Structure-based design of novel dihydroalkoxybenzyloxopyrimidine derivatives as potent nonnucleoside inhibitors of the human immunodeficiency virus reverse transcriptase

Article abstract of DOI:10.1128/aac.42.12.3225

Two highly potent dihydroalkoxybenzyloxopyrimidine (DABO) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket and tested for anti-HIV activity. Our lead DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]- 6-(benzyl)-pyrimidin-4-(1H)-one, elicited potent inhibitory activity against purified recombinant HIV RT and abrogated HIV replication in peripheral blood mononuclear cells at nanomolar concentrations (50% inhibitory concentration, <1 nM) but showed no detectable cytotoxicity at concentrations as high as 100 μM.

Full text of DOI:10.1128/aac.42.12.3225

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1998, p. 3225–3233
0066-4804/98/$04.00ϩ0
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 42, No. 12
Structure-Based Design of Novel Dihydroalkoxybenzyloxopyrimidine
Derivatives as Potent Nonnucleoside Inhibitors of the Human
Immunodeficiency Virus Reverse Transcriptase
ELISE A. SUDBECK,1,2,3 CHEN MAO,1,3 RAKESH VIG,1,2 T. K. VENKATACHALAM,1,2
LISA TUEL-AHLGREN,4 AND FATIH M. UCKUN1,4
*
Drug Discovery Program,1 Departments of Chemistry,2 Structural Biology,3 and Virology,4
Hughes Institute, St. Paul, Minnesota 55113
Received 7 April 1998/Returned for modification 5 June 1998/Accepted 5 October 1998
Two highly potent dihydroalkoxybenzyloxopyrimidine (DABO) derivatives targeting the nonnucleoside in-
hibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been
designed based on the structure of the NNI binding pocket and tested for anti-HIV activity. Our lead DABO
derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one, elicited potent inhibitory
activity against purified recombinant HIV RT and abrogated HIV replication in peripheral blood mononuclear
cells at nanomolar concentrations (50% inhibitory concentration, <1 nM) but showed no detectable cytotox-
icity at concentrations as high as 100 ␮M.
different structures of RT complexed with 1-[(2-hydroxyethoxy)methyl]-6-(phe-
nylthio)thymine (HEPT), 6-benzyl-2-(ethoxymethyl)-5-isopropyluracil (MKC),
6-benzyl-1-[(benzyloxy)methyl]-5-isopropyluracil (TNK), ␣-anilinophenylacet-
amide (APA), nevirapine (dipyridodiazepinone derivative), N-ethyl nevirapine
The nonnucleoside inhibitor (NNI) binding site of human
immunodeficiency virus type 1 (HIV-1) reverse transcriptase
(RT) has been studied extensively (20, 21, 29, 32, 40, 41, 44,
46). Several crystal structures of HIV-1 RT complexed with
NNIs have been determined and have provided important
structural information about the NNI binding site. These RT-
NNI crystal structures have shown distinct properties of the
NNI binding pocket which can be utilized for structure-based
rational drug design (20, 21, 29, 32, 40, 41, 44, 46). However,
each reported structure revealed a unique binding pattern,
indicating that rational drug design efforts should not rely on
one particular crystal structure. Therefore, we have used the
NNI binding site coordinates of nine RT-NNI structures to
generate a composite binding pocket revealing a molecular
surface which defines a larger-than-presumed NNI binding
pocket and serves to summarize critical features unique to the
NNI binding site (35, 48). The composite binding pocket al-
lowed a clear identification of features which could be success-
fully exploited in new inhibitor designs. In the present study, we
have utilized this composite pocket, together with a computer
docking procedure and a structure-based semiempirical score
function, for the design of novel derivatives of dihydroalkoxy-
benzyloxopyrimidine (DABO) as potent NNIs of HIV-1 RT.
Here, we report two highly potent DABO derivatives which
were found to inhibit HIV RT. Our most potent DABO de-
rivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-benzyl)pyri-
midin-4-(1H)-one (3c), showed potent inhibitory activity
against purified recombinant HIV RT and abrogated HIV
replication in peripheral blood mononuclear cells (PBMNC)
(50% inhibitory concentration at nanomolar concentrations
[IC50], Ͻ1 nM) but showed no detectable cytotoxicity at con-
centrations as high as 100 ␮M.
derivative, 9-Cl-tetrahydrobenzodiazepine derivative (9-Cl TIBO) (41), 9-Cl
TIBO (17), and 8-Cl-TIBO (protein data bank [PDB] access codes, 1rti, 1rt1,
1rt2, 1hni, 1vrt, 1rth, 1rev, 1tvr, and 1hnv, respectively). The “thumb” region of
RT complexes is relatively variable compared with the “palm” region. Therefore,
a total of 117 C␣ atoms of residues 97 to 213, which cover part of the NNI
binding site and the palm region, were used for a least-squares superimposing
procedure within program O (30). The RMS values for the superimposed coor-
dinates were 1.00, 0.98, 0.99, 0.62, 0.80, 0.87, 0.94, and 0.65 Å for HEPT, MKC,
TNK, APA, cyclopropanyl nevirapine, N-ethyl nevirapine derivative, and the two
9-Cl TIBO complexes, respectively. The coordinates of the corresponding inhib-
itor molecules were then transformed according to the same matrices derived
from the superimposition. Lastly, the overlaid coordinates of all inhibitors were
read into the program GRASP (38), from which an overall molecular surface was
generated and provided a binding pocket encompassing all inhibitors. The mo-
lecular surface was used to better visualize the available space within the NNI
binding pocket of RT and to more easily identify potentially usable space in a
qualitative way. The generated binding pocket, referred to as the composite
binding pocket, was used as a basis for the analysis of inhibitor binding.
Molecular docking and Ki prediction. Fixed docking in the Affinity program
within InsightII (37) was used for docking of small molecules into the NNI
binding site, which was taken from a crystal structure (PDB code, 2rt1; RT-MKC
complex) (17, 29). As the modeling calculations progressed, the residues within
a defined radius (5 Å) of the NNI molecule were allowed to move in accordance
with energy minimization. Ten final docking positions were initially chosen for
each inhibitor modeling calculation but failed to reveal more than two promising
positions. Later, only two calculated positions were set for the search target. The
limited number of suitable docked positions is consistent with the observation
that the NNI binding site of RT is sufficiently enclosed so that inhibitors can only
bind in a small number of specific orientations, unlike other proteins, where the
binding site is relatively open and molecules can bind in a number of orienta-
tions. Binding calculations were carried out on an SGI INDIGO2 by using the
CVFF force field in the Discover Program and a Monte Carlo Search Strategy in
Affinity (33). No solvation procedures were used. Since the total number of
movable atoms exceeded 200, conjugated gradient minimization was used in-
stead of the Newton minimization method to conserve computation time. The
initial coordinates of the compounds were generated by using the Sketcher
module within InsightII, which calculates an energy-minimized conformation for
the molecule. InsightII was also used to calculate intermolecular energy (van der
Waals and Coulombic interaction energy) for some compounds to compare
energy differences between different conformers. The largest conformational
differences can result from a rotation around the bond joining the two rings in
DABO compounds. For our study, several conformers were generated which
differed in the orientation of the phenyl ring around this bond and were used to
calculate an energy score for each conformer. Calculated interaction scores for
inhibitor conformations were considered which differed from the conformation
we eventually used for docking. Our chosen conformation was based on the
RT-HEPT and RT-MCK442 crystal structures and was found to be one of the
MATERIALS AND METHODS
Construction of the NNI composite binding pocket. Modeling studies required
the construction of a binding pocket which encompassed the superimposed
crystal structure coordinates of all known RT-NNI complexes, including nine
* Corresponding author. Mailing address: Hughes Institute, 2665
Long Lake Road, Suite 330, St. Paul, MN 55113. Phone: (651) 697-
9228. Fax: (651) 697-1042. E-mail: fatih_uckun@mercury.ih.org.
3225

3226
SUDBECK ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
tested was added to the reaction mixture at 0.001 to 100 ␮M concentrations.
Addition of 10 mU of HIV rRT and incubation at 37°C for 1 h resulted in
extension of the primer by incorporation of [3H]TTP. The reaction was stopped
by adding of 0.2 ml of 120 mM EDTA. The samples were counted in an open
window by using a Beckman LS 7600 instrument, and IC50s were calculated by
comparing the measurements to untreated samples.
TABLE 1. Hydrogen bond geometry definitions
used in Ludi program
Hydrogen
bonding group
Distance from H to
acceptor (Å)
Hydrogen bond angle
between H and A–X (°)
(ii) p24 assays for anti-HIV activity. Normal human PBMNC from HIV-
negative donors were cultured for 72 h in RPMI 1640 medium supplemented
with 20% (vol/vol) heat-inactivated fetal bovine serum, 3% interleukin-2, 2 mM
L-glutamine, 25 mM HEPES, 2-g/liter NaHCO3, 50-␮g/ml gentamicin, and 4-
␮g/ml phytohemagglutinin prior to exposure to HIV-1 at a multiplicity of infec-
tion of 0.1 during a 1-h adsorption period at 37°C in a humidified 5% CO2 at-
mosphere. Subsequently, cells were cultured in 96-well microtiter plates (100 ␮l/
well; 2 ϫ 106 cells/ml, triplicate wells) in the presence of various inhibitor con-
centrations, and aliquots of culture supernatants were removed from the wells on
day 7 after infection for antigen p24 enzyme immunoassays (EIA), as previously
described (22, 47, 49). The applied p24 EIA was the unmodified kinetic assay
commercially available from Coulter Corporation/Immunotech, Inc. (Westbrooke,
Maine), which utilizes a murine monoclonal antibody to HIV core protein used
to coat microwell strips to which the antigen present in the test culture super-
natant samples binds. Percent inhibition of viral replication was calculated by
comparing the p24 values from the test substance-treated infected cells with p24
values from untreated infected cells (i.e., virus controls). In parallel, the effects
of various treatments on cell viability were also examined as previously described
(22, 49). In brief, noninfected PBMNC were treated with each compound for 7
days under identical experimental conditions. A microculture tetrazolium assay
(MTA), using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-car-
bonyl]-2H-tetrazolium hydroxide, was performed to quantitate cellular prolifer-
ation.
X-ray crystallography. Yellow rectangular plates of 3b and colorless plates of
3c were grown from tetrahydrofuran in separate experiments by slow evaporation
at room temperature. The crystals were mounted on glass fibers by using epoxy,
and X-ray diffraction data for a crystal (0.5 by 0.2 by 0.08 mm) of 3b and a crystal
(0.3 by 0.2 by 0.1 mm) of 3c were collected at 22°C by using a SMART charge-
coupled device X-ray detector (Bruker Analytical X-Ray Systems, Madison, Wis.).
Structure solution and refinement were performed by using the SHELXTL suite
of programs (Bruker Analytical X-Ray Systems). All nonhydrogen atoms were
refined by using anisotropic displacement parameters. Hydrogen atoms were
placed at ideal positions and refined as riding atoms with relative isotropic
displacement parameters.
CAO
1.9
1.9
1.8
1.8
1.9
1.9
1.9
110–180
150–180
150–180
100–140
150–180
100–140
90–130
NH or OH
NH (charged)
COOϪ
ANO
R-O-R (sp2)
R-O-R (sp3)
lowest-energy conformers, which probably represents the most reasonable con-
formation for binding. This conformation was then compared with our small-
molecule crystal structure of the compound, which was found to be confor-
mationally similar. The CVFF force field was used to obtain a low-energy
conformation of a DABO compound representing a local minimum. After the
docking run was completed, by using the chosen conformer, each final position
was then evaluated by an interaction score function using the Ludi module.
Therefore, a final docked position was chosen for each DABO compound which
was based on a combination of both the CVFF energy minimum and the best
Ludi score (the final position of the DABO compound in the RT active site was
first minimized and evaluated by using the CVFF force field and then scored
based on the Ludi scoring function). The top-scoring model was then compared
with the constructed binding pocket and the known crystal structures of similar
compounds and used for further analyses, which involved an evaluation of the
compound in the composite binding pocket. A qualitative assessment was made
of how well the compound fit the binding site relative to other DABO com-
pounds or other NNIs. This was accomplished by superimposing the compounds
into the binding site and visually inspecting unoccupied regions, favorable inter-
actions with RT, and unfavorable interactions. This analysis was done to see if
the final Ludi scores were reasonable in light of biological data or known binding
modes of NNIs based on crystal structures.
The calculated inhibitory constants (Ki values) of the positioned DABO com-
pounds were evaluated by using the Ludi score function (6, 7). (The InsightII
program used for docking and Ludi scoring employed a calibration procedure
during its establishment which involved calculation of the Ki values of 45 protein-
ligand complexes having known Ki values and known crystal structures and
comparing the calculated Ki values to the experimentally determined Ki values.
The Ki value has a correlation with the free energy of binding, ⌬G, where ⌬G ϭ
ϪRTlnKi.) We imposed several modifications during the calculation. First, the
molecular surface areas were directly calculated from the coordinates of the
compounds in docked conformations by using the MS program (13). (The mo-
lecular surface area is used to calculate a Lipo score, which is an important
component of binding affinity in the Ludi score function.) Second, we reevalu-
ated the number of rotatable bonds, which was assessed inaccurately by InsightII.
Third, we assumed that the conserved hydrogen bond with RT did not deviate
significantly from the ideal geometry (the backbone carbonyl of RT residue 101
interacts with a hydrogen bond acceptor on the inhibitor, as observed in the
RT-HEPT, RT-MKC442, RT-TIBO, and RT-APA crystal structures). This as-
sumption was supported by the fact that in the known crystal structures of RT
complexes, all hydrogen bonds between NNIs and RT are near ideal in geometry.
Consequently, the Ki values for our modeled compounds were more predictable
than they would be otherwise without such constraints (6, 7).
Chemical synthesis. All chemicals were purchased from Aldrich Chemical
Company (Milwaukee, Wis.) and used as received. Anhydrous solvents were
obtained from Aldrich in Sure Seal bottles and transferred to reaction vessels via
cannula under nitrogen. All reactions were carried out under nitrogen. Nuclear
magnetic resonance (NMR) spectra were recorded on a Varian (Palo Alto,
Calif.) 300-MHz instrument, and chemical shifts (␦) are reported in parts per
million (ppm) relative to tetramethylsilane, which was the internal standard. 13
C
NMR spectra were recorded in CDCl3 on the same instrument by using the
proton decoupling technique. The chemical shifts reported for 13C NMR spectra
are referenced to chloroform at 77.0 ppm. Melting points were obtained by using
a Fisher-Johns melting apparatus and left uncorrected. Mass spectral analyses
were conducted by using a Finnigan (Madison, Wis.) MAT 95 mass spectrome-
ter. Column chromatography was performed by using EM Science silica gel 60.
The 5-alkyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one de-
rivatives 3a to 3d were prepared as shown in Fig. 4. Ethyl-2-alkyl-4-(phenyl)-3-
oxobutyrates 1a to 1d were obtained from commercially available phenylaceto-
nitrile by a method previously described (15, 16, 34). The ␤-ketoesters were
condensed with thiourea in the presence of sodium ethoxide to furnish the
corresponding thiouracils 2a to 2d. Subsequent reaction of thiouracil with meth-
ylthiomethyl chloride in N,N-dimethylformamide (DMF) in the presence of
potassium carbonate afforded compounds 3a to 3d in moderate yields.
In the Ludi scoring function used to evaluate the binding of NNIs to RT, an
ideal hydrogen bond corresponds to a maximum hydrogen bond score of 85. Our
analysis showed that all of the RT-NNI crystal structures studied can be assigned
a hydrogen bond score of 50 or greater. Six of nine complexes showed a hydrogen
bond score of 80 or greater, indicating nearly ideal hydrogen bond geometry for
these complexes, except in the case of the RT-APA structure. For modeling
studies, we used the same definitions for ideal hydrogen bonds that are used in
the Ludi program, which are briefly summarized in Table 1 (6, 7).
General procedure for synthesis of compounds 3a to 3d. A mixture of thio-
uracil 2 (1 mmol), methylchloromethylsulfide (1 mmol), and potassium carbon-
ate (1 mmol) in anhydrous DMF (5 ml) was stirred overnight at room temper-
Biological assays. (i) Purified RT assays for anti-HIV activity. 5-Methyl-2-
[(methylthiomethyl)thio]-6-benzylpyrimidin-4-1H-one (compound 3a), 5-ethyl-2-
[methylthiomethyl)thio]-6-benzylpyrimidin-4-1H-one (compound 3b), 5-iso-
propyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1H-one (compound 3c),
and 5-isopropyl-2-[(methylthiomethyl)thio]-6-(3,5-dimethylbenzyl)-pyrimidin-4-
1H-one (compound 3d) were tested for RT inhibitory activity (IC50[rRT])
against purified HIV recombinant RT (rRT) by using the cell-free Quan-T-RT
system (Amersham, Arlington Heights, Ill.), which utilizes the scintillation prox-
imity assay (SPA) principle (8). In the assay, a DNA-RNA template is bound to
SPA beads via a biotin-strepavidin linkage. The primer DNA is a 16-mer oli-
god(T) which has been annealed to a poly(rA) template. The primer-template is
bound to a strepavidin-coated SPA bead. [3H]TTP is incorporated into the
primer by reverse transcription. In brief, [3H]TTP, at a final concentration of 0.5
␮Ci/sample, was diluted in RT assay buffer (49.5 mM Tris-Cl [pH 8.0], 80 mM
KCl, 10 mM MgCl2, 10 mM dithiothreitol, 2.5 mM EGTA, 0.05% Nonidet P-40)
and added to annealed DNA-RNA bound to SPA beads. The compound being
FIG. 1. Structures of nevirapine and delavirdine.

VOL. 42, 1998
NNIs OF HIV RT
3227
FIG. 2. View of composite NNI binding pocket of HIV-1 RT. Blue grid lines represent the collective van der Waals surface of nine different inhibitor crystal
structures superimposed on the active site and highlight the space available for binding (inhibitor structures included HEPT, MKC, TNK, APA, nevirapine, N-ethyl
nevirapine derivative, 8-Cl TIBO, and two 9-Cl TIBO compounds with PDB access codes 1rti, 1rt1, 1rt2, 1hni, 1vrt, 1rth, 1hnv, 1rev, and 1tvr, respectively). (A)
Compound 3c superimposed on the composite NNI binding site of the crystal structure of the RT-MKC442 complex (hydrogen atoms are not shown; PDB access code,
1rt1). MKC442 (from crystal structure) is shown in pink, and compound 3c (from docking calculations) is multicolored. Compound 3c was docked into the active site
of the RT-MKC442 complex and then superimposed onto the composite NNI binding pocket based on the matrix used in pocket construction. The S2 substituent of
DABO analog 3c occupies the same region of the binding pocket as the N1 substituent of HEPT analog MKC442. (B) X-ray crystal structure of compound 3b (yellow)
superimposed on the docked model of compound 3d in the composite NNI binding pocket of RT, demonstrating their similar conformations.
ature. After treatment with water (50 ml), the solution was extracted with ethyl
acetate (3 ϫ 50 ml). The combined extracts were washed with saturated NaCl
(2 ϫ 50 ml), dried (MgSO4), filtered, and concentrated in vacuo to give the crude
products 3a to 3d, which were purified by column chromatography (hexane-ethyl
acetate eluent). Selected analytical data for compounds 3a to 3d are shown as
follows. Compound 3a yield, 62%; mp, 148 to 149°C; 1H NMR (CDCl3) ␦ 2.10
(s, 3H), 2.14 (s, 3H), 3.91 (s, 2H), 4.29 (s, 2H), 7.29–7.26 (m, 5H), 12.20 (s, 1H);
13C NMR (CDCl3) ␦ 10.7 (CH3), 15.5 (SCH3), 36.6 (CH2Ph), 41.0 (SCH2), 116.7
(C-5), 137.6 to 126.4 (Ph), 155.2 (C-6), 162.0 (C-4), 165.1 (C-2); CI-MS (chemical
ionization mass spectrometry), 293.1 (Mϩ1). Compound 3b yield, 65%; mp, 124
to 126°C; 1H NMR (CDCl3) ␦ 1.08 (t, 3H), 2.12 (s, 3H), 2.58 (q, 2H), 3.91 (s, 2H),
4.26 (s, 2H), 7.28-7.26 (m, 5H), 12.30 (s, 1H); 13C NMR (CDCl3) ␦ 13.1 (CH3),

3228
SUDBECK ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Interaction scores, calculated Ki values, and biological assay data for inhibitors of HIV RT
MSa
(Å2)
BSb
(%)
Lipo
score
Ludic Ki
(␮M)
IC50[rRT]d
(␮M)
CC50[MTA]e
Compound
R1
R2
IC50[p24]d (␮M)
Minimum SIf
(␮M)
3a
3b
3c
H
H
H
Meg
Eth
i-
275
283
301
88
88
89
709
730
785
3.3
28.8 Ϯ 10.0
12.0 Ϯ 2.3
5.6 Ϯ 2.4
3.4 Ϯ 1.1
0.8 Ϯ 0.2
Ͼ100
Ͼ100
Ͼ100
32 Ϯ 10
2.0
146 Ϯ 38
0.56
Ͻ0.001 Ϯ 0.000j
100,000 Ϯ 0
Pri
3d
Me
i-Pr
NA
NA
NA
329
NDl
ND
ND
89
875
ND
ND
ND
0.05
ND
ND
ND
7.0 Ϯ 2.2
0.8 Ϯ 0.1
2.3 Ϯ 1.0
19.8 Ϯ 5.5
0.016 Ϯ 0.011
0.004 Ϯ 0.001
ND
Ͼ100
Ͼ100
ND
31,520 Ϯ 31,310
25,830 Ϯ 4,383
ND
MKC442
Delavirdine
Nevirapine
NAk
NA
NA
ND
ND
ND
ND
ND
ND
a MS, molecular surface area calculated by using Connolly’s MS program (13), is defined as the boundary of the volume within any probe sphere (meant to represent
a water molecule) with a given radius sharing no volume with the hard sphere atoms which make up the molecule. The values are slightly lower than those approximated
by the Ludi program.
b BS, the buried surface, is the percentage of the molecular surface in contact with protein calculated by Ludi relative to docked positions. Based on published crystal
structures of RT complexes, our calculation shows that these values can be as low as 77% (in an RT-HEPT complex) and as high as 90% (in an RT-APA complex),
but most of them, including that for RT-MKC, average around 84%.
c Ludi Ki values were calculated based on the empirical score function in the Ludi program (6, 37). Ideal hydrogen bond distances and angles between compounds
and protein are assumed in all cases for Ludi Ki and Ludi score calculations. In published crystal structures of RT complexes, hydrogen bond geometries are indeed
close to ideal; the amide carbonyl of residue A101 on a loop demonstrates substantial flexibility, which can accommodate the best geometry for hydrogen bonding. The
number of rotatable bonds (two) is used in the Ludi calculation to reflect the loss of binding energy due to freezing of internal degrees of freedom.
d Mean Ϯ standard error of three or four independent experiments (two experiments for 3a), each performed in triplicate.
e Even at an inhibitor concentration of 100 ␮M, the viability of cells was minimally affected, with 50% cytotoxic concentration (CC50[MTA]) values of Ͼ100 ␮M in
each of three or four independent experiments.
f SI, the selectivity index, is equal to the ratio of CC50[MTA] to IC50[p24]. Since the CC50 values for the inhibitors tested were Ͼ100 ␮M, only the minimum selectivity
index estimates could be determined for these noncytotoxic agents.
g Me, methyl.
h Et, ethyl.
i i-Pr, isopropyl.
j In each of four independent experiments, at a 1 nM concentration of 3c, the percent inhibition of p24 production was Ͼ50%, with a mean value of 67% Ϯ 4%. In
a dose-response study using picomolar and nanomolar concentrations of compound 3c, the subnanomolar IC50 was determined to be 130 pM.
k NA, not applicable.
l ND, not determined.
15.4 (SCH3), 18.7 (CH2), 36.4 (CH2Ph), 40.3 (SCH2), 122.4 (C-5), 138.0 to 126.3
position of the thymine ring, which led to our design and
(Ph), 155.4 (C-6), 161.5 (C-4), 165.2 (C-2); CI-MS, 307.1 (Mϩ1). Compound 3c
synthesis of compounds 3b, 3c, and 3d. The inhibition con-
stants of the docked molecules were calculated based on an
interaction score function and are listed in Table 2. The trend
of the calculated Ki values predicted that compounds having a
slightly larger R2 group would show stronger inhibition of RT;
this general trend was also observed for the measured IC50s
(Table 2).
yield, 57%; mp, 116 to 117°C; 1H NMR (CDCl3) ␦ 1.22 (d, 6H), 2.07 (s, 3H), 3.03
(q, 1H), 3.88 (s, 2H), 4.21 (s, 2H), 7.24-7.13 (m, 5H), 12.43 (s, 1H); 13C NMR
(CDCl3) ␦ 15.4 (SCH3), 19.6 (CH3), 28.0 (CH), 36.3 (CH2Ph), 40.9 (SCH2),
125.3 (C-5), 138.3 to 126.3 (Ph), 155.5 (C-6), 161.1 (C-4), 164.5 (C-2); CI-MS,
321.1 (Mϩ1). Compound 3d yield, 67%; mp, 116 to 120°C; 1H NMR (CDCl3) ␦
1.28 (d, 6H), 2.15 (s, 3H), 2.27 (s, 6H), 3.10 (q, 1H), 3.88 (s, 2H), 4.31 (s, 2H),
6.84 (s, 3H), 12.42 (s, 1H); 13C NMR (CDCl3) ␦ 15.3 (SCH3), 19.6 (CH3), 21.2
(CH3), 28.0 (CH), 36.3 (CH2Ph), 40.8 (SCH2), 125.2 (C-5), 138.0 to 126.5 (Ph),
155.4 (C-6), 161.3 (C-4), 164.7 (C-2); CI-MS; 349.2 (Mϩ1).
The refined small-molecule X-ray crystal structures of com-
pounds 3b and 3c are shown in Fig. 3. Table 3 lists crystal data
and structure refinement statistics, and Tables 4 and 5 list
atomic coordinates for 3b and 3c. The molecular coordinates
of DABO compounds which were energy minimized and
docked into the NNI binding site adopted a conformation
remarkably similar to that of the crystal structure of compound
3b. Fig. 2B shows the model coordinates of 3d superimposed
on the crystal structure coordinates of 3b and illustrates their
conformational similarity. Compound 3c adopted a different
conformation in the crystal structure relative to the molecular
model of 3c used for docking. The conformational differences
of 3c include a 174° rotation of the isopropyl group around the
C5-C4 bond (Fig. 3B) (roughly the equivalent of twofold ro-
tation) and a 130° rotation of the sulfur tail around the S1-C17
bond (the equivalent of a mirror reflection through the S1-
C17-S2 plane). This suggests that the two different molecular
conformations did not differ significantly in energy, and both
represent low-energy conformers. The main difference is due
to a 45° rotation of the phenyl ring around the C6-C7 bond,
RESULTS
Modeling and design. Our detailed analysis of HEPT and
the HEPT derivative MKC442, both of which are active NNIs
of HIV RT, revealed that the N1 substituents of HEPT deriv-
atives occupy the same region of the binding site as the thio
(S2) substituents of DABO compounds (Fig. 2A). We there-
fore designed new DABO derivatives and modeled their bind-
ing into the NNI site of RT by using the crystal structure
coordinates of the RT-MKC complex (PDB access code, 1rt1)
and a molecular docking procedure in the Affinity module of
the InsightII program (37) (Table 2). We then superimposed
the final coordinates of the docked molecules on the composite
binding pocket to evaluate the fit within the RT NNI pocket.
Notably, multiple sterically allowed unoccupied spatial gaps in
the binding site were identified from the docking studies which
could be filled by strategically designed functional groups (Fig.
2B). The docked DABO molecule (compound 3a) showed
significant space surrounding the 6-benzyl ring and the fifth

VOL. 42, 1998
NNIs OF HIV RT
3229
3a in both cell-free and cellular RT inhibition assays. By com-
parison, MKC442 inhibited p24 production consistently at
slightly higher concentrations than our lead compound 3c. The
individual IC50s of MKC442 in the four independent experi-
ments were 0.006, 0.005, 0.003, and 0.003 ␮M, with a mean
value of 0.004 Ϯ 0.001 ␮M. Larger compounds, which better
fill the composite binding pocket and have lower calculated Ki
values, showed better IC50[rRT]s. This is reflected by the en-
hancement of the inhibitory activity with the addition of pro-
gressively larger groups, such as methyl (3a), ethyl (3b), and
isopropyl (3c) groups, at the C-5 position of the thymine ring
(Table 2). The same general trend was also observed for
IC50[p24]s. Compound 3d, which differs from compound 3c by
the addition of two methyl groups to the 6-benzyl ring, pro-
vides more hydrophobic contact with the NNI binding pocket
but showed potency similar to that of 3c in enzymatic assays.
As shown in Table 2, compound 3d, despite its comparable
IC50[rRT] in cell-free assays, failed to inhibit HIV replication
in HTLV-IIIB-infected cells as effectively as compound 3c,
contrary to our predictions. Our calculations indicate that com-
pounds 3a to 3d have progressively larger molecular surface
areas but still maintain approximately the same percentage of
the molecular surface area in contact with the protein residues.
Consequently, the calculated contact surface area between the
protein and the compound increases in the order 3a, 3b, 3c,
and 3d. This increased surface area, in turn, dictates a decrease
in calculated Ki values, with 3a having the worst value and 3d
the best. The trend of calculated Ki values for compounds 3a to
3c were predictive of the general trend of their measured
IC50[p24]s (Table 2).
TABLE 3. Crystal data and refinement statistics for X-ray crystal
structures of compounds 3b and 3ca
Parameter
3b (SD)
3c (SD)
Unit cell
a (Å)
8.9531 (9)
4.7893 (4)
b (Å)
23.374 (2)
8.862 (9)
90
10.8709 (10)
30.040 (3)
90
c (Å)
␣ (°)
FIG. 3. X-ray crystal structures of DABO analogs 3b (A) and 3c (B) (30%
ellipsoids; temperature, 22°C).
␤ (°)
␥ (°)
112.271 (2)
90
92.474 (2)
90
Space group
P21/c
1,717.4 (3)
4
P21/c
1,562.5 (2)
4
Unit cell vol (Å3)
Z
which in our model can be stabilized by favorable contacts with
binding site residues.
Predictable activities. Compounds 3a to 3d were tested for
RT inhibitory activity in cell-free assays using purified HIV RT
(listed as IC50[rRT] in Table 2), as well as by in vitro assays of
anti-HIV activity in human T-cell leukemia virus IIIB (HTLV-
IIIB)-infected PBMNC (22, 47, 49) (IC50[p24] in Table 2).
Compound 3a, which showed the lowest activity and the worst
interaction scores, inhibited rRT in two independent experi-
ments with IC50s of 38.8 and 18.8 ␮M (mean IC50[rRT], 28.8 Ϯ
10.0 ␮M), whereas our lead compound, 3c, inhibited rRT in
three independent experiments with IC50s of 6.1, 1.3, and 9.5
␮M (mean IC50[rRT], 5.6 Ϯ 2.4 ␮M). As shown in Table 2, the
IC50[p24]s for the inhibition of HIV-infected cells were signif-
icantly lower than the IC50[rRT]s, which measure the inhibi-
tion of rRT. Compound 3a inhibited p24 production at micro-
molar concentrations with a mean IC50 of 3.4 Ϯ 1.1 ␮M from
three independent experiments, whereas the lead compound
had an IC50 of Ͻ0.001 ␮M in each of four independent exper-
iments (percent inhibition at 1 nM in the four experiments,
58.3, 76.5, 63.3, and 70.0%). Thus, the lead compound had
consistently and significantly higher potency than compound
␪ range for data collection (°)
Limiting indices
1.74 to 24.99
Ϫ11 Յ h Յ11
Ϫ17 Յ k Յ 31
Ϫ11 Յ l Յ 11
8,167
2,990 (0.0676)
2,990/0/194
1.029
R1, 0.0578; wR2, R1, 0.0666; wR2,
0.0856 0.1384
R1, 0.1207; wR2, R1, 0.1114; wR2,
0.1028 0.1569
0.310 0.338
0.4894, 0.4456
0.0023 (6)
1.36 to 28.27
Ϫ6 Յ h Յ 6
Ϫ8 Յ k Յ 14
Ϫ39 Յ l Յ 37
8,744
3,507 (0.0486)
3,507/0/183
1.095
Reflections collected
Independent reflections (Rint
Data/restraints/parameters
Goodness of fit on F2
)
Final R indices [IϾ2␴(I)]
R indices (all data)
Absorption coefficient (mmϪ1
Max, min transmission
Extinction coefficient
)
0.8356, 0.6542
0.0004 (11)
0.279, Ϫ0.211
Largest difference peaks (eÅϪ3
)
0.227, Ϫ0.204
b
a Data were collected at 22°C (␭ ϭ 0.71073 Å), refined by using full-matrix
least-squares refinement on F2, and corrected for absorption by using semiem-
pirical psi-scan data. Rint ϭ ¥͉Fo2 Ϫ ϽFo ͉/¥͉Fo ͉, F1 ϭ ¥ʈFo͉ Ϫ ͉Fcʈ/¥͉Fo͉. R1 ϭ
2
2
⌺ʈFo͉ Ϫ ͉Fcʈ/⌺͉Fo͉. wR3 ϭ {¥[w(Fo Ϫ Fc2)2]/¥[w(Fo2)2]}1/2. GooF ϭ S ϭ
2
{¥[w(Fo Ϫ FFc2)2]/(n Ϫ p)}1/2, where n is reflections and p is parameters.
2
b eÅ, Ϫ3, electrons/Å3.

3230
Atom
SUDBECK ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 4. Atomic coordinates and equivalent isotropic
displacement parameters for 3b based on its X-ray
crystal structure at 22°C
pounds. Observed RT mutations such as L100I, V106A, and
Y181C (46) involve substituting a slightly smaller residue at the
active site. Our model suggests that DABO compounds 3a to
3d would not be prohibited from binding to these mutant RTs.
The mutations can accommodate each compound’s slightly
larger molecular volume (larger than that of the parent DABO
compound and, in some cases, MKC442). Inhibition would
probably not be as effective as for wild-type RT, however, due
to loss of hydrophobic contacts. In this context, it is noteworthy
that a novel PETT derivative, designed by using our composite
HIV RT binding pocket, exhibited potent activity against the
MKC442-resistant V106A RT mutant strain of HIV (data not
shown). The activity of compound 3c against various NNI-
resistant HIV-1 strains will be examined in future studies. In a
clinical setting, the currently applied approach to handling the
problem of drug resistance by RT is to use a combination of
drugs which have different mechanisms of action (viz., nucle-
Atomic coordinate (104) and equivalent isotropic
displacement parameter (Å, 103)b
x
y
z
U(eq)a
S1
8,080 (2)
5,831 (2)
2,787 (5)
4,538 (5)
5,010 (5)
5,613 (6)
3,231 (7)
2,015 (6)
2,693 (6)
113 (8)
1,773 (10)
1,436 (7)
3,421 (7)
5,121 (8)
6,938 (10)
7,025 (11)
5,312 (13)
3,557 (10)
8,199 (7)
7,394 (10)
5,764 (5)
Ϫ1,100 (8)
Ϫ1,054 (8)
515 (10)
2,998 (1)
5,424 (1)
Ϫ801 (2)
2,146 (2)
1,011 (2)
1,980 (3)
84 (3)
532 (1)
871 (1)
347 (1)
1,133 (1)
481 (1)
748 (1)
601 (1)
1,029 (1)
1,268 (1)
1,164 (1)
1,341 (2)
1,708 (1)
2,104 (1)
2,234 (1)
2,609 (1)
2,850 (2)
2,730 (2)
2,355 (1)
952 (1)
60 (1)
79 (1)
74 (1)
49 (1)
53 (1)
47 (1)
55 (1)
52 (1)
50 (1)
69 (1)
93 (1)
60 (1)
56 (1)
76 (1)
96 (1)
106 (2)
117 (2)
91 (1)
62 (1)
93 (1)
64
S2
O1
N1
N3
C2
C4
C5
238 (3)
C6
1,264 (3)
Ϫ794 (3)
Ϫ1,884 (4)
1,572 (3)
1,473 (3)
2,421 (4)
2,331 (5)
1,282 (6)
345 (5)
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
H1
TABLE 5. Atomic coordinates and equivalent isotropic
displacement parameters for 3c based on its X-ray
crystal structure at 22°C
432 (4)
Atomic coordinate (104) and equivalent isotopic
4,179 (3)
6,198 (4)
969 (2)
displacement parameter (Å, 103)b
Atom
420 (1)
227 (1)
1,392 (1)
909 (1)
x
y
z
U(eq)a
H2
Ϫ501 (3)
Ϫ1,048 (3)
Ϫ2,524 (4)
Ϫ1,637 (4)
Ϫ2,184 (4)
2,406 (3)
1,026 (3)
3,146 (4)
2,991 (5)
1,208 (6)
Ϫ368 (5)
Ϫ237 (4)
3,799 (3)
4,511 (3)
6,948 (15)
5,678 (12)
6,383 (26)
83
S1
2,787 (1)
2,938 (1)
1,819 (3)
3,649 (3)
4,643 (3)
2,724 (4)
3,739 (4)
2,741 (4)
1,849 (4)
777 (5)
1,450 (1)
2,175 (1)
759 (1)
4,913 (1)
7,812 (1)
6,769 (3)
5,748 (3)
6,250 (3)
5,931 (4)
6,458 (4)
7,391 (4)
7,500 (4)
8,499 (5)
8,675 (5)
7,542 (5)
7,721 (6)
9,039 (7)
10,179 (5)
10,006 (5)
8,215 (4)
7,015 (5)
9,555 (5)
5,816 (4)
7,388 (5)
5,163 (3)
7,992 (5)
9,579 (5)
6,637 (5)
6,937 (6)
9,150 (7)
11,088 (5)
10,809 (5)
8,740 (4)
7,576 (7)
6,515 (22)
6,191 (17)
10,340 (17)
9,094 (6)
10,079 (22)
5,107 (4)
5,853 (4)
8,389 (6)
6,685 (26)
6,862 (29)
51 (1)
65 (1)
47 (1)
43 (1)
58 (1)
39 (1)
40 (1)
37 (1)
43 (1)
78 (1)
50 (1)
75 (1)
90 (2)
81 (1)
70 (1)
59 (1)
44 (1)
70 (1)
80 (1)
49 (1)
80 (1)
52
H3
83
S2
H4
1,425 (2)
1,597 (2)
1,115 (2)
1,692 (1)
1,751 (1)
2,069 (1)
2,692 (1)
3,097 (2)
2,901 (2)
2,272 (1)
1,236 (1)
973 (1)
139
N1
H5
2,906 (10)
2,952 (10)
713 (7)
139
N3
383 (1)
H6
139
O1
Ϫ515 (1)
808 (1)
H7
72
C2
H8
Ϫ131 (7)
5,072 (8)
8,083 (10)
8,254 (11)
5,314 (13)
2,441 (10)
7,847 (7)
10,080 (7)
6,399 (42)
7,313 (60)
9,309 (21)
72
C4
Ϫ147 (1)
Ϫ220 (1)
239 (1)
H9
91
C5
H10
H11
H12
H13
H14
H15
H16
H17
H18
115
C6
127
C7
213 (2)
140
C8
24 (5)
766 (2)
110
C9
Ϫ1,424 (6)
Ϫ2,116 (5)
Ϫ1,367 (6)
51 (6)
938 (2)
74
C10
1,445 (2)
1,786 (2)
1,617 (2)
1,110 (2)
Ϫ789 (1)
Ϫ1,278 (2)
Ϫ907 (2)
1,924 (1)
2,638 (2)
445 (1)
74
C11
355 (7)
140
C12
161 (3)
140
C13
740 (4)
501 (4)
140
C14
2,770 (4)
2,224 (5)
4,395 (5)
1,768 (4)
4,318 (5)
4,210 (3)
Ϫ77 (5)
a U(eq) is defined as one-third of the trace of the orthogonalized Uij tensor.
b Estimated standard deviations are given in parentheses.
C15
C16
C17
DISCUSSION
C18
H3
Recent translational research efforts against AIDS have fo-
cused on the development of potent inhibitors of HIV RT (18,
26, 36). Two NNIs of HIV RT that have been approved by the
U.S. Food and Drug Administration for licensing and sale in
the United States are nevirapine (27) and delavirdine [bis
(heteroaryl)piperazine derivative U-90152] (23, 42, 43) (Fig.
1). Other promising NNI derivatives that have been studied
include DABO derivatives (14–16, 34), HEPT derivatives (2,
14, 39, 45), TIBO (20), 2Ј,5Ј-bis-O-(tert-butyldimethylsilyl)-3Ј-
spiro-5Љ-(4Љ-amino-1Љ,2Љ-oxathiole-2Љ,2Ј-dioxide)pyrimidine (3,
4), oxathiin carboxanilide derivatives (9–11), quinoxaline de-
rivatives (24, 31), thiadiazole derivatives (25), and phenethyl-
thiazolylthiourea (PETT) derivatives (1, 5, 12, 28). NNIs have
been found to bind to a specific allosteric site of HIV-1 RT
near the polymerase site and interfere with reverse transcrip-
tion by altering either the conformation or the mobility of RT,
thereby leading to noncompetitive inhibition of the enzyme
(20, 21, 32, 40, 41, 44).
H7A
H7B
H9A
H10A
H11A
H12A
H13A
H14A
H15A
H15B
H15C
H16A
H16B
H16C
H17A
H17B
H18A
H18B
H18C
Ϫ63 (2)
93
1,421 (5)
Ϫ1,949 (6)
Ϫ3,101 (5)
Ϫ1,830 (6)
564 (6)
1,714 (4)
1,987 (4)
2,142 (28)
2,995 (15)
1,190 (14)
4,660 (15)
5,206 (7)
4,349 (11)
1,384 (4)
828 (4)
71 (2)
93
709 (2)
89
1,557 (2)
2,131 (2)
1,845 (2)
997 (2)
108
98
84
71
Ϫ761 (1)
Ϫ1,621 (3)
Ϫ1,335 (7)
Ϫ1,190 (5)
Ϫ603 (6)
Ϫ931 (11)
Ϫ1,262 (6)
2,253 (1)
1,727 (1)
2,796 (9)
2,942 (6)
2,424 (3)
53
104
104
104
120
120
120
58
58
5,064 (20)
3,731 (5)
4,900 (23)
120
120
120
a U(eq) is defined as one-third of the trace of the orthogonalized Uij tensor.
b Estimated standard deviations are given in parentheses.
The crystal structure of RT was analyzed to determine how
certain mutations might affect the binding of DABO com-

VOL. 42, 1998
NNIs OF HIV RT
3231
FIG. 4. Preparation of 5-alkyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one derivatives 3a to 3d. Reagents and conditions: a, R2CHBrCOOEt/Zn/
tetrahydrofuran; b, HCl (aqueous); c, (H2N)2CS-Na-ethanol; d, DMF, K2CO3, and chloromethyl methyl sulfide; 15 h. Me, methyl; Et, ethyl; i-Pr, isopropyl.
oside inhibitors, NNIs, and protease inhibitors). Another strat-
egy is to use a sufficiently high concentration of a single NNI to
eradicate the virus in the host before mutations can occur (19).
The more potent the drug, the more effective this approach
would be, if the drug has sufficiently low toxicity.
the nonpolar region predicted by docking, this less favorable
interaction would lead to weaker RT binding than was esti-
mated. We have described similar observations for halogen-
substituted PETT derivatives, which also contact this interfa-
cial region of the NNI binding pocket of HIV RT (48).
Our lead DABO derivative, compound 3c, elicited signifi-
cant anti-HIV activity with an IC50 of less than 1 nM for inhi-
bition of HIV replication (measured by p24 production in
HIV-infected human PBMNC) and showed no detectable cy-
totoxicity (inhibition of cellular proliferation occurred at Ͼ100
␮M, as measured by MTA) (Table 2 and Fig. 5). In contrast to
all previously published data for DABO and S-DABO deriva-
tives, which were less active than zidovudine and MKC442 (15,
16, 34) and showed selectivity indices of Ͻ1,000, compound
3c was more than fourfold more active than zidovudine and
MKC442 and abrogated HIV replication in PBMNC at nano-
molar concentrations with an unprecedented selectivity index
(CC50[MTA]/IC50[p24]) of Ͼ100,000.
Our modeling studies showed that the trend of calculated Ki
values for compounds 3a to 3c were predictive of the general
trend of their measured IC50[p24]s (Table 2). However, 3d was
predicted to be significantly more potent than 3c (based on
calculated Ki values), which was not observed experimentally.
The IC50[rRT]s of 3c and 3d were, in fact, similar, and the
IC50[p24] of 3c was Ͼ100-fold better than of 3d. Compound 3c
was not predicted to be quite so potent; the calculated Ki value
for compound 3c differs from the measured IC50 by several
orders of magnitude. This is in contrast to the other, less active,
compounds in the series, which exhibit reasonably predictable
Ki values. These results prompt the hypothesis that the binding
interactions taken into consideration in our modeling studies
may not fully account for the superior anti-HIV activity of our
lead compound, 3c. For example, one of the methyl groups on
the phenyl ring of 3d is in a position to contact a region of the
binding pocket which is at an interface between a polar region
and a nonpolar region. If the actual binding position of 3d
allows the methyl group to contact the polar region rather than
An additional consideration for NNI binding is the Tyr183
residue of the HIV RT located in the catalytic region which has
a conserved YMDD motif characteristic of RTs. The displace-
ment of this conserved tyrosine residue can interfere with
catalysis and render the HIV-1 RT protein inactive. It has been
suggested that bulky substituents at the fifth position of the
thymine ring could indirectly accomplish such inactivation by
displacing Tyr181, which is near Tyr183 (29). Our composite
binding pocket shows sufficient room for at least a three-car-
bon group at the fifth position. Modeling shows that the addi-
tion of a methyl, ethyl, or isopropyl group at the fifth position
of the thymine ring would lead to higher affinity for the rela-
tively hydrophobic environment at this location of the binding
pocket. The favorable hydrophobic contact increases as the
hydrophobic group at the fifth position gets bulkier. As it binds
to the site, the ethyl or isopropyl group can also cause the
nearby Tyr181 residue to rotate away from the inhibitor. Our
modeling showed that this change in conformation, in turn,
affects the positions of neighboring Tyr183 and Tyr188, which
may contribute to the inactivation of HIV-1 RT. The 6-benzyl
ring of compounds 3a to 3d is located near a region surrounded
by the hydrophobic ring planes of residues Trp229, Pro95,
Y188, and Y181. The analysis of compounds 3a to 3c in the
composite binding pocket suggests that the 6-benzyl ring would
be located on the boundary of the pocket, near residue Y188.
The para position of the ring is situated perpendicular to the
ring plane of nearby Trp229, within van der Waals contact, and
thus would be an unsuitable location for large-group substitu-
tion. However, there is significant unfilled space between the
compound and Pro95. With slight conformational rotation of
the 6-benzyl ring, compound 3d, with the addition of two
methyl groups, was found to better fill the composite binding

3232
SUDBECK ET AL.
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FIG. 5. Anti-HIV activity of compound 3c. The anti-HIV activity of DABO
derivative 3c was evaluated in four independent experiments (each performed in
triplicate) by measuring its ability to inhibit HIV replication in HTLV-IIIB-
infected PBMNC by using the p24 EIA as previously reported (22, 47, 49). The
results depicted are from one representative experiment. CC, uninfected control
cell cultures; VC, HTLV-IIIB-infected cultures not exposed to compound 3c;
S.E., standard error. Me, methyl.
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pocket (Fig. 2B). Such observations suggest that further mod-
ification of the 6-benzyl ring could lead to even more potent
inhibitors.
The goal of our modeling studies was to understand the
interactions between RT and its inhibitors so that better in-
hibitors could be designed. The results of our studies are useful
for a more precise design of inhibitors. A qualitative design
choice may be possible without any modeling but would prob-
ably result in significantly fewer highly effective inhibitors.
Quantitating how well a design might interact with the binding
site is our greater goal and is more useful for designing a new
generation of compounds highly effective against HIV RT. It is
important to note, however, that computer modeling of NNIs,
while providing valuable guidance to direct the chemical syn-
thesis of new inhibitors, can offer only an “educated guess”
regarding the biologic activity of new designs until experimen-
tally verified. In future studies, we will continue to use our
composite binding pocket geometry, in conjunction with bio-
logical testing, for the purpose of structure-based design of
other potent NNIs of HIV RT.
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