mares-Guia, M.; Cohen, W. Biochemistry 1965, 4, 2219.
4. IUPAC-IUB Commission on Biochemical Nomenclature (CBN) Biochemistry
1972, 11, 1726.
18. Independent digestion assay of compound 7 and 1: For the direct assay of PK
inactivation, 50 ll of proteinase K (100 lg/ml) solution was mixed with 5 ll of
stop solution with varying amounts of compound 7 and 1 separately and
incubated for 10 min at room temperature. Ten microliters of Ultrapure BSA
(50 mg/ml) was added to each sample and the samples were incubated for
10 min at room temperature. The reaction mixture was heated for 30 min at
95 °C to inactivate any remaining functional PK and the sample mixes analyzed
5. Powers, J. C.; Asgian, J. L.; Ekici, Z. D.; James, E. Chem. Rev. 2002, 102, 4639.
6. Harper, J. W.; Hemmi, K.; Powers, J. C. Biochemistry 1985, 24, 1831.
7. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157.
8. Poulos, T.; Alden, R. A.; Freer, S. T.; Birktoft, J. J.; Kraut, J. J. Biol. Chem. 1976, 251,
1097.
using precast protein gels. For each 65
mixed with 5 l of gel-loading dye and further heated at 95 °C for 5 min. Then,
the samples were kept on ice for 2 min and 15 l of reaction mixture was
loaded on the gel along with ladder. For the control reactions, 50 l of PK
l of BSA were mock-incubates and analyzed in a
ll of total reaction mixture, 10 ll was
9. Data for MeOSuc-AAPF-CH2Cl 7: 1H NMR (D2O, 400 MHz) d 7.76 (m, 2H), 7.26–
7.11 (m, 5H), 6.43 (d, J = 7.6 Hz, 1H), 4.78 (m, 1H), 4.64 (m, 2H), 4.49 (m, 1H),
4.10 (m, 2H), 3.72 (m, 1H), 3.65 (s, 3H), 3.56 (m, 1H), 3.11 (m, 1H), 2.95 (m, 1H),
2.59 (m, 2H), 2.47 (m, 2H), 2.14–1.93 (m, 4H), 1.35 to 1.19 (m, 6H); MS (m/z):
551 [M+H]+.
l
l
l
solution without BSA, and 10
similar fashion.
l