J.-T. Nguyen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 366–370
369
IC50 = 88 nM). A substitution study with Tle and Phg
at the respective P2 and P3 positions, followed by re-
moval of the P02–P30 residue and optimization of the P10
cap moiety from the structural design of KNI-10166,
led to the intuitive discovery of KNI-10516 (27), which
is a tetrapeptidic HTLV-I PR inhibitor, composing of
only non-natural amino acid residues and exhibiting
highly potent inhibition (IC50 = 107 nM) against
HTLV-I PR.
Acknowledgements
This research was supported by The Frontier
Research Program; The 21st Century COE Program
from The Ministry of Education, Culture, Sports,
Science and Technology, Japan; and Japan Society
for the Promotion of Science’s Post-Doctoral Fellow-
ship for Foreign Researchers. We are grateful to Mr.
T. Hamada for mass spectra and HIV-1 PR assay
determinations.
Figure 1. Computer model of KNI-10516 (27) in the active site of
HTLV-I PR. Dotted lines represent possible hydrogen bond interac-
tions throughout the backbone of the inhibitor and HTLV-I PR’s
Asp32A, Gly34A, Asp36A, Leu57A, Ala59A, Asp32B, and Ala59B.
Interactions between Ala59A and Ala59B in the hairpin regions of
HTLV-I PRs flaps and the inhibitor is mediated by a water molecule.
The inhibitor’s transition-state mimic HMC isostere interacts with
catalytic Asp32A and Asp32B.
References and notes
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Soares, B. C.; Murphy, E. L. Retrovirology 2005, 24, 6058.
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J. Chem. Biol. 2003, 10, 373.
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R. A.; Shuker, S. B. Bioorg. Chem. 2002, 30, 138.
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Hayashi, Y.; Kiso, Y. Bioorg. Med. Chem. Lett. 2004, 14,
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9. Kimura, T.; Nguyen, J.-T.; Maegawa, H.; Nishiyama, K.;
Arii, Y.; Matsui, Y.; Hayashi, Y.; Kiso, Y. Bioorg. Med.
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10. KNI-10167 (6), Ac-Ile-Apns-Dmt-Ile-Met-NH2; 39%
HTLV-I PR inhibition at 1 lM; 89% HIV-1 PR inhibition
at 50 nM. KNI-10472, Ac-Tle-Apns-Dmt-Ile-NH2; 5%
HTLV-I PR inhibition at 600 nM; 30% HIV-1 PR
inhibition at 50 nM. KNI-10471, Ac-Phg-Tle-Apns-Dmt-
NH2; 8% HTLV-I PR inhibition at 600 nM; 36% HIV-1
PR inhibition at 50 nM.
11. Naka, H.; Teruya, K.; Bang, J. K.; Aimoto, S.; Tatsumi,
T.; Konno, H.; Nosaka, K.; Akaji, K. Bioorg. Med. Chem.
Lett. 2006, 16, 3761.
12. A synthesized HTLV-I PR gene was introduced into
Escherichia coli BL21(DE3)-pLysS cells as described in
Ref. 8. The gene was extracted and mutated to construct a
plasmid as follows: (1) an NdeI restriction site was added to
the 50 end; (2) the Leu40 codon was mutated to an Ile codon
to block autolysis; and (3)a stopcodon anda PstI restriction
site were introduced 30 to the Leu125 codon. The gene was
cloned into pColdI (Takara-Bio) by using the NdeI and PstI
restriction sites to afford pColdI/HTLV-I PR, and intro-
duced into E. coli BL21 cells. Expression of the gene and
ensuing procedures were performed as described in Ref. 8.
13. The HTLV-I PR inhibitory activity was determined by
measuring the rate of hydrolysis of APQVL(p-nitrophe-
nylalanine)VMHPL. The enzyme reaction mixture (50 lL)
contained 1 lg protease (OD595 ꢀ 0.050, as a dimer),
HOBt (1,3-diisopropylcarbodiimide; 1-hydroxybenzotri-
azole) method. Piperidine (20%) in DMF was employed
to remove the Fmoc protection group, while coupling
with an appropriate Fmoc-protected amino acid was
performed using the DIPCDI-HOBt method for each
chain-elongation step. N-Acetylation was performed
with acetic anhydride and Et3N in DMF. Cleavage from
the resin was performed with trifluoroacetic acid, m-cre-
sol, thioanisole, and water. Compounds 21–28 were syn-
thesized by standard solution phase peptide synthesis in
which sequential coupling of a respective amine to a cor-
responding Boc-protected amino acid was performed in
DMF with EDCÆHCl-HOBt (1-ethyl-3-(3-dimethylami-
nopropyl)carboiimide hydrochloride) or BOP (ben-
zotriazol-1-yl-oxy-tris-dimethylaminophosphonium hexa-
fluorophosphate) as coupling reagents in the presence
of Et3N. Coupling with BOP was often more efficient
than with EDCÆHCl-HOBt. Removal of the Boc protec-
tion group was achieved with 4 N HCl in dioxane during
each chain-elongation step. Similarly, P3 N-acetylation
was performed with acetic acid, BOP, and Et3N in
DMF. After preparative HPLC purification, all target
compounds (6–28) were >98% pure by analytical HPLC.
The identities of the compounds were confirmed by ESI-
Q MS and/or TOF MS. To improve the reliability of our
HTLV-I PR assay, we developed a more proteolytic
HTLV-I PR L40I mutant12 against a substrate, which
consequently dramatically reduced incubation time
from 6 h to 30 min when compared to the former meth-
od.8 This more efficient mutant permitted a cost-effective
means of determining IC50 values.13 Recombinant HIV-
1 PR assay8 and computer-assisted docking experi-
ments9 were performed in similar manners as previously
described.
In the current study, rational drug design was applied to
the potent lead hexapeptidic inhibitor, KNI-10166 (5,