Modulation of Retinoic Acid Receptor Subtypes by 5- and 8-Substituted (Naphthalen-2-yl)-based Arotinoids.

Retinoid receptors (RARs and RXRs) transduce the signals of their natural and synthetic ligands (retinoids and rexinoids) to cellular transcriptional machinery to induce gene programs that control diverse biological and physiological effects on organisms. All-trans-retinoic acid, the natural ligand for RARs, is used therapeutically for the treatment of acute promyelocytic leukemia (APL), whereas the synthetic rexinoid bexarotene (a representative member of the aromatic retinoids or arotinoids) is approved for the treatment of cutaneous T-cell lymphoma (CTCL). Other retinoids have found applications in the topical treatment of skin disorders. In continuation of previous work on the naphthalene-based arotinoid scaffold, we synthesized a new series of (3-halo)benzoic acids connected to C5- or C8-substituted naphthyl rings via (E)-ethenyl and amide and, for the C5 series, (E)-chalcone linkers. These compounds were evaluated as RAR modulators in comparison with previously described dihydronaphthalene arotinoids with the same substitution pattern. Transactivation studies in this series revealed an absence of synergy between small halogen atoms (F, Cl) at C3 and the groups at C5 or C8, as had been observed on some of the dihydronaphthalene analogues. Instead, non-halogenated 4-(2-naphthamido)benzoic acid derivatives transactivated toward the RARß subtype in preference to the paralogues. The derivatives with bulkier substituents at C8 were characterized as dual RARß/RARα antagonists, and (E)-4-[(8-(phenylethynyl)naphthalene-2-yl)ethenyl]benzoic acid (11 c), with an ethenyl connector, was shown to be a potent antagonist of RARα.

Dual RXR Agonists and RAR Antagonists Based on the Stilbene Retinoid Scaffold.

Arotinoids containing a C5,C8-diphenylnaphthalene-2-yl ring linked to a (C3-halogenated) benzoic acid via an ethenyl connector (but not the corresponding naphthamides), which are prepared by Horner-Wadsworth-Emmons reaction of naphthaldehydes and benzylphosphonates, display the rather unusual property of being RXR agonists (15-fold induction of the RXR reporter cell line was achieved at 3- to 10-fold lower concentration than 9-cis-retinoic acid) and RAR antagonists as shown by transient transactivation studies. The binding of such bulky ligands suggests that the RXR ligand-binding domain is endowed with some degree of structural elasticity.

Indole-derived psammaplin A analogues as epigenetic modulators with multiple inhibitory activities.

A SAR study has been carried out around a modified scaffold of the natural product psammaplin A obtained by replacing the o-bromophenol unit by an indole ring. A series of indole psammaplin A constructs were generated in a short synthetic sequence that starts with the functionalization of the C3 indole position with in situ generated nitrosoacrylate, and this is followed by protection of the ß-indole-α-oximinoesters, saponification, condensation with symmetrical diamines, and deprotection. Biochemical and cellular characterization using U937 and MCF-7 cells confirmed that many of these analogues displayed more potent actitivies than the parent natural product. Moreover, in addition to the reported HDAC and DNMT dual epigenetic inhibitory profile of the parent compound, some analogues, notably 4a (UVI5008), also inhibited the NAD(+)-dependent SIRT deacetylase enzymes. The SAR study provides structural insights into the mechanism of action of these multiple epigenetic ligands and paves the way for additional structural exploration to optimize their pharmacological profiles. Because of their multi(epi)target features and their action in ex vivo samples, the indole-based psammaplin A derivatives are attractive molecules for the modulation of epigenetic disorders.

Epigenetic profiling of the antitumor natural product psammaplin A and its analogues.

A collection of analogues of the dimeric natural product psammaplin A that differ in the substitution on the (halo)tyrosine aryl ring, the oxime and the diamine connection has been synthesized. The effects on cell cycle, induction of differentiation and apoptosis of the natural-product inspired series were measured on the human leukaemia U937 cell line. Epigenetic profiling included induction of p21(WAF1), effects on global H3 histone and tubulin acetylation levels as well as in vitro enzymatic assays using HDAC1, DNMT1, DNMT3A, SIRT1 and a peptide domain with p300/CBP HAT activity. Whereas the derivatives of psammaplin A with modifications in the length of the connecting chain, the oxime bond and the disulfide unit showed lower potency, the analogues with changes on the bromotyrosine ring exhibited activities comparable to those of the parent compound in the inhibition of HDAC1 and in the induction of apoptosis. The lack of HDAC1 activity of analogues modified on the disulfide bond suggests that its cleavage must occur in cells to produce the monomeric Zn(2+)-chelating thiol. This assumption is consistent with the molecular modelling of the complex of psammaplin A thiol with h-HDAC8. Only a weak inhibition of DNMT1, DNMT3A and residual activities with SIRT1 and a p300/CBP HAT peptide were measured for these compounds.

C3 halogen and c8'' substituents on stilbene arotinoids modulate retinoic Acid receptor subtype function.

The synthesis and biological evaluation of the entire series of C3-halogenated derivatives and bulkier substituents at the C8'' position of the parent stilbene-based RARbeta-selective agonist BMS641 4 c was undertaken. The synthesis uses an E-selective Horner-Wadsworth-Emmons (HWE) condensation of C8-substituted C5-dimethyl dihydronaphthaldehyde and the benzylic phosphonates derived from the C3-halogenated benzoates to construct the stilbene skeleton. Transactivation studies revealed the synergistic effect of small halogen atoms at C3 (F, Cl) and the moderately bulky phenyl group at C8'' (in 4 b and 4 c) to achieve RARbeta selectivity. Our results, supported by computational studies, provide a structural rationale for the mixed agonist-antagonist activities of these arotinoids, which are potent agonists of the RARbeta subtype and antagonists of the RARalpha paralogue. Moreover, transitions from partial agonists to inverse agonists and antagonists can be accomplished with the incorporation of the same halogen atoms into the structures of known modulators BMS701 (5 a) and BMS493 (6 a), which have bulkier substituents than phenyl (p-tolyl and phenylethynyl, respectively) at C8''. Conversely, incorporation of halogen atoms in 6 a converted the ligand from an RARbeta inverse agonist (6 b) to an antagonist (6 c) or an agonist (6 d). Amazingly, 6 a-c commonly acted as inverse agonists for RARalpha, while 6 d and 6 e acted as regular RARalpha antagonists, not affecting co-repressor interaction. In the case of the mixed agonist/antagonist 5 a, C3-halogenation yields inverse RARalpha and RARbeta agonists (5 b-d) with the exception of iodinated 5 e, which is a regular antagonist for both these receptors. Because RARbeta gene expression is frequently deleted or epigenetically silenced in several tumor cells, the novel repertoire of receptor and function-selective RAR agonists, mixed agonist/antagonists, regular antagonists, and inverse agonists will be useful in the elucidation of the mechanism of tumor suppression by retinoids.

Retinoid receptor subtype-selective modulators through synthetic modifications of RARgamma agonists.

A series of retinoids designed to interfere with the repositioning of H12 have been synthesized to identify novel RARgamma antagonists based on the structure of known RARgamma agonists. The transcriptional activities of the novel ligands were revealed by cell-based reporting assays, using engineered cells containg RAR subtype-selective fusions of the RAR ligand-binding domains with the yeast GAL4 activator DNA-binding domain and the cognate luciferase reporter gene. Whereas none of the ligands exhibited features of a selective RARgamma antagonist, some of them are endowed with interesting activities. In particular 24a acts as a pan-RAR agonist that induces at high concentration a higher transactivation potential on RARalpha than TTNPB and synergizes at low concentration with TTNPB-bound RARalpha but not RARbeta or RARgamma. Similarly, 24c synergizes with TTNPB-bound RARgamma and exhibits RARalpha,beta antagonist activity. Compounds 24b and 25b are strong RARalpha,beta-selective antagonists without agonist or antagonist activities for RARgamma. Compounds 24b and 24c display weak RXR antagonist activity. In addition several pan-antagonists and partial agonist/antagonists have been defined.

Modulating retinoid X receptor with a series of (E)-3-[4-hydroxy-3-(3-alkoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)phenyl]acrylic acids and their 4-alkoxy isomers.

Rexinoids are ligands for the retinoid X receptor (RXR) that have great promise for both the prevention and treatment of cancer and metabolic diseases. In this regard, synthetic, functional, and structural investigations into the structure-activity relationships of derivatives of the potent RXR agonist (E)-3-[3-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-4-hydroxyphenyl]acrylic acid (CD3254, 9) have been conducted. We recently reported on the characterization of a series of C3'-substituted alkyl ether analogues of 9 (10a-f), which display activities ranging from partial agonists to pure antagonists. The importance of the position of the alkoxy side chain for ligand activity has been further explored with the synthesis of C4'-substituted analogues (11a-f). Here we describe the synthesis of compounds 11a-f, which appear functionally different from their isomeric counterparts, as judged from transactivation assays and fluorescence anisotropy experiments. We also report on the 2.0 A resolution structure of RXR in complex with the parent compound 9, which helps understanding of the impact of the alkyl side chain location on ligand activity.

Selective, potent PPARgamma agonists with cyclopentenone core structure.

A series of analogues of the PPARgamma ligand 15-deoxy-Delta(12,14)-PGJ(2) have been synthesized by functionalization of a 5-alkyl-4-hydroxycyclopentenone core structure obtained by Piancatelli rearrangement of precursor furylcarbinol. Transient transactivation assays indicate that analogues 18 and 20 are selective nanomolar agonists of PPARgamma. This subtype selectivity is lost in derivatives (23, 24) with an alkynyl (oct-1-yn) chain at the C3 position, although the cyclopentenone derivative with cis relative configuration (23) showed greater affinity for PPARalpha.

Highly twisted adamantyl arotinoids: synthesis, antiproliferative effects and RXR transactivation profiles.

Retinoid-related molecules with an adamantyl group (adamantyl arotinoids) have been described with selective activities towards the retinoid receptors as agonists for NR1B2 and NR1B3 (RARbeta,gamma) (CD437, MX3350-1) or RAR antagonists (MX781) that induce growth arrest and apoptosis in cancer cells. Since these molecules induce apoptosis independently of RAR transactivation, we set up to synthesize novel analogs with impaired RAR binding. Here we describe adamantyl arotinoids with 2,2'-disubstituted biaryl rings prepared using the Suzuki coupling of the corresponding fragments. Those with cinnamic and naphthoic acid end groups showed significant antiproliferative activity in several cancer cell lines, and this effect correlated with the induction of apoptosis as measured by caspase activity. Strikingly, some of these compounds, whereas devoid of RAR binding capacity, were able to activate RXR.

Modulators of the structural dynamics of the retinoid X receptor to reveal receptor function.

Retinoid X receptors (RXRalpha, -beta, and -gamma) occupy a central position in the nuclear receptor superfamily, because they form heterodimers with many other family members and hence are involved in the control of a variety of (patho)physiologic processes. Selective RXR ligands, referred to as rexinoids, are already used or are being developed for cancer therapy and have promise for the treatment of metabolic diseases. However, important side effects remain associated with existing rexinoids. Here we describe the rational design and functional characterization of a spectrum of RXR modulators ranging from partial to pure antagonists and demonstrate their utility as tools to probe the implication of RXRs in cell biological phenomena. One of these ligands renders RXR activity particularly sensitive to coactivator levels and has the potential to act as a cell-specific RXR modulator. A combination of crystallographic and fluorescence anisotropy studies reveals the molecular details accounting for the agonist-to-antagonist transition and provides direct experimental evidence for a correlation between the pharmacological activity of a ligand and its impact on the structural dynamics of the activation helix H12. Using RXR and its cognate ligands as a model system, our correlative analysis of 3D structures and dynamic data provides an original view on ligand actions and enables the establishment of mechanistic concepts, which will aid in the development of selective nuclear receptor modulators.

Structure, function and modulation of retinoic acid receptor beta, a tumor suppressor.

Only one of the three-retinoic acid receptors, RARbeta, is frequently deleted or epigenetically silenced at early stages in tumor progression and there is compelling evidence that RARbeta corresponds to a tumor suppressor. Recent discoveries may help to reveal the molecular basis of the tumor suppressive action of this retinoic acid receptor subtype and provide new tools for its analysis and, possibly, therapeutic exploitation. The first concerns the recent elucidation of the crystal structure of the ligand-binding domain of the agonist-bound receptor. The second is the discovery of selective agonists, including isoform selective ligands, which are important tools to facilitate the pharmacological analysis of the tumor suppressor function of this protein in vivo. Lastly, its involvement in a retinoic acid-induced tumor-specific apoptosis program mediated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Herein we describe the structure, function and ligand-dependent transcription mechanism of retinoic acid receptor beta, and use rational drug design to understand the selectivity of these modulators.

N1-substituted thymine derivatives as mitochondrial thymidine kinase (TK-2) inhibitors.

Novel N1-substituted thymine derivatives related to 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine have been synthesized and evaluated against thymidine kinase-2 (TK-2) and related nucleoside kinases [i.e., Drosophila melanogaster deoxynucleoside kinase (Dm-dNK) and herpes simplex virus type 1 thymidine kinase (HSV-1 TK)]. The thymine base has been tethered to a distal triphenylmethoxy moiety through a polymethylene chain (n = 3-8) or through a (2-ethoxy)ethyl spacer. Moreover, substitutions at position 4 of one of the phenyl rings of the triphenylmethoxy moiety have been performed. Compounds with a hexamethylene spacer (18, 26b, 31) displayed the highest inhibitory values against TK-2 (IC50 = 0.3-0.5 microM). Compound 26b competitively inhibited TK-2 with respect to thymidine and uncompetitively with respect to ATP. A rationale for the biological data was provided by docking some representative inhibitors into a homology-based model of human TK-2. Moreover, two of the most potent TK-2 inhibitors (18 and 26b) that also inhibit HSV-1 TK were able to reverse the cytostatic activity of 1-(beta-D-arabinofuranosyl)thymine (Ara-T) and ganciclovir in HSV-1 TK-expressing OST-TK-/HSV-1 TK+ cell cultures.

Involvement of novel human immunodeficiency virus type 1 reverse transcriptase mutations in the regulation of resistance to nucleoside inhibitors.

We characterized 16 additional mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) whose role in drug resistance is still unknown by analyzing 1,906 plasma-derived HIV-1 subtype B pol sequences from 551 drug-naïve patients and 1,355 nucleoside RT inhibitor (NRTI)-treated patients. Twelve mutations positively associated with NRTI treatment strongly correlated both in pairs and in clusters with known NRTI resistance mutations on divergent evolutionary pathways. In particular, T39A, K43E/Q, K122E, E203K, and H208Y clustered with the nucleoside analogue mutation 1 cluster (NAM1; M41L+L210W+T215Y). Their copresence in this cluster was associated with an increase in thymidine analogue resistance. Moreover, treatment failure in the presence of K43E, K122E, or H208Y was significantly associated with higher viremia and lower CD4 cell count. Differently, D218E clustered with the NAM2 pathway (D67N+K70R+K219Q+T215F), and its presence in this cluster determined an increase in zidovudine resistance. In contrast, three mutations (V35I, I50V, and R83K) negatively associated with NRTI treatment showed negative correlations with NRTI resistance mutations and were associated with increased susceptibility to specific NRTIs. In particular, I50V negatively correlated with the lamivudine-selected mutation M184V and was associated with a decrease in M184V/lamivudine resistance, whereas R83K negatively correlated with both NAM1 and NAM2 clusters and was associated with a decrease in thymidine analogue resistance. Finally, the association pattern of the F214L polymorphism revealed its propensity for the NAM2 pathway and its strong negative association with the NAM1 pathway. Our study provides evidence of novel RT mutational patterns that regulate positively and/or negatively NRTI resistance and strongly suggests that other mutations beyond those currently known to confer resistance should be considered for improved prediction of clinical response to antiretroviral drugs.

Mitochondrial thymidine kinase inhibitors.

Mitochondrial thymidine kinase or TK-2 belongs to the family of mammalian deoxynucleoside kinases (dNKs) that catalyze the phosphorylation of deoxynucleosides to their corresponding deoxynucleoside monophosphates by gamma-phosphoryl transfer of ATP. These enzymes are instrumental in the activation of deoxynucleoside analogues with biological and therapeutic properties. Moreover, dNKs are fundamental to maintain dNTPs pools for DNA synthesis and repair. TK-2 has a mitochondrial localization and is the only thymidine kinase that is physiologically active in non-proliferating and resting cells. Several recent investigations point to an important role of TK-2 in the maintenance of mitochondrial dNTPs pools. Indeed, mutations in the gene encoding TK-2 have been associated with mitochondrial DNA (mtDNA) depletion that mostly affects skeletal muscle. Moreover, TK-2 has been suggested to be implicated in mitochondrial toxicity associated to prolonged treatments with nucleoside analogues (i.e AZT for the treatment of AIDS patients). In this scenario, TK-2 inhibitors could be a useful tool to further clarify both the physiological role of TK-2 in the maintenance of mitochondrial dNTP pools, and the possible contribution of TK-2 to the mitochondrial toxicity of pyrimidine nucleoside analogues. In the present article we review the most recent literature covering different aspects of TK-2 as well as published TK-2 inhibitors, with special emphasis on acyclic nucleoside analogues that have been described by our research groups and whose prototype compound is 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine.

High sequence conservation of human immunodeficiency virus type 1 reverse transcriptase under drug pressure despite the continuous appearance of mutations.

To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs.

The role of Thr139 in the human immunodeficiency virus type 1 reverse transcriptase sensitivity to (+)-calanolide A.

The coumarins represent a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that were isolated from tropical plants. (+)-Calanolide A, the most potent compound of this class, selects for the T139I resistance mutation in HIV-1 reverse transcriptase (RT). Seven RTs mutated at amino acid position 139 (Ala, Lys, Tyr, Asp, Ile, Ser, and Gln) were constructed by site-directed mutagenesis. The mutant T139Q enzyme retained full catalytic activity compared with wild-type RT, whereas the mutant T139I, T139S, and T139A RTs retained only 85 to 50% of the activity. Mutant T139K, T139D, and T139Y RTs had seriously impaired catalytic activities. The mutations in the T139I and T139D RTs were shown to destabilize the RT heterodimer. (+)-Calanolide A lost inhibitory activity (up to 20-fold) against the mutant T139Y, T139Q, T139K, and T139I enzymes. All of the mutant enzymes retained marked susceptibility toward the other NNRTIs, including nevirapine, delavirdine, efavirenz, thiocarboxanilide UC-781, quinoxaline GW867420X, TSAO [[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)] derivatives, and the nucleoside inhibitor, ddGTP. The fact that the T139I RT 1) proved to be resistant to (+)-calanolide A, 2) represents a catalytically efficient enzyme, and 3) requires only a single transition point mutation (ACA-->ATA) in codon 139 seems to explain why mutant T139I RT virus strains, but not virus strains containing other amino acid changes at this position, predominantly emerge in cell cultures under (+)-calanolide A pressure.

The molecular basis of resilience to the effect of the Lys103Asn mutation in non-nucleoside HIV-1 reverse transcriptase inhibitors studied by targeted molecular dynamics simulations.

A series of targeted molecular dynamics simulations have been carried out in an attempt to assess the effect that the common Lys103Asn mutation in HIV-1 reverse transcriptase (RT) has on the binding of three representative non-nucleoside RT inhibitors (NNRTI), nevirapine, efavirenz, and etravirine. We have shown previously that, in the absence of an incoming inhibitor, creation of the NNRTI binding pocket is hampered due to the existence of a hydrogen bond between the side chains of Asn103 and Tyr188 for which no equivalent exists in the wild-type enzyme. As an extension of this work, we now apply the same methodology to drive the enzyme's conformation from the unbound state to the drug-bound state in the presence of the NNRTI. The location of each drug outside the binding pocket was determined by an automated docking program, and steering into the binding pocket followed a route that is likely to represent the actual entrance pathway. The additional hurdle to inhibitor entry imposed by the extra Asn103-Tyr188 hydrogen bond is seen to affect each NNRTI differently, with the ability to disrupt this interaction increasing in the order etravirine >> efavirenz > or = nevirapine, in good accord with the experimental findings. This coherent picture strongly suggests that attempts to overcome resistance through structure-based drug design may be considerably more successful if dynamic structural aspects of the type studied here are considered, particularly in cases where binding energy-based structure-activity relationship methods are unable to provide the required information.

The amino acid Asn136 in HIV-1 reverse transcriptase (RT) maintains efficient association of both RT subunits and enables the rational design of novel RT inhibitors.

The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.07 to 2.1% of wild-type activity is retained, depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by second-generation NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturating) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the beta7-beta8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity.

The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti-HIV-1 drugs.

Amino acids N137 and P140 in the p51 subunit of HIV-1 reverse transcriptase (RT) are part of the beta7-beta8-loop that contributes to the formation of the base of the non-nucleoside RT inhibitor (NNRTI)-binding pocket and makes up a substantial part of the dimerization interface. Amino acid P95 in p66 also markedly contributes to the dimerization binding energy. Nine RT mutants at amino acid 137 were constructed bearing the mutations Y, K, T, D, A, Q, S, H or E. The prolines at amino acid positions 95 and 140 were replaced by alanine in separate enzymes. We found that all mutant RT enzymes showed a dramatically decreased RNA-dependent DNA polymerase activity. None of the mutant RT enzymes showed marked resistance against any of the clinically used NNRTIs but they surprisingly lost significant sensitivity for NRTIs such as ddGTP. The denaturation analyses of the mutant RTs by urea are suggestive for a relevant role of N137 in the stability of the RT heterodimer and support the view that the beta7-beta8 loop in p51 is a hot spot for RT dimerization and instrumental for efficient polymerase catalytic activity. Consequently, N137 and P140 in p51 and P95 in p66 should be attractive targets in the design of new structural classes of RT inhibitors aimed at compromising the optimal interaction of the beta7-beta8 loop in p51 at the p66/p51 dimerization interface.

Understanding the basis of resistance in the irksome Lys103Asn HIV-1 reverse transcriptase mutant through targeted molecular dynamics simulations.

Results of targeted molecular dynamics simulations confirm the existence of a higher energy barrier for creation of the pocket where non-nucleoside reverse transcriptase inhibitors bind in the K103N mutant enzyme relative to wild-type.