Báo cáo Y học: Structural basis for the inhibitory efficacy of efavirenz (DMP-266), MSC194 and PNU142721 towards the HIV-1 RT K103N mutant doc

Structural basis for the inhibitory efficacy of efavirenz (DMP-266), MSC194 and PNU142721 towards the HIV-1 RT K103N mutant Jimmy Lindberg 1 , Snævar Sigurðsson 1 , Seved Lo¨ wgren 1 , Hans O. Andersson 1 , Christer Sahlberg 2 , Rolf Nore ´ en 2 , Kerstin Fridborg 1 , Hong Zhang 2 and Torsten Unge 1 1 Department of Cell and Molecular Biology, Uppsala Biomedical Center, Uppsala University, Sweden; 2 Medivir AB, Huddinge, Sweden The K 103N 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 r ecent generation of NNRTIs characterized by an improved resistance profile to the most common single point mutations within HIV-1 R T, including t he K103N mutation. In the present study we present structural obser- vations from Efavirenz i n complex with wild-type p rotein 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 t he binding pocket. Thus, compared to t he 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 o f the substituted N103 residue. T his i s s upported by c hanges 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 c hain. 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. Mo l. Biol. 30 9, 4 37–445] 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 N 103 and Y188 side chains. Keywords: drug-resistance; HIV; NNRTI; reverse tran- scriptase. The u se of highly active ant iretroviral t herapy (HAART) involving multidrug combinations has s ignificantly 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 w ith inhibitors of the H IV-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 o f RT i n complex with template/primer and NNRTIs together with native R T 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 o f residues in the no n- 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 in itial 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 ne w generation NNRTIs, e.g. Efavirenz, s elect for a panel of resistance mutations K103N, V106I, V108I, Y181C, Y188H Y188L, G190S, P225H, and F227L, indicating that a majority of t he NNIBP residues are potential sites for drug-resistant mutations [6]. The Ôfirst generationÕ NNRTIs, such as the c urrently marketed drugs N evirapine and Delavirdine show orders of magnitude decreases in binding as a r esult 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] de mon- strate more favorable resistance p rofiles. Efforts are now Correspondence to T. Unge, Department of Cell and Molecular Biology, Uppsala Bi omedical C enter, Uppsala University, Box 596, SE-751 24 Up psala, Sweden. Fax: + 4618536971, Tel.: + 46184714985, E-mail: torsten.unge@icm.uu.se Abbreviations: HAART, h ighly a ctive 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 b een deposited i n 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) Eur. J. Biochem. 269, 1670–1677 (2002) Ó FEBS 2002 being put on the design o f n ew inhibitors with improved resistance profiles to the most frequently drug-induced mutations generated within RT. The K 103N mutation is the most frequent mutation observed within R T resulting from therapeutic interventions involving NNRTIs [6,13–15]. As indicated above K 103 is one of the NNIBP residues. The position of t he K103 residue is close t o 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 t he inhibitor compounds [16]. The combination of a broad cross-resistance to the K103N mutant and the fact that previous crystal s tructures o f a number of inhibitors did not indicate a direct contact to this residue, h ave 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 s teric 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 t he structural implications for the inhibitory efficacy of the inhibitors Efavirenz, PNU 142721, and MSC194 against the K103N mutant. The results are deduced from comparisons of crystal s tructures of inhibitor complexes of wild-type a nd the K103N mutant. MATERIALS AND METHODS Protein expression, purification, and crystallization The RT gene (HIV-1, BH10 isolat e, nucleotides 1908–3587) was isolated by PCR, and ligated into the pET 11a expression vector at the NdeI/BamHIsitesaspreviously described [19]. Through this construct, th e 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, r esidue 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 h eterodimer, 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 SandPOROSÒ HE (PerSeptive B iosystems) were used. Purified protein was used in evaluating the antiviral activity of the three inhibitors in th e HIV-1 RT enzyme assay described previously [20]. Prior to crystalliza- tion, RT was concentrated by precipitation with 2 M (NH 4 ) 2 SO 4 and red issolved in distilled water. C rystalliza- tion was performed by vapor diffusion as f ollows. Drops consisting of 5 lLpremixedRT(20mgÆmL )1 ) and twofold molar access inhibitor (30 m M in dimethylsulfoxide) toge- ther with 5 lL of crystallization buffer [1.4 M (NH 4 ) 2 SO 4 , 50 m M Hepes pH 7.2, 5 m M MgCl 2 , 300 m M 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 w ithin two mon ths. Crystals belong to the orthorhombic space group, C222 1 . 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 B M14. Indexing and integration of data were performed using DENZO ,the data were merged together with SCALEPACK [21], and further processing was performed with the CCP 4 program suite [22]. Essential details of data collection and processing are given in Table 1. T he structures were refined b y e mploying the software program CNS [23]. The protein model coordinates from 1hni were used for rotation and translation functions. The r ms deviations for the C a between the initial model and the final structures were in the range of 1.9–2.1 A ˚ . Inhibitory parameters were generated with XPLO 2 D [24]. The refinement proceeded with energy minimization, simu- lated annealing, and individual B-factor refinemen t and were monitored by the statistical values R work /R free [25]. Model building was carried out using the software program MAPMAN [26], LSQMAN [27] and O [28]. Difference F ourier maps were calculated with ligand and residue 103 omitted employing the omit-map option in CNS . All figures were produced u sing SWISS - PDB VIEWER v. 3.51 [29] and 3D-ren- dered with POV - RAY v. 3.1 [30]. RESULTS 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 analogue, PNU142721 at 3.0 and 2.8 A ˚ resolution, respectively. The mean temperature factors was t ypically 55 A ˚ 2 for all at oms, 30–60 A ˚ 2 for the inhibitor atoms and lining residues. The temperature factors for K103 a nd N103 atoms were i n the range of 50–60 A ˚ 2 . All complexes crystallized with the symmetry of space group C222 1 . 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 r esidues 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 R T/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). Ó FEBS 2002 The HIV-1 RT K103N mutant and inhibitor efficacy (Eur. J. Biochem. 269) 1671 Antiviral activity The three NNRTIs in this study, Efavirenz, MSC194, and PNU142721, were t ested in HIV-RT enzyme assays wit h wild-type RT and the K103N mutant [20]. The IC 50 values 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 fo r 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 p ronounced for E favirenz where t here is a 200- fold reduction in efficacy compared t o wild-type RT. This corresponds to a 5–10-fold larger v alue than obtained by others [9]. T his 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 IC 50 value of 9 n M .In contrast the first generation NNRTI, Nevirapine is 20-fold lesspotentcomparedtowild-typewithanIC 50 value of 3800 n M . Wild-type and K103N mutant RT/Efavirenz complexes are structurally conserved Structural analysis of Efavirenz i n complex with the K103N mutant revealed that the overall position of the inhibitor as well as the r esidues lining the NNIBP corresponds to the wild-type–Efavirenz complex (Fig. 3). C omparison between the two complexes shows an rms-deviation of 0.4 A ˚ (all atoms) and 0.20 A ˚ (Ca atoms) fo r r esidues w ithin 4.0 A ˚ of the i nhibitor. 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 s ide chain of D192 Table 1. C rystallographic 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 Image plate MAR-research MAR-research MAR-research MAR-research Space group C222 1 C222 1 C222 1 C222 1 Wavelength (A ˚ ) 1.0232 1.0232 0.931 1.0159 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 Resolution range (A ˚ ) 25–3.0 25–3.0 50–3.0 25–2.8 Observations 254 669 48 782 261 528 289,120 Unique reflections 30 024 29 948 32 188 36,534 Completeness (%) 96.4 97.6 85.2 90.3 Reflections with F/rF > 3 19 795 16 687 24 601 27,906 R merge a 0.113 0.152 0.114 0.079 Outer resolution shell Resolution range (A ˚ ) 3.11–3.00 3.11–3.00 3.16–3.09 2.90–2.80 Unique reflections 2873 2856 1170 3,306 Completeness (%) 97.9 97.8 73.6 92.3 Reflections with F/rF > 3 765 383 124 1,488 Refinement statistics Resolution range (A ˚ ) 25.0–3.0 25.0–3.0 25–3.0 25–2.8 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 bond length deviation c (A ˚ ) 0.008 0.008 0.008 0.007 Rms bond angle deviation (°) 1.4 1.4 1.4 1.3 Rms dihedral angle deviation c (°) 22.6 23.1 22.7 22.6 Rms improper angle deviation c (°) 0.96 0.95 0.93 0.93 Mean B-factor (A ˚ 2 ) d 75.3 53.0 60.6 54.9 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. Struc tures of NNRTIs. Chemical structure o f t he NN RTIs ( A) Efavirenz, (B) PNU142721, and (C) MSC194. Atom numbering was included for clarification of Table 3. 1672 J. Lindberg et al. (Eur. J. Biochem. 269) Ó FEBS 2002 forming 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 i n 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 c ontact distances from the back- bone N a nd the C b and Cc atoms to th e inhibitor a re all within optimal van der Waals d istance for close packing interactions. T he interactions of Efavirenz to the substituted N103 residue is conserved compared to t he wild-type except for the contact with Cc. The K103 Cc methylene group is replaced by a bulky amide of N 103 that consequently abolish the interaction. Introduction of the asparagine at position 103 in the NNIBP induces a m inute orientational shift of Efavirenz. The effect on the i nhibitor is observed a s a minor rotation around the branching carbon of Efavire nz. Consequently, the trifluoromethyl g roup 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 w ild-type RT and K103N mutant NNIBPs. (A) Simulated annealing o mit electron density map covering E favirenz and residue K103 (green) i n the wild-type NNIB P. In ( B) the same v iew is shown for E favirenz an d residue N103 (maroon) in the mutated N NIBP. Re sidue s ide-chains characteristic of the wild-type and K103N mutant NNIBPs are colored a ccordingly. The map was calculated w ith ligand and residu e 103 om itted employing the o mit-map op tion in CN S and contou red at 1.5 r. Table 2. Inhibition of HIV-1 RT. HIV-1RT (rCdG), IC 50 (n M ) a Wild-type K103N MSC194 5.5 52 PNU142721 2.5 7.0 Efavirenz 2.5 520 Nevirapine 170 3800 a The HIV-1 RT assay which used (poly)rCÆ(oligo)dG as the tem- plate/primer is described in [23]. Fig. 3. Superimposition o f Efavirenz bound to wild-type a nd K 103N mutant RT NNIBPs. Stereoview of the superimposition of Efavirenz bound t o the NNIBP of wild-type RT and the K103N mu tant. Residue side chain s c haracteristic of the NNIBP are included from e ach inhibitor com plex a nd colored green for wild-type and m aroon for the K103N mu tant. T he superimposition was c arried 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). Ó FEBS 2002 The HIV-1 RT K103N mutant and inhibitor efficacy (Eur. J. Biochem. 269) 1673 Similar 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 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 t hat several pivotal inter- actions are shared to key residues i n 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 w ith 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 s imilarities in the NNIBP of the K103N mutant structures t he 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 minu te displacements of Y181 and Y 188 away from the inhibitors compared to Efavirenz. These rear- rangements allow for a rotation o f the side chain of F227 with respect to t he 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 c hemical struc- ture of wing2 in PNU142721 with respect to MSC194 and Efavirenz induces a change in t he rotamer of D192, thereby disrupting the negative patch. DISCUSSION Anti-HIV compounds belonging to the new generation of NNRTIs have increased inhibitory efficacy with respect to wild-type and a number of d rug resistant RT mutants. Table 3. I nter-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 clarifi ed in Fig. 1. Residue 103 WT-Efavirenz K103N-Efavirenz K103N-MSC194 K103N-PNU142721 Cb C6 4.2 C6 4.0 C6 3.8 C5 3.9 N8 3.9 N2 3.8 N C6 4.1 C6 3.9 C6 3.7 N18 3.4 Cc N8 3.7 N8 3.7 N2 4.0 F1 3.8 C9 3.9 S1 4.0 Fig. 4. Supe rimposition of three K103N mutant NNIBPs. Stere oview of the sup erimposition of Efavirenz (m aroon), 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 a nd colored accordingly. The superimposition was carried ou t using all atom s from the residu es w ithin 4.0 A ˚ from the inhibitors ( V189, K101, K103N, V179, Y181, Y188, F227, W229, L234, H235, Y318 and E1138). 1674 J. Lindberg et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Accordingly, the NNRTIs Efavirenz, PNU142721, and MSC194 have IC 50 -values in the nanomolar range t o wild- type RT as well as to the f requently occurring K103N mutant. Not unexpectedly the efficacy towards this mutant differs significantly among the three inhibitors. The IC 50 -values range from 7.0 n M (PNU142721) to 520 n M (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 i nto the structural basis f or 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 t he 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] c oncluded t hat the e fficacy of Efavirenz against t he K103N mutant was du e to this novel binding mode. The data presented herein indicate that the K103N mutation induces minute repositions of the inhibitors PNU142721, MSC194 and efavirenz, w ith minor readjust- ments in t he positions of residues lining t he bind ing 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 inh ibitor and lining amino-acid residues, the compound needs t o b e s mal- 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 b inding mode to the K103N mutant a s the chemically related inhibitors MSC204 and M SC215 h ave to wild-type R T [11]. Superimposition o f 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 an d lining r esidues. 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 t he structures of PNU142721 and MSC194. Thus, the reasons for resistance, and the individual differences in efficacy exhibited by t hese 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 t he 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 t hree compounds. Though t he electron density for the a mide 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 e fficacy of these compounds compared to Efavirenz, shown by the antiviral data (Table 2). Changes in electrostatic interactions induced by the K103N mutation The e lectron density fo r Cc of the K 103N mut ant st ructures is well defined but the quality of t he map does n ot allow assessment of the orientation of the amide p lane. 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 N 103 am ide i s orientated w ith N d2 positioned in the negatively charged patch composed of D192, w hile the backbone carbonyls of G191 and K102 impose a stabilizing effect on that regio n of RT. A similar orientation in the p 66 subunit positions O d1 in close proximity t o the highly electronegative trifluoromethyl moiety of Efavirenz and the sulfur atoms of PNU142721 and MSC194, with repulsion as a c onsequence. However, in the cases of MSC194 and PNU142721 the position of the sulfur atom is such that the repulsive forces are less apparent. In t he MSC194 mutant complex the rotational f reedom of the amide is reduced by the s tacking of O d1 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 r eflect 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 i n agreement Ó FEBS 2002 The HIV-1 RT K103N mutant and inhibitor efficacy (Eur. J. Biochem. 269) 1675 with the r anking of the compounds with respect to their inhibitory efficacy. However, these factors may not solely account for t he 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 h ydrogen bonds that was not present in the wild- type enzyme [17]. In particular the hydrogen bond between N103 and Y 188 was suggested to s tabilize 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 p roposal that individual differences in efficacy between related NNRTIs can arise from d ifferential 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 f or competition with the hydrogen-bond network i n the K103N mutant remains to b e 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 t hese changes for NNRTI-binding is described as changes among two properties influencing the inhibitory efficacy: hydrophobic and electrostatic factors. The potent i nhibitor 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. ACKNOWLEDGEMENTS This work was supported by the Swedish Medical Research Council (MFR, K79-16X-09505-07A), the Swedish National Board for Indus- trial a nd Technical Develop ment (NUTEK). We thank the staffs of station 711 of the MAX synchrotron, L und, Sweden, and the beam l ine BM14, ESRF, 6 rue Jules Horowitz, BP 220, F-38043 G renoble Cedex, France, fo r their assistance. Terese Bergfors is addressed t hanks for proofreading the manuscript. REFERENCES 1. 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The cyclopropyl- group of MSC194 and the methyl group of PNU142721 partly overlap the trifluoromethyl group of Efavirenz. Furthermore,