An interesting class of RHRTIs is the hydrazone derivatives, whose first reported analog was N
-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) (). Unlike other NNRTIs or RHRTIs, BBNH inhibits both the polymerase and the RNase H activities of HIV-1 RT [143
] and therefore can be classified as dual NNRTI (DNNRTI). In addition, BBNH inhibits both RT-associated RNase H and RDDP activities of K103N, Y181I, Y188H, and Y188L mutant RTs with potency similar to wt RT, while, when assayed on Y181C mutant RT, it inhibits only the RDDP function and is inactive on the RNase H function [144
]. This information, together with the data on other hydrazone derivatives that chelate the metal ion cofactor in the RNase H site [145
], led to propose that two BBNH molecules could bind RT in two different sites, the first one in the polymerase domain, possibly near the NNRTI-binding site, and the second one possibly located in the RNase H domain. Subsequently, another derivative, (E)-3,4-dihydroxy-N′-((2-hydroxynaphthalen-1-yl)methylene)benzohydrazide (DHBNH) (), has been reported to bind near the polymerase active site in a pocket different from the NNRTI-binding site and also >50
Å away from the RNase H active site () [146
]. Hence, it was hypothesized that DHBNH may either perturb the trajectory of the template primer, so that RNase H cannot operate on its substrate, or that it may also bind to a second site, in or near the RNase H domain, that was not seen in the crystal. More recently, molecular docking studies on a series of hydrazone analogs proposed that they bind to a pocket that includes residues Y405, W406, Q500, and Y501 of p66 subunit, and, hence, they form hydrophobic interactions with RT and with base pairs in the groove of the RNA:DNA substrate [147
]. In fact, residues D499 and A502, adjacent to Q500, which were perturbed by the hydrazone derivatives presence [147
], are part of the primer grip of the RNase H domain and play a role in aligning the DNA:RNA substrate with the active site. Therefore, the hydrazones binding to Q500 may disrupt the primer grip's role in the activity of RNase H.
Chemical structures of dual RNase H and polymerase inhibitors.
A second class of DNNRTI is a series of emodin [148
] and alizarine anthraquinone derivatives [149
] such as 1-acetoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl 4-bromobenzoate (KNA-53) (), that inhibits both RT-associated functions of wt and K103N RTs and only the RNase H function of Y181C RT. Mode of action studies and molecular dynamic simulation led to proposing that the anthraquinone derivatives bind to the site adjacent to the NNRTI pocket, which was originally reported [146
] for the hydrazones derivatives () [149
]. Accordingly, it has been suggested that the anthraquinone inhibition of the RNase H function may be due to a change in the RNA:DNA hybrid RT accommodation, induced by their binding, which results in a possible variation in the nucleic acid trajectory toward the RNase H catalytic site [149
A third class of DNNRTI is the naphthalenesulfonic acid derivatives that were originally reported to have a selective activity on the RT-associated RDDP function [151
] and were further developed by structure-based design, molecular similarity, and combinatorial medicinal chemistry to obtain compound 2-Naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4 cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]-carbonyl]amino]-3-[(4-cinnamylphenyl)]azo (KM-1) (), that inhibits both RT functions in the nanomolar range [152
]. Subsequently, KM-1 was shown to weaken the RT DNA-binding affinity and to displace DNA from the enzyme [153
]. Hence, it has been proposed to preclude the proper alignment of DNA at the polymerase active site, depleting the active DNA-bound RT complex required for nucleotide incorporation [153
It is important to note that questions have been raised regarding the use of combinations between RHRTIs and NRTIs. In fact, RHRTIs have been proposed to lead to an increase in NRTIs resistance by mimicking the RNase H-dependent mechanism of NRTI resistance of some connection domain mutations [43
]. Recently, however, studies on the effects of some RHRTIs on the HIV-1 susceptibility to AZT and 3TC have shown that none of the tested RHRTIs decreased NRTI susceptibility, while only one DNNRTI decreased AZT susceptibility by 5-fold [154
]. More studies are needed to fully understand the interplay between RNase H inhibition and NRTIs susceptibility as well as its clinical relevance.