The nucleic acid binding cleft of HIV-1 RT is formed by the fingers, palm, thumb, connection, and RNase H subdomains of the p66 subunit. The p51 thumb and connection subdomains form the floor of the cleft (Figure 1). In all crystal structures of RT in complex with nucleic acids, the DNA/DNA or RNA/DNA contact simultaneously both the polymerase and the RNase H domains of RT, separated by 17–18 base pairs of the nucleic acid substrate. While the 3′-end of the primer strand is always engaged at the polymerase active site, the RNase H active site is not seen to contact the scissile phosphate in any of the crystallized complexes. Nonetheless, biochemical data suggest that it is possible for RT to engage a template/primer at both active sites simultaneously [35].

The interactions of RT with the nucleic acid involve primarily the sugar-phosphate backbone and therefore these interactions are non-specific. Most interactions are between the six primer, and eight template nucleotides proximal to the polymerase active site, with the fingers, palm, and thumb subdomains of p66. Residues of the p66 fingers form the ‘template grip’ (V75, R78, N81, E89, P157, and G93), have close contacts with base-paired residues of the template strand and help position the template at the polymerase active site [8,9], while others (W24, F61) help to bend the unpaired 5’ template overhang away from the helical axis of the nucleic acid duplex by making hydrophobic contacts with nucleotide bases [12,20,23]. Interactions of the nucleic acid with the p66 palm subdomain involve amino acid residues from the polymerase active site region, including M184, which interacts with the 3’-OH primer terminus and is part of the highly conserved YMDD motif present in all retroviral RTs [36-38]. The primer strand interacts with p66 palm domain residues that form the “DNA primer grip”, a highly conserved structural motif close to the polymerase active site [39], which comprises the p66 β12–β13 hairpin in HIV-1 RT and helps position the 3′-OH end of the primer strand at the polymerase active site. In addition, residues of the “RNase H primer grip” in the p66 connection subdomain interact with the primer strand 11–15 nucleotides upstream of the primer 3′-terminus in all RT complexes with nucleic acid. Interactions of the p66 thumb with the nucleic acid involve helix αH of the p66 thumb, which is partly inserted into the minor groove of the dsDNA and helix αI, which is directly adjacent to the backbone of the template strand. Biochemical studies have shown that changes in residues of the αH, αI helices or in the DNA primer grip alter nucleic acid binding and may affect the polymerase and/or RNase H activities of RT [8,20,39-47].

The contacts between RT and the primer strand are very similar in RT structures with various DNA/DNA substrates. However, in the RT-RNA/DNA structure almost half of the RNA template residues have additional interactions with RT through their 2’-OH group (Figure 2). Finally, an RNA template has additional interactions with p51 residues at the floor of the nucleic acid binding cleft. The more extensive contacts between RT and RNA/DNA versus DNA/DNA may account for the increased polymerization activity and processivity of the enzyme with RNA templates.

Comparison of the structures of binary (RT-DNA/DNA or RT-RNA/DNA) [8-13,20] and ternary (RT-DNA/DNA-dNTP or RT-DNA/DNA-TFV diphosphate) complexes [12,13,23] suggests that the overall conformation of the nucleic acid is maintained after a dNTP or an analog binds at the nucleotide binding site. The part of the template/primer that is close to the polymerase active site has bases in the C3’-endo (North) conformation (A-form), as is the case with DNA bound to other DNA polymerases [48-50]. This type of geometry allows optimal alignment of the primer 3’-OH for nucleophilic attack on the α−phosphate of the incoming dNTP (Figure 3A) [51]. Interactions of Y115 with the ribose ring of the incoming dNTP or its analogs, impose strict conformational requirements for substrate binding. Hence, only nucleotides with ribose bases in the North conformation can be accommodated at the dNTP-binding site (Figure 3B). Moreover, Y115 acts as a steric gate, hindering binding and incorporation of ribonucleotides [52,53] and affecting the processivity of the enzyme [54].

Three aspartates form the catalytic center of the RT polymerase active site, the 3’-OH group of the primer strand and the phosphate groups of the incoming dNTP, bind two divalent metal ions (Figure 4). They are part of a β-sheet composed of β-strands 7, 9, and 10. Two of the three aspartates, D185 and D186, belong to the conserved YMDD motif. Moreover, M184 also interacts with the 3’ primer terminus and the incoming dNTP and is also involved in NRTI drug resistance [10,56,57]. Binding of dNTP induces a major conformational change of the p66 fingers subdomain, which folds over the incoming nucleotide triphosphate (Figure 5). As a result, conserved residues R72 and K65 of the fingers bind the β- and γ-phosphates of the dNTP, respectively [12], completing the “engulfment” of the nucleotide by residues of the palm and thumb subdomains. The 3’-OH of dNTP interacts with residues of the 3’ ‘pocket’ [12]. This pocket is formed by conserved residues D113, A114, Y115, F116, and Q151 (Figure 4).

The first anti-AIDS drugs were 3’-OH-modified NRTIs aimed at exploiting the requirement of free 3’-OH by HIV RT. Currently, all approved NRTIs (Figure 6) lack a 3’-OH and act as chain terminators after their incorporation into viral DNA by RT. The potency of NRTIs is primarily affected by three major factors: (i) the efficiency by which they are converted to the active species, which is the triphosphate form [71,72]. Activation of NRTIs is carried out by cellular kinases that add the α-, β-, and γ- phosphates to the nucleoside prodrugs [73]. Tenofovir (TFV) already contains a phosphonate group and requires the addition of only two phosphates; (ii) NRTIs must be reasonably stable to catabolic enzymes present in the host cell environment, and (iii) their triphosphate form must be an efficient inhibitor of HIV RT. Notably, the absence of a 3’-OH from all approved NRTIs significantly decreases their activation efficiency, binding interactions with RT, and overall potency [74,75].

RT incorporates NRTI triphosphates with variable efficiencies. For example, RT incorporates zidovudine-5’-triphosphate (AZT-TP), a thymidine analog (Figure 6), very efficiently [76]. In contrast, lamivudine-5’-triphosphate (3TC-TP), an L-enantiomer analog of cytidine, is incorporated considerably less efficiently [77]. The crystal structures of RT bound to zidovudine-5’-monophosphate (AZT-MP)-terminated primer in pre- and post-translocation conformations revealed interactions of the 3’-azido of AZT-MP with residues of the active site (D113 and A112) that are likely to be responsible for the efficient binding of zidovudine (AZT) at the dNTP binding site [11]. On the other hand, modeling 3TC-TP instead of dTTP at the active site of the catalytic complex of RT-DNA/DNA-dTTP suggested that the L-enantiomer would be less favorable than the canonical substrates [10].

The crystal structures of the catalytic complex of RT with DNA/DNA and tenofovir diphosphate highlighted the role of K65 and R72 in binding the phosphonate and phosphate moieties of the incoming inhibitor [13,23]. In the same structure, Q151 and Y115 were shown to interact with the aliphatic (acyclic) component of TFV.

Recently, it was shown that 4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA) (Figure 6), a nucleoside analog that unlike known chain-terminators retains a 3'-hydroxyl moiety, can inhibit HIV-1 RT with an unprecedented potency (EC₅₀ = 50 pM in Peripheral Blood Mononuclear Cells), several orders of magnitude better than any known NRTI [78]. This exceptional antiviral activity is the result of multiple factors, including: a) an efficient phosphorylation of the prodrug due to the presence of a 3’OH; b) a resistance to catabolic degradation by adenosine deaminase [79], and c) a mechanism of action that is different from all other approved NRTIs. RT can use EFdA-5'-triphosphate (EFdA-TP) as substrate even more efficiently than the natural substrate dATP [79]. Surprisingly, despite the presence of a 3'-OH, the incorporated EFdA-5’-monophosphate (EFdA-MP) acts mainly as a de facto terminator of further RT-catalyzed DNA synthesis, due to difficulty of RT translocation on the nucleic acid primer possessing 3'-terminal EFdA-MP. Thus, EFdA is a Translocation Defective RT Inhibitor (TDRTI) that blocks HIV replication by a novel mechanism of inhibition [79].

NNRTIs are important components of several combination therapies. They bind in a hydrophobic pocket of HIV-1 RT, close to the polymerase active site and at the base of the p66 thumb. This pocket is formed by residues L100, K101, K103, V106, T107, V108, V179, Y181, Y188, V189, G190, F227, W229, L234, and Y318 of p66, and E138 of p51 [5,17] (Figure 7). Comparison of structures in the presence and absence of NNRTIs showed that the NNRTI-binding pocket (NNIBP) does not exist in the absence of NNRTIs [14,21,28,80]. Instead, it is created upon the binding of NNRTIs by large-scale conformational changes in the side chains of RT residues, including Y181 and Y188, and by moving the “primer grip” to an extended conformation [80]. NNRTI binding renders constraints upon the conformation of the p66 thumb such that it stays in an over-extended conformation [17,21]. It has been reported that some potent NNRTIs enhance the dimerization of HIV-1 RT [81]. There is no NNRTI binding pocket in p51 because the relative position of the RT subdomains is different than in p66.

Biochemical data have shown that NNRTIs are non-competitive RT inhibitors with respect to either dNTP or nucleic acid substrates. Based on transient kinetics data, it has been proposed that binding of an NNRTI interferes with the chemical step of DNA synthesis [83,84]. A number of different mechanisms have been proposed for NNRTI inhibition of RT. Based on the early RT-nevirapine structure [17], it was postulated that nevirapine somehow alters the precise geometry of the polymerase active site, or restricts the mobility of the p66 thumb subdomain. The support for this hypothesis stems from the comparison of the RT-nevirapine structure with RT-DNA/DNA complex [20,80]. The comparison showed that NNRTI-binding causes local distortions of polymerase active site residues at the β9-β10 hairpin, as well as residues of the “primer grip” (β12-β13 hairpin) (Figure 8). These distortions may prevent proper positioning of the primer strand relative to the polymerase active site. Esnouf et al. reiterated that displacement of active site residues is likely to affect DNA synthesis [15]. Furthermore, Ding et al. [14,28] as well as Ren et al. [21] showed that other RT-NNRTI structures have striking similarity in the binding modes of diverse non-nucleoside inhibitors and remarkable consistency of a butterfly-like shape adopted by the inhibitor molecules suggesting the impact on polymerase activity by NNRTIs via a common mechanism. More recently, structural evidence was presented to demonstrate that the binding of NNRTIs restrict the flexibility of the YMDD loop and prevent the catalytic aspartate residues from adopting the metal-binding conformation seen in the RT-DNA/DNA-dTTP complex [12]. In summary, structural studies have shown that NNRTIs alter the geometry of the thumb, of the polymerase active site (YMDD motif and metal binding residues), and of the primer grip. Structural differences in the binding of various NNRTIs suggest that is possible that different NNRTIs have differences in their inhibition mechanism.

The above mechanisms of action of NNRTIs have been extrapolated from the comparison of apo RT, NNRTI-bound RT and ternary complex (containing nucleic acid and nucleotide) of RT. A key missing structure of RT complexed with template-primer, dNTP, and NNRTI would elucidate the structural changes that NNRTIs cause to the catalytic complex.

All approved NRTIs lack the 3′-OH found in the canonical dNTP substrates. Once incorporated, NRTIs act as chain terminators, blocking further DNA synthesis [71,72,85]. The NRTI-resistant RTs prevent NRTIs from blocking DNA synthesis, and are still able to incorporate normal dNTPs (or the virus would not be able to replicate). So far, HIV RT has used two main strategies for developing resistance to NRTIs (reviewed in [86] and references within):

Residues that interfere with the incorporation of NRTIs reside in the p66 fingers or palm subdomains of RT; all are in positions that could affect the binding of an incoming dNTP or NRTI. A classic example of this mechanism is the high-level resistance to lamivudine (3TC) and emtricitabine (FTC), which is imparted by a single mutation at codon 184 of the RT polymerase active site (M184V or M184I) [87]. The M184V and M184I mutations also decrease viral replication capacity, particularly in the presence of low concentrations of dNTP [88,89]. In addition, M184V confers low resistance to zalcitabine (ddC), didanosine (ddI), and abacavir (ABC) [57,90-93]. Structural work has suggested that resistance to 3TC is caused by unfavorable (steric) interactions between β-branched amino acids (Val, Ile, Thr) and the β-L-oxathialone ring of 3TC triphosphate [10]. These interactions interfere less with the incorporation of dNTPs; thus the β-branched amino acids act as a molecular filter against 3TC [94]. Based on structural work, Sarafianos et al. proposed that NRTIs with a decreased footprint would have a more favorable profile against M184 mutants [11]. This prediction is confirmed by the favorable resistance profile of the FDA-approved acyclic drug TFV, and three more drugs currently in clinical trials (amdoxovir [95], elvucitabine [96,97] and apricitabine [98]) (Figure 6).

Other examples of mutations affecting the incorporation of NRTIs include the Q151M complex (Q151Mc: Q151M followed by A62V, V75I, F77L and F116Y), L74V, and K65R. The Q151M complex mutations cause multidrug resistance to AZT, ddI, ddC, stavudine (d4T), and ABC, but the mutant enzyme remains sensitive to adefovir-diphosphate (an acyclic NRTI related to tenofovir-diphosphate) and to 3TC-triphosphate [99-102]. Crystal structures of RT in complex with substrates [10-12,20,23] have provided insights into the molecular mechanism by which mutations at these residues alter the interactions of the incoming dNTP (or NRTI triphosphate) and cause resistance. The mechanism of Q151Mc multi-drug resistance involves a reduction of the catalytic rate (k_pol) of NRTI incorporation, as compared to the canonical dNTP. The decrease in k_pol may be due to the loss of the hydrogen bond network involving the 3′-OH group of the incoming dNTP, residues Q151 and Y115, and the leaving PPi group [12,13,23,103].

The L74V mutation causes resistance to ddC, ddI [104,105], and ABC [92,93] by decreasing the incorporation rate of the inhibitor, as shown by steady-state kinetics studies [105]. The discrimination appears to be the effect of reduced catalytic rate (k_pol) by L74V compared to WT enzyme [106]. Similar to Q151, L74 is important for the structural integrity of the dNTP-binding site, as it stabilizes the templating base opposite the incoming dNTP or NRTI substrates [9,12,20]. Changes in these interactions selectively reduce the incorporation rate of the nucleotide analogs and cause NRTI resistance [106]. Mutation at a neighboring residue (V75T) causes d4T resistance also by affecting the stability of the nucleotide-binding site through its proximity to residue 74 [107-109].

The K65 residue is located in the fingers subdomain of RT and interacts with the γ-phosphate of the incoming nucleotide substrate in the ternary complexes of HIV-1 RT [12]. This residue also interacts with the terminal phosphate of TFV-DP [13,23]. The K65R mutation has been found to confer resistance to TFV [110], but also to ddI and ABC, and to a lesser extent to 3TC and ddC [90,92,93,111]. Pre-steady state kinetic characterization of the K65R mutant suggested the discrimination against ddC-TP, ddA-TP, 3TC-TP and TFV-DP was due to either higher K_d or lower k_pol or combination of both [106,112-115]. In the case of TFV-DP, the discrimination by K65R results in a reduced k_pol[116,117] but for ddC-TP both K_d and k_pol are altered [114].The recent crystal structures of K65R RT with tenofovir diphosphate (TFV-DP) or dATP have shown that the guanidinium planes of the arginines K65R and R72 stack and restrict the conformational adaptability of both the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation. Furthermore, the guanidinium planes of K65R and R72 are in two different rotameric conformations in TFV-DP and dATP-bound structures, which may help explain how K65R RT discriminates the drug from substrates [23].

In the case of some NRTIs, the resistant enzymes readily accept the inhibitor as a substrate for incorporation into the DNA chain. However, these mutant RTs have an enhanced capacity for removing the chain-terminating nucleotide from the DNA terminus to generate a free 3’-OH and allow elongation of the formerly terminated DNA strand to resume [118,119]. The excision reaction is related to the normal DNA polymerization but in the reverse direction [118,119], and it requires a pyrophosphate donor which RT joins to the NRTI at the^{3' primer terminus, excising it from the primerDNA. Although both ATP or PPi can serve as pyrophosphate donors [118-122] it is now widely accepted that resistance is caused by the nucleophilic attack of ATP at the primer terminus to generate a free 3’-OH and a 5’-5’ dinucleoside-tetraphosphate [119]. Interestingly, the dinucleotide tetraphosphate product of ATP-mediated excision of chain-terminating AZT-MP is a potent chain-terminating substrate for HIV-1 RT [123].}

AZT and d4T-resistance mutations M41L, D67N, K70R, L210W, T251Y/F and K219Q/E are the most common examples of excision enhancing mutations (EEM). Although not selected by TFV, these mutations also confer cross-resistance to this inhibitor. These residues are not located at the dNTP-binding site [12,13,23]. Biochemical data have shown that the presence of EEMs does not affect the incorporation of nucleotide analogs [124]. Instead, they facilitate the ATP-dependent removal of the incorporated NRTI that occurs at the same active site as the polymerization reaction [121].

A structural model has been proposed to address the biochemical data of the excision reaction [116]. In this model, the ATP binding cleft is surrounded by residues involved in the excision-based resistance to NRTIs (41,44,67,70,210,215 and 219) (Figure 9). The aromatic ring of ATP interacts with the aromatic ring of Y215 through π-π interactions. This interaction changes the binding of ATP and affects its orientation. A recently presented crystal structure of WT and EEM-containing RTs complexed with a DNA/DNA primer/template and the ATP-dependent excision product of AZT (AZTppppA) confirmed this model [125].

For excision to occur, the chain-terminating nucleotide must be located in the nucleotide-binding site (the N-site) (Figure 9), in a pre-translocation conformation [11,126,127]. Structural and biochemical evidence have provided insights on the various factors that affect excision of NRTIs.

Excision of NRTIs is susceptible to the presence of the next incoming dNTP. This is because the presence of an incoming dNTP at the N-site stabilizes the NRTI-terminated primer to the post-translocation priming site (P-site) where it is not accessible for excision by nucleophilic attack by ATP [126,128]. If the NRTI is a dideoxy- inhibitor, then the resulting complex of RT with dideoxy-teminated DNA and incoming dNTP forms a stable “dead-end complex”, as shown in band mobility shift assays [129] and in the crystal structure of the corresponding complex [12] (Figure 4). However, the RT complex with AZT-MP terminated nucleic acid and incoming dNTP is not as stable [129]. Modeling of an incoming nucleotide in the crystal structure of the post-translocation complex of RT with AZT-terminated DNA (DNA _{AZT-MP (P)}) predicts steric hindrance between the azido group of DNA _{AZT-MP (P)and the incoming nucleotide. This suggests that the azido group prevents the next complementary nucleotide from inhibiting the excision reaction [11]. Primers terminated with a 3’ azido nucleotide (including AZT) have also been shown to bind preferentially in the pre-translocation state in the absence of dNTPs, properly positioning the chain-terminator for excision [128,130]. Binding of AZT-terminated primers in this mode appears to be enhanced by interactions of the azido group with RT residues of the N-site, as seen in the crystal structure of RT in complex with AZT-terminated DNA in pre-translocation mode [11].}

The excision reaction is also affected by the presence of specific mutations. When added to RT with EEM mutations, the M184V mutation causes a decrease in AZT excision [131-133] and resensitization to AZT [134]. The decreased rate of excision in the presence of mutations at residue 184 may be related to a repositioning of the nucleic acid in the mutant enzyme, which may affect the alignment of the excision reaction components [10].

The K65R and L74V mutations [135-137], as well as the NNRTI resistance mutations L100I and Y181C [138-140], and the foscarnet resistance mutations W88G and E89K [141] also cause decreases in the excision reaction. While the structural details of this inhibition are not clear, some of the mutated residues (residues 74, 88, and 89) are in a position to affect the binding of the nucleic acid substrate and affect the alignment of the NRTI-terminated primer at the excision site.

The “fingers insertion complex” consists of an EEM mutation backbone with an additional T69S mutation and an insertion of two or more amino acids (usually SS, SA, or SG) between residues 69 and 70 of the fingers subdomain. This mutational pattern confers resistance to all NRTIs [142,143] through an increase in the rate of chain-terminator removal [144,145]. There is an apparent destabilization of the dNTP-induced dead-end complexes for primers terminated with a variety of NRTIs and an increase in their excision [146-148]. Site-specific foot-printing assays also showed that there was an increased access to the pre-translocational state [126] associated with a decreased binding for NRTIs and dNTPs [144].

Other RT mutations affect the specificity of the excision reaction in a different way. Such mutations are usually selected during HIV-1 treatment with multiple NRTIs. For example, while the excision enhancing mutations (EEM: M41L, D67N, K70R, L210W, T215Y/F, and K219Q) cause resistance to AZT, addition of the E44D/A mutation in the EEM background confers additional moderate resistance to 3TC [149]. It was shown that 3TC resistance due to mutation E44D/A is associated with the excision mechanism [150]. Such mutations are expected to affect the alignment of the ATP substrate of excision in the ATP-binding pocket (Figure 9).

Mutations at the connection subdomain of HIV-1 RT have been reported to affect drug resistance [151,152]. Specifically, N348I was reported to cause resistance to both the NRTI AZT, and the NNRTI nevirapine [153,154]. The AZT resistance mechanism appears to involve unblocking of AZT-terminated primers [155]. It has been proposed that due to their reduced RNase H activity, connection-domain mutant enzymes can excise incorporated AZT before the RNA template is degraded and the substrate becomes unusable by RT [155].

Wild-type HIV-2 RT has reduced ATP-dependent phosphorolytic activity in comparison with HIV-1 RT. When HIV-2 is challenged with AZT, it acquires resistance mutations that cause reduced AZT incorporation rather than enhanced excision of AZT-MP [156]. Comparison of the crystal structures of HIV-1 and HIV-2 RT [157] suggested that there are differences in the putative ATP binding sites of the two enzymes that explain why HIV-1 RT binds ATP more effectively and is more efficient in causing resistance to AZT through the excision mechanism [156].

Amino acids Y181 and Y188 are key residues of the NNIBP that interact with “Wing I” of the butterfly-like NNRTIs (Figure 11). Upon binding of nevirapine, loviride (α-APA), and tetrahydroimidazo[4,5,1-jkj][1,4]benzodiazepin-2(1H)-one (TIBO) [14,21,26,28,160] both tyrosines assume a “flipped” conformation (Figure 11) [154,156]. For weaker inhibitors such as 1-(2-2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) and {3-[4-(2-methyl-imidazo[4,5-c]pyridin-1-yl)-benzyl]-3H-benzothiazol-2-one} (CP-94,707) only one chain is repositioned [31,161]. The large contribution of Y181 and Y188 to the binding energy of these NNRTIs explains why mutations at these residues result in a drastic decrease in inhibitor binding. Similarly, F227L also appears to cause resistance through loss of interactions with NNRTIs. Mutations L100I, V108A, and V18I have been proposed to cause minor changes, which in turn diminish the interactions of Y181 and Y188 with the bound NNRTI [33].

The L100I mutation can cause steric interference between the β-branched isoleucine and an incoming NNRTI. Similarly, the G190A mutation can cause resistance through steric conflict of the alanine side chain and the bound NNRTI. For example, in the crystal structure of the WT RT/[(S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-dihydroquinoxaline-2(1H)-thione] (HBY097) complex [29] the bulky and rigid quinoxaline ring of HBY097 is near residue 190. A G190A mutation would cause the Cβ atom of A190 to have a steric clash with the quinoxaline moiety and reduce the binding of HBY097 [29].

Amino acids K101 and K103 are thought to be at the site of NNRTI entry to the pocket. Mutations K103N and K101E cause strong resistance to multiple NNRTIs, including the first generation NNRTI nevirapine. Comparison of the crystal structures of WT and K103N RTs in complexes with efavirenz [30], or HBY097 [29] demonstrated that the K103N mutation has minimal influence on the bound conformation of an NNRTI. However, in the crystal structure of K103N in the absence of NNRTI, a hydrogen bond between Tyr188 and Asn103 closes the entrance to the NNRTI binding pocket, efficiently reducing its access to multiple NNRTIs [16,24,30]. Moreover, this interaction results in a stabilized unbound state of RT and hence affects the kinetics of the inhibitor-binding process [16]. However, K103N has no significant resistance to etravirine, because this inhibitor was designed to interact with this residue, hence this mutation enhances its binding [161]. The kinetic of NNRTI binding have also been studied by the surface plasmon resonance technique showing that the resistance to NNRTIs may also be related to changes in the affinity for inhibitor either by reducing the association rate or by increasing the dissociation rate [162].

In summary, NNRTI-resistance mutations appear to affect NNRTI binding directly, by altering the size, shape, and polarity of different parts of the NNIBP or, indirectly, by affecting access to the pocket. To overcome the binding deficiency of NNRTIs to resistance mutants, more flexible NNRTIs were designed with so-called strategic flexibility [25,163]. Flexible NNRTIs such as TMC-278 (rilpivirine) have compensatory interactions with RTs that have mutations causing resistance to the first-generation NNRTIs. TMC-278 uses its cyanovinyl group to recruit Y183 and help compensate for the loss of interactions caused by the Y181C mutation (Figure 11). This flexibility has been called “wiggling and jiggling” and allows NNRTIs to adapt to changes in the NNIBP caused by resistance mutations. Structural studies of WT, K103N/Y181C, and L100I/K103N HIV-1 RT complexes with TMC-278 [25] have shown that the side chains of the flexible NNIBP act as a molecular “shrink wrap” that makes a shape complementary to the optimized TMC-278 in WT and drug-resistant forms of HIV-1 RT.