Docking studies indicated that the nitrofuran group of 14 chelates the two Mg2+ions and orients toward His539, possibly to form a hydrogen bond, while its carbonyl oxygen atom in the amide group may interact with the side chain of Ser553 and its adamantan group can make hydrophobic contacts with the Lys550 side chain. transcriptase (RT), in addition to nonspecifically hydrolyzing the RNA strand of the RNA/DNA replication intermediate, catalyzes highly specific hydrolytic events that are critical to synthesis of integration-competent double-stranded proviral DNA from the RNA genome of the infecting particle [1]. Prominent among these is precise removal of the RNA primers that initiate (-) and (+) strand DNA synthesis (a host-coded tRNA and the polypurine tract, respectively), since these events ultimately define 5 and 3 long terminal repeat sequences essential for efficient integration of viral DNA. With respect to (+) strand synthesis, generating the polypurine tract 3 terminus also mandates a mechanism whereby this sequence is accurately recognized TAK-632 when embedded within the replication intermediate. The observation over two decades ago that mutating active site residues of the RNaseH domain of HIV-1 RT eliminates activity [2] and results in loss of virus infectivity [3] demonstrates the necessity for this function and that the retrovirus-associated activity cannot be complemented by a host enzyme. Together, these observations define the C-terminal RT-associated RNaseH domain as an additional and important target in the development of future combination antiretroviral regimens. For the nucleoside- and non-nucleoside-derived DNA polymerase inhibitors (NRTIs and NNRTIs, respectively), there is a wealth of data to guide structure-based drug design since the complex of HIV-1 RT containing the NNRTI nevirapine was TAK-632 solved in 1992 by Kohlstaedt and Steitz [4]. In contrast, high-resolution structures of HIV-1 RT containing an inhibitor bound to the RNaseH active site have only recently become available following KRT17 the initial report in 2009 2009 by Himmel [5]. Although the ease with which the current generation of RNaseH active site inhibitors can be displaced from their binding site in the presence of the nucleic acid substrate represents a major obstacle, the recently-reported structure of HIV-1 RT containing an RNA/DNA hybrid and an NNRTI (Figure 1A) [6] provides a plausible model where the hybrid has ready access to the RNaseH active site (Figure 1B). This model indicates that TAK-632 significant structural alterations within and between the p66 and p51 subunits of the parental p66/p51 heterodimer (Figure 1) are a prerequisite to correctly accommodating the duplex, thus, it may be possible to identify non-active site inhibitors that occupy a site within, or close to, the RNaseH domain and restrict conformational flexibility. Indeed, although a high-resolution co-crystal structure is unavailable, recent data suggest that vinylogous ureas and thienopyrimidinones might fulfill this requirement. The goal of this article is to extend previous reviews by providing an TAK-632 updated account of progress towards developing HIV-1 RNaseH inhibitors that interact outside the RNaseH active site. The reader is also encouraged to read recent reviews by Tramontano and Di Santo [7], and Ilina [8]. Open in a separate window Figure 1. p66/p51 HIV-1 reverse transcriptase containing an RNA/DNA hybrid.(A) Fingers, palm, thumb and connection subdomains are color coded blue, red, green and yellow, respectively, with the darker and lighter colors representing the p66 and p51 subunits, respectively. The p66 C-terminal RNaseH domain is depicted in gold. RNA and DNA strands of the cross are depicted as magenta and sand-colored spheres, respectively. (B) Close-up of the p66 RNaseH website containing portions of the RNA/DNA cross explained by Lapkouski [6]. Structural elements have been defined, and catalytic residues (cyan) are: D1: Asp498; D2: Asp549; D3: Asp443; E: Glu478. RNA and DNA strands of the RNA/DNA cross are depicted in reddish and blue, respectively. RNaseH: Ribonuclease H. Metal-chelating active site inhibitors RNase HI, and inhibited RNA-dependent DNA polymerase activity of HIV-1 RT only at significantly higher concentrations. Initial structureCactivity relationship (SAR) data suggested that substitutions within the phenyl moiety improved both potency and selectivity [10] while crystallographic studies indicate the flexible His-loop near the RNaseH C-terminus was stabilized in the presence of 1 [11]. Open in a separate window Number 2. RNaseH scaffold and the HIV-1 RNaseH website (p15-EC) with an IC50 value of 4.7 M; lack inactivity within the RNaseH. Isothermal titration calorimetry showed a BTDBA dissociation constant of 8.9 M, assisting the hypothesis.