In this study we present the structural indications for the role of K103 and N103 in drug binding and the structural implications for the inhibitory efficacy of the inhibitors Efavirenz,
Trang 1Structural basis for the inhibitory efficacy of efavirenz (DMP-266), MSC194 and PNU142721 towards the HIV-1 RT K103N mutant
Jimmy Lindberg1, Snævar Sigurðsson1, Seved Lo¨wgren1, Hans O Andersson1, Christer Sahlberg2,
Rolf Nore´en2, Kerstin Fridborg1, Hong Zhang2and Torsten Unge1
1
Department of Cell and Molecular Biology, Uppsala Biomedical Center, Uppsala University, Sweden;2Medivir AB, Huddinge, Sweden
The K103N substitution is a frequently observed HIV-1 RT
mutation in patients who do not respond to
combination-therapy The drugs Efavirenz, MSC194 and PNU142721
belong to the recent generation of NNRTIs characterized by
an improved resistance profile to the most common single
point mutations within HIV-1 RT, including the K103N
mutation In the present study we present structural
obser-vations from Efavirenz in complex with wild-type protein
and the K103N mutant and PNU142721 and MSC194 in
complex with the K103N mutant The structures
unani-mously indicate that the K103N substitution induces only
minor positional adjustments of the three inhibitors and the
residues lining the binding pocket Thus, compared to the
corresponding wild-type structures, these inhibitors bind to
the mutant in a conservative mode rather than through
major rearrangements The structures implicate that the
reduced inhibitory efficacy should be attributed to the
changes in the chemical environment in the vicinity of
the substituted N103 residue This is supported by changes in hydrophobic and electrostatic interactions to the inhibitors between wild-type and K103N mutant complexes These potent inhibitors accommodate to the K103N mutation by forming new interactions to the N103 side chain Our results are consistent with the proposal by Hsiou et al [Hsiou, Y., Ding, J., Das, K., Clark, A.D Jr, Boyer, P.L., Lewi, P., Janssen, P.A., Kleim, J.P., Rosner, M., Hughes, S.H & Arnold, E (2001) J Mol Biol 309, 437–445] that inhibitors with good activity against the K103N mutant would be expected to have favorable interactions with the mutant asparagines side chain, thereby compensating for resistance caused by stabilization of the mutant enzyme due to a hydrogen-bond network involving the N103 and Y188 side chains
Keywords: drug-resistance; HIV; NNRTI; reverse tran-scriptase
The use of highly active antiretroviral therapy (HAART)
involving multidrug combinations has significantly reduced
the death rates of HIV-1 infected individuals receiving such
treatment [1] Inhibitors of the HIV-1 reverse transcriptase
(RT) constitute a cornerstone in this therapy and are
commonly used in combination with inhibitors of the HIV-1
protease The RT inhibitors belong to two classes, the
nucleoside inhibitors and the non-nucleoside inhibitors
(NNRTI) Whereas the NRTIs are nucleoside analogues
with chain-terminating properties and affinity to active site
residues, the NNRTIs include a wide range of series of
chemical compounds characterized by noncompetitive binding to an allosteric site some 10 A˚ away from the active site Structural comparison of RT in complex with template/primer and NNRTIs together with native RT complexes have shown that the NNRTIs inhibit the polymerase activity through long-range and short-range structural distortions in several of the RT subdomains The distortions involve repositioning of residues in the non-nucleoside binding pocket (NNIBP) that impose steric impediments on the thumb subdomain flexibility forcing it
to remain in the open conformation In addition, the RNase H activity as well as initiation of polymerization may be affected by these NNRTI-induced distortions [2,3] Despite the initial efficacy in combating HIV infection, NNRTIs select for multidrug resistant strains of HIV over time [4,5] The mutations occur exclusively among the residues in the NNIBP The new generation NNRTIs, e.g Efavirenz, select for a panel of resistance mutations K103N, V106I, V108I, Y181C, Y188H Y188L, G190S, P225H, and F227L, indicating that a majority of the NNIBP residues are potential sites for drug-resistant mutations [6]
The Ôfirst generationÕ NNRTIs, such as the currently marketed drugs Nevirapine and Delavirdine show orders of magnitude decreases in binding as a result of single point mutations [7,8] The so-called Ôsecond generationÕ NNRTIs such as Efavirenz (DMP-266) [9], carboxanilides [10], PETT analogues [11] and the recent member S-1153 [12] demon-strate more favorable resistance profiles Efforts are now
Correspondence to T Unge, Department of Cell and Molecular
Biology, Uppsala Biomedical Center, Uppsala University, Box 596,
SE-751 24 Uppsala, Sweden.
Fax: + 4618536971, Tel.: + 46184714985,
E-mail: torsten.unge@icm.uu.se
Abbreviations: HAART, highly active antiretroviral therapy;
HIV-1, human immunodeficiency virus type 1; RT, reverse
transcriptase; rms, root mean square; NNRTI, non-nucleoside
RT inhibitor; NNIBP, non-nucleoside inhibitor NNIBP.
Note: The coordinates have been deposited in the Protein Data Bank
(PDB) with accession codes 1IKW, 1IKV, 1IKY and 1IKX for
wild-type RT-Efavirenz, K103N RT-Efavirenz, K103N RT-MSC194 and
K103N RT-PNU142721, respectively.
(Received 8 October 2001, revised 28 December 2001, accepted
24 January 2002)
Trang 2being put on the design of new inhibitors with improved
resistance profiles to the most frequently drug-induced
mutations generated within RT
The K103N mutation is the most frequent mutation
observed within RT resulting from therapeutic interventions
involving NNRTIs [6,13–15] As indicated above K103 is
one of the NNIBP residues The position of the K103
residue is close to the entrance of the pocket and contributes
through its aliphatic carbons to the hydrophobic character
of that part of the pocket that interacts with wing2 of the
inhibitor compounds [16] The combination of a broad
cross-resistance to the K103N mutant and the fact that
previous crystal structures of a number of inhibitors did not
indicate a direct contact to this residue, have encouraged
additional explanations to the resistance phenomenon
In support of an alternative resistance mechanism is the
observation of a hydrogen-bond network as a direct result
of the mutation The network could stabilize the closed
conformation of the NNIBP [17] This result is consistent
with kinetic data, which indicate the presence of a steric
barrier in the K103N mutant affecting NNRTI entrance to
the NNIBP [8] Despite these negative effects on drug
binding, no reduction in viral replication capacity has been
observed [18]
In this study we present the structural indications for the
role of K103 and N103 in drug binding and the structural
implications for the inhibitory efficacy of the inhibitors
Efavirenz, PNU142721, and MSC194 against the K103N
mutant The results are deduced from comparisons of
crystal structures of inhibitor complexes of wild-type and
the K103N mutant
M A T E R I A L S A N D M E T H O D S
Protein expression, purification, and crystallization
The RT gene (HIV-1, BH10 isolate, nucleotides 1908–3587)
was isolated by PCR, and ligated into the pET 11a
expression vector at the NdeI/BamHI sites as previously
described [19] Through this construct, the protein sequence
of 560 amino acids was provided with an N-terminal
methionine However, the methionine was processed by
bacterial proteases and never detected in the electron
density In order to extinguish the RNase H activity, residue
E478 (GAG) was mutated to Q (CAG) by site-directed
mutagenesis RT was expressed in the Escherichia coli, strain
BL21 (DE3) and purified as described previously [19] with
the following modifications Instead of allowing HIV-1
protease or bacterial proteases to process the RT p66/p66
homodimer to the p66/p51 heterodimer, processing was
performed by chymotrypsine D digestion of the total
bacterial lysate for 60 min immediately prior to purification
(1 mg to 30 mL lysate) In the chromatographic steps
the ion exchange and affinity matrices POROSÒ HQ,
POROSÒ S and POROSÒ HE (PerSeptive Biosystems)
were used Purified protein was used in evaluating the
antiviral activity of the three inhibitors in the HIV-1 RT
enzyme assay described previously [20] Prior to
crystalliza-tion, RT was concentrated by precipitation with 2M
(NH4)2SO4 and redissolved in distilled water
Crystalliza-tion was performed by vapor diffusion as follows Drops
consisting of 5 lL premixed RT (20 mgÆmL)1) and twofold
molar access inhibitor (30 m in dimethylsulfoxide)
toge-ther with 5 lL of crystallization buffer [1.4M(NH4)2SO4,
50 mM Hepes pH 7.2, 5 mM MgCl2, 300 mM KCl] were equilibrated against the same buffer at room temperature Typically, crystals appeared within two weeks and grew to a size of 0.3· 0.2 · 0.2 mm within two months Crystals belong to the orthorhombic space group, C2221
Data collection and processing, structure solution and refinement
X-ray data were collected at 4°C using the Max-Labora-tory beam line 711 and ESRF beam line BM14 Indexing and integration of data were performed usingDENZO, the data were merged together withSCALEPACK[21], and further processing was performed with theCCP4 program suite [22] Essential details of data collection and processing are given
in Table 1 The structures were refined by employing the software programCNS[23] The protein model coordinates from 1hni were used for rotation and translation functions The rms deviations for the Ca between the initial model and the final structures were in the range of 1.9–2.1 A˚ Inhibitory parameters were generated with XPLO2D [24] The refinement proceeded with energy minimization, simu-lated annealing, and individual B-factor refinement and were monitored by the statistical values Rwork/Rfree [25] Model building was carried out using the software program MAPMAN [26],LSQMAN[27] andO[28] Difference Fourier maps were calculated with ligand and residue 103 omitted employing the omit-map option in CNS All figures were produced usingSWISS-PDB VIEWERv 3.51 [29] and 3D-ren-dered withPOV-RAYv 3.1 [30]
R E S U L T S
We have determined the X-ray structures of wild-type and the K103N mutant in complex with Efavirenz at 3.0 A˚ resolution Two additional K103N mutant complexes were structurally determined together with a PETT ana-logue (MSC194) and a pyrimidine thioether anaana-logue, PNU142721 at 3.0 and 2.8 A˚ resolution, respectively The mean temperature factors was typically 55 A˚2for all atoms, 30–60 A˚2for the inhibitor atoms and lining residues The temperature factors for K103 and N103 atoms were in the range of 50–60 A˚2 All complexes crystallized with the symmetry of space group C2221
Overall hydrophobic NNRTI interactions to wild-type and K103N mutant RT
The interactions of Efavirenz, MSC194 and PNU142721 to wild-type RT and the K103N mutant correspond to previously described RT/NNRTI complexes [11,31] The NNRTI interactions are predominantly of hydrophobic nature to pivotal residues from p66 and p51 lining the NNIBP The aromatic residues Y181, Y188 and W229 surround wing1 whereas wing2 is sandwiched between L100 and V106, while also making edge-on contacts with V179 and Y318A A prominent nonhydrophobic contact is the hydrogen bond formed between the NNRTIs and the backbone carbonyl of K101 Omit electron density maps of the RT/NNRTI complexes clearly show the orientation and conformation of the inhibitors in the NNIBP, including the mutated side-chain at position 103 (Fig 2A,B)
Trang 3Antiviral activity
The three NNRTIs in this study, Efavirenz, MSC194, and
PNU142721, were tested in HIV-RT enzyme assays with
wild-type RT and the K103N mutant [20] The IC50values
from these assays are presented in Table 2 The activity measurements rank the inhibitors MSC194, PNU142721 and Efavirenz according to potency The inhibitory efficacy
to wild-type RT is within subnanomolar range for all three inhibitors PNU142721 and MSC194 show a 3–10-fold reduction in efficacy for the K103N mutant The effect of the mutation is more pronounced for Efavirenz where there
is a 200-fold reduction in efficacy compared to wild-type
RT This corresponds to a 5–10-fold larger value than obtained by others [9] This may be due to the use of homopolymeric rC-dG template in the assay The assay shows that PNU142721 is the most potent of the three towards the K103N mutant with an IC50value of 9 nM In contrast the first generation NNRTI, Nevirapine is 20-fold less potent compared to wild-type with an IC50 value of
3800 nM
Wild-type and K103N mutant RT/Efavirenz complexes are structurally conserved
Structural analysis of Efavirenz in complex with the K103N mutant revealed that the overall position of the inhibitor as well as the residues lining the NNIBP corresponds to the wild-type–Efavirenz complex (Fig 3) Comparison between the two complexes shows an rms-deviation of 0.4 A˚ (all atoms) and 0.20 A˚ (Ca atoms) for residues within 4.0 A˚ of the inhibitor The only significant difference is the K103N substitution In wild-type RT K103 is protruding into a negatively charged patch composed of the backbone carbonyls of K102 and G191 and the side chain of D192
Table 1 Crystallographic structure determination statistics.
WT-Efavirenz K103N-Efavirenz K103N-MSC194 K103N-PNU142721 Data collection details
Data collection site MAX-lab beam line 711 MAX-lab beam line 711 ESRF beam line BM14 MAX-lab beam line 711
Unit cell dimensions (A˚) 119.54, 157.31, 157.17 119.63, 157.17, 156.19 120.34, 156.54, 156.47 119.90, 156.40, 156.90
R merge
a
Outer resolution shell
Refinement statistics
Reflections (working/test) 29,828/1505 25,992/1309 27,346/1385 32,921/1,661
R-factor b (R work /R free ) 0.218/0.272 0.229/0.292 0.208/0.266 0.210/0.273
Rms dihedral angle deviation c
a R merge ¼ S|I–<I>|/S<I> b R-factor ¼ S|F o –F c |/SF o c Ideal parameters are those defined by Engh and Huber d Mean B-factor for main chain, side chain, inhibitor and water atoms, respectively.
Fig 1 Structures of NNRTIs Chemical structure of the NNRTIs (A)
Efavirenz, (B) PNU142721, and (C) MSC194 Atom numbering was
included for clarification of Table 3.
Trang 4forming a weak hydrogen bond interaction to D192
(3.16 A˚) However, in the K103N mutant complexes the
orientation of the N103 amide is undefined Modeling of the
amide in the Efavirenz complex resulted in an optimal
distance to residue D192 of 3.5 A˚
The binding mode of Efavirenz to wild-type RT only
allows a few contacts between the K103 residue and the
inhibitor (Table 3) The contact distances from the
back-bone N and the Cb and Cc atoms to the inhibitor are all
within optimal van der Waals distance for close packing
interactions The interactions of Efavirenz to the substituted
N103 residue is conserved compared to the wild-type except
for the contact with Cc The K103 Cc methylene group is
replaced by a bulky amide of N103 that consequently
abolish the interaction
Introduction of the asparagine at position 103 in the
NNIBP induces a minute orientational shift of Efavirenz
The effect on the inhibitor is observed as a minor rotation
around the branching carbon of Efavirenz Consequently,
the trifluoromethyl group is repositioned 0.2 A˚ away and the O10 of the benzoaxine-2-one ring 0.3 A˚ towards the N103 amide These subtle changes are accompanied by repositioning of the side chain of V179 0.4 A˚ towards and of D192 0.7 A˚ away from the inhibitor with respect to the wild-type-Efavirenz complex
Fig 2 Orientation and Conformation of Efavirenz and Residue 103 in the wild-type RT and K103N mutant NNIBPs (A) Simulated annealing omit electron density map covering Efavirenz and residue K103 (green) in the wild-type NNIBP In (B) the same view is shown for Efavirenz and residue N103 (maroon) in the mutated NNIBP Residue side-chains characteristic of the wild-type and K103N mutant NNIBPs are colored accordingly The map was calculated with ligand and residue 103 omitted employing the omit-map option in CNS and contoured at 1.5 r.
Table 2 Inhibition of HIV-1 RT.
HIV-1RT (rCdG), IC 50 (n M )a
a The HIV-1 RT assay which used (poly)rCÆ(oligo)dG as the tem-plate/primer is described in [23].
Fig 3 Superimposition of Efavirenz bound to wild-type and K103N mutant RT NNIBPs Stereoview of the superimposition of Efavirenz bound to the NNIBP of wild-type RT and the K103N mutant Residue side chains characteristic of the NNIBP are included from each inhibitor complex and colored green for wild-type and maroon for the K103N mutant The superimposition was carried out using all atoms from the residues within 4.0 A˚ from the inhibitors (V189, K101, K103N, V179, Y181, Y188, F227, W229, L234, H235, Y318 and E138).
Trang 5Similar overall NNRTI binding mode to K103N mutant RT
In Fig 4 the binding modes of the three inhibitors are
superimposed in the mutant NNIBP The
cyclopropyl-group of MSC194 and the methyl cyclopropyl-group of PNU142721
partly overlap the trifluoromethyl group of Efavirenz
Furthermore, wing2 composed of the heterocyclic ring
structure of MSC194 and the substituted pyrimidine
functionality of PNU142721 occupy the same part of the
NNIBP as the benzoaxine-2-one ring of Efavirenz In a
similar manner, the position of wing1 composed of the
substituted phenyl ring of MSC194 and the fused
ring-structure of PNU142721 overlap the less bulky cyclopropyl
group of Efavirenz
The overall similarity in binding mode of the inhibitors
to the K103N mutant means that several pivotal
inter-actions are shared to key residues in the NNIBP,
des-pite the chemical differences among the inhibitors This
similarity is apparent when the three structures are
superimposed (Fig 4) Only subtle changes in the
over-all positioning and orientation of residues lining the
NNIBP can be observed among the three complexes
A structural comparison of the K103N mutant-MSC194
and mutant-PNU142721 complexes with Efavirenz shows
an rms-deviation of 0.9/0.4 A˚ (all/Ca atoms) and 0.7/0.3 A˚
(all/Ca atoms), respectively, for residues within 4.0 A˚ of the inhibitors
Regardless of the overall similarities in the NNIBP of the K103N mutant structures the chemically different inhibitors affect the position and orientation of particular residues differently This is apparent in the K103N mutant-MSC194 complex where a flip of E138 in p51 to a downward rotamer
is observed in contrast to Efavirenz and PNU142721 Furthermore, both MSC194 and PNU142721 bound to the mutant show minute displacements of Y181 and Y188 away from the inhibitors compared to Efavirenz These rear-rangements allow for a rotation of the side chain of F227 with respect to the position in the K103N mutant-Efavirenz complex and the phenyl ring is observed with the partially positively charged side pointing towards MSC194 and PNU142721 In addition, the difference in chemical struc-ture of wing2 in PNU142721 with respect to MSC194 and Efavirenz induces a change in the rotamer of D192, thereby disrupting the negative patch
D I S C U S S I O N
Anti-HIV compounds belonging to the new generation of NNRTIs have increased inhibitory efficacy with respect to wild-type and a number of drug resistant RT mutants
Table 3 Inter-atomic distances for the inhibitors Efavirenz, MSC194, PNU142721 to residue 103 in the wild-type and K103N mutant NNIBPs Distance units are in A˚ Interactions to Od and Nd of the N103 amide have been left out due to the undefined amide orientation The atom numbering is clarified in Fig 1.
C5 3.9 N8 3.9
N2 3.8
S1 4.0
Fig 4 Superimposition of three K103N mutant NNIBPs Stereoview of the superimposition of Efavirenz (maroon), MSC194 (light blue) and PNU142721 (yellow) bound to the K103N mutant NNIBP Residue side chains characteristic of the NNIBP are included from each inhibitor complex and colored accordingly The superimposition was carried out using all atoms from the residues within 4.0 A˚ from the inhibitors (V189, K101, K103N, V179, Y181, Y188, F227, W229, L234, H235, Y318 and E1138).
Trang 6Accordingly, the NNRTIs Efavirenz, PNU142721, and
MSC194 have IC50-values in the nanomolar range to
wild-type RT as well as to the frequently occurring K103N
mutant Not unexpectedly the efficacy towards this mutant
differs significantly among the three inhibitors The
IC50-values range from 7.0 nM (PNU142721) to 520 nM
(Efavirenz) (Table 2) These inhibitory constants are
orders-of-magnitude lower than the corresponding values for the
first generation NNRTI, Nevirapine A detailed structural
analysis of the binding mode of the inhibitors in complex
with wild-type RT and the K103N mutant should give
insight into the structural basis for the inhibitory efficacy
and the resistance phenomenon Previously, a few studies of
inhibitors in complex with RT mutants have been reported
[17,32] The analysis of the inhibitors HBY 097 in complex
with the Y188L mutant and 8-Cl TIBO in complex with the
Y181C mutant, revealed that the retained efficacy of these
inhibitors was due to only minor alterations in the binding
mode compared to wild-type RT [32,33] A significantly
different result was obtained by Ren et al for the inhibitor
Efavirenz in complex with the K103N mutant determined to
2.9 A˚ resolution [34] In this case the binding mode was
different from the previous inhibitor complex structures
Indicating the flexibility of the RT structure Accompanying
this alteration in binding mode was a repositioning of Y181
into a position close to what has been found in the
RT/DNA complex [35] In addition, the structure of the p66
subunit displayed a more open conformation compared to
our K103N mutant structure Ren et al [34] concluded that
the efficacy of Efavirenz against the K103N mutant was due
to this novel binding mode
The data presented herein indicate that the K103N
mutation induces minute repositions of the inhibitors
PNU142721, MSC194 and efavirenz, with minor
readjust-ments in the positions of residues lining the binding site
Thus, compared to the corresponding wild-type structures,
these inhibitors bind to the mutant in a conservative mode
rather than through major rearrangements of the inhibitor
and binding site
The consequences of a conserved binding mode
on inhibitory efficacy
In order to allow for bigger readjustments of the inhibitor and
lining amino-acid residues, the compound needs to be
smal-ler than the accessible volume This requirement is fulfilled
for Efavirenz, whereas MSC194 and PNU142721 effectively
occupy the binding volume with extensive interactions
Accordingly, MSC194 has a similar binding mode to the
K103N mutant as the chemically related inhibitors MSC204
and MSC215 have to wild-type RT [11] Superimposition of
the structure complexes of MSC194 and PNU142721
show that these inhibitors bind to the K103N mutant in
essentially the same way Only minor adjustments are seen
in the positioning of the inhibitors and lining residues
Interestingly, our structural studies of efavirenz in
complex with wild-type RT and the K103N mutant
revealed the same conservative binding mode for Efavirenz
as observed for PNU142721 and MSC194 In addition, the
Efavirenz complexes superimpose well with the structures
of PNU142721 and MSC194 Thus, the reasons for
resistance, and the individual differences in efficacy
exhibited by these inhibitors, should be found among the
minor local structural and chemical differences in the vicinity of the mutation The substitutions of a charged and linear lysine for a uncharged and branched asparagine
at position 103 result in a drastic change in the chemical environment in the proximity of the mutation This has mainly two consequences for the binding of NNRTIs: changed hydrophobic and electrostatic properties of the NNIBP
Changes in hydrophobic interactions induced
by the K103N mutation The aliphatic carbons of K103 make hydrophobic close packing contacts with wing2 of the inhibitor compounds The extent of these interactions is more abundant for MSC194 than for PNU142721 and Efavirenz (Table 3) The effects of the K103N mutation on the hydrophobic interactions reveal individual differences among the three compounds Though the electron density for the amide part
of the residue is not very well defined for any of the inhibitor complexes, the inhibitors can still be clearly ranked with respect to the extent of the van der Waals interactions: MSC194, PNU142721 and Efavirenz The more extensive interactions of MSC194 and PNU142721 to the asparagine residue are in agreement with the higher efficacy of these compounds compared to Efavirenz, shown by the antiviral data (Table 2)
Changes in electrostatic interactions induced
by the K103N mutation The electron density for Cc of the K103N mutant structures
is well defined but the quality of the map does not allow assessment of the orientation of the amide plane There are, however, marginal differences in the quality of the electron density, with the most featured density for MSC194 In the case of Efavirenz, it is difficult to model the amide dipole in such a way that electrostatic repulsion will not occur with neighboring amino-acid residues In the p51 subunit the N103 amide is orientated with Nd2 positioned in the negatively charged patch composed of D192, while the backbone carbonyls of G191 and K102 impose a stabilizing effect on that region of RT A similar orientation in the p66 subunit positions Od1 in close proximity to the highly electronegative trifluoromethyl moiety of Efavirenz and the sulfur atoms of PNU142721 and MSC194, with repulsion
as a consequence However, in the cases of MSC194 and PNU142721 the position of the sulfur atom is such that the repulsive forces are less apparent In the MSC194 mutant complex the rotational freedom of the amide is reduced by the stacking of Od1 in between the plane of the thiourea moiety and the Ca of G190
Hence, the undefined orientation of the N103 amide Od1 and Nd2 atoms may reflect the repulsive forces exerted on the inhibitors
Other factors of importance for resistance induced
by the K103N mutation
In conclusion, our results indicate that the K103N mutation leads to changes in hydrophobic and electrostatic interac-tions Moreover, the significance of these changes on binding, for the individual compounds, is in agreement
Trang 7with the ranking of the compounds with respect to their
inhibitory efficacy However, these factors may not solely
account for the total reduction in inhibitory efficacy caused
by the K103N mutation An additional factor was presented
by Hsiou et al were they showed, in a study of unliganded
RT, that the K103N mutation led to the formation of a
network of hydrogen bonds that was not present in the
wild-type enzyme [17] In particular the hydrogen bond between
N103 and Y188 was suggested to stabilize the closed form of
the NNIBP Hsiou et al suggested that this stabilization of
the closed conformation of the RT structure could interfere
with NNRTI binding by imposing an energy barrier for
NNRTI entrance, consistent with kinetic data Our results
are complementary to those of Hsiou et al and support
their proposal that individual differences in efficacy between
related NNRTIs can arise from differential interactions
between the inhibitors and the N103 side chain [8,36]
The compounds Efavirenz, MSC194 and PNU142721
have one property in common, namely that they contain a
hydrogen-bond donor in wing2 This property is of general
importance for the efficacy of the inhibitor Substitution of
the hydrogen-donating amide group for a methylene group
completely abolishes the activity of a MSC194-related
compound (unpublished results) Whether this property is
of importance for competition with the hydrogen-bond
network in the K103N mutant remains to be shown An
interesting observation is, however, that the first generation
inhibitor Nevirapine lacks this property
We have presented new insights in drug resistance that
could explain the reduced susceptibility of the K103N
mutant to NNRTIs The mutation leads to changes in the
chemical environment of the NNIBP which affect the
interactions to NNRTIs The implication of these changes
for NNRTI-binding is described as changes among two
properties influencing the inhibitory efficacy: hydrophobic
and electrostatic factors The potent inhibitor compounds
accommodate the K103N mutation by the formation of
new interactions to the N103 side chain and minor
rearrangements of the inhibitor position in the binding site
These results should be useful for design of improved
NNRTIs to the K103N mutant
A C K N O W L E D G E M E N T S
This work was supported by the Swedish Medical Research Council
(MFR, K79-16X-09505-07A), the Swedish National Board for
Indus-trial and Technical Development (NUTEK) We thank the staffs of
station 711 of the MAX synchrotron, Lund, Sweden, and the beam line
BM14, ESRF, 6 rue Jules Horowitz, BP 220, F-38043 Grenoble Cedex,
France, for their assistance Terese Bergfors is addressed thanks for
proofreading the manuscript.
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