Binding characteristics of small molecules that mimic nucleocapsid protein-induced maturation of stem-loop 1 of HIV-1 RNA.

As a retrovirus, the human immunodeficiency virus (HIV-1) packages two copies of the RNA genome as a dimer in the infectious virion. Dimerization is initiated at the dimer initiation site (DIS) which encompasses stem-loop 1 (SL1) in the 5'-UTR of the genome. Study of genomic dimerization has been facilitated by the discovery that short RNA fragments containing SL1 can dimerize spontaneously without any protein factors. On the basis of the palindromic nature of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via standard Watson-Crick base pairs and then converted into a more stable extended dimer by the viral nucleocapsid protein (NCp7). This dimer maturation in vitro is believed to mimic initial steps in the RNA maturation in vivo, which is correlated with viral infectivity. We previously discovered a small molecule activator, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates dimer maturation in vitro, and determined aspects of its structure-activity relationship. In this report, we present measurements of the binding affinity of the activators and characterization of their interactions with the SL1 RNA. Guanidinium groups and increasing positive charge on the side chain enhance affinity and activity, but features in the aromatic ring at least partially decouple affinity from activity. Although KA-AMC can bind to multiple structural motifs, the NMR study showed KA-AMC preferentially binds to unique structural motifs, such as the palindromic loop and the G-rich internal loop in the SL1 RNA. NCp7 binds to SL1 only 1 order of magnitude more tightly than the best small molecule ligand tested. This study provides guidelines for the design of superior small molecules that bind to the SL1 RNA that have the potential of being developed as an antiviral by interfering with SL1-NCp7 interaction at the packaging and/or maturation stages.

Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole.

BACKGROUND: Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14alpha-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding. METHODOLOGY/PRINCIPAL FINDINGS: We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 A and 2.27 A), and from the related pathogen T. brucei (resolutions of 2.7 A and 2.6 A), co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180 degrees depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target. CONCLUSIONS/SIGNIFICANCE: The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for rationally designed anti-Chagasic drugs with improved efficacy and reduced toxicity.

Discovery of ligands for a novel target, the human telomerase RNA, based on flexible-target virtual screening and NMR.

The human ribonucleoprotein telomerase is a validated anticancer drug target, and hTR-P2b is a part of the human telomerase RNA (hTR) essential for its activity. Interesting ligands that bind hTR-P2b were identified by iteratively using a tandem structure-based approach: docking of potential ligands from small databases to hTR-P2b via the program MORDOR, which permits flexibility in both ligand and target, with subsequent NMR screening of high-ranking compounds. A high percentage of the compounds tested experimentally were found via NMR to bind to the U-rich region of hTR-P2b; most have MW < 500 Da and are from different compound classes, and several possess a charge of 0 or +1. Of the 48 ligands identified, 24 exhibit a decided preference to bind hTR-P2b RNA rather than A-site rRNA and 10 do not bind A-site rRNA at all. Binding affinity was measured by monitoring RNA imino proton resonances for some of the compounds that showed hTR binding preference.

A small molecule, Lys-Ala-7-amido-4-methylcoumarin, facilitates RNA dimer maturation of a stem-loop 1 transcript in vitro: structure-activity relationship of the activator.

The type 1 human immunodeficiency virus (HIV-1), like all retroviruses, contains two copies of the RNA genome as a dimer. A dimer initially forms via a self-complementary sequence in the dimer initiation site (DIS) of the genomic RNA, but that dimer is converted to a mature dimer in a process generally promoted by the viral nucleocapsid (NC) protein. Formation of the mature dimer is correlated with infectivity. Study of genomic dimerization has been facilitated by discovery of short RNA transcripts containing the DIS stem-loop 1 (SL1), which can dimerize spontaneously without any protein factors in vitro as well as via the NC protein. On the basis of the palindromic nature of the apical loop of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via a loop-loop interaction and then converted into a more stable extended dimer by the NC protein. This dimerization process in vitro is believed to mimic the in vivo RNA maturation. During experimental screening of potential inhibitors, we discovered a small molecule, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates the in vitro conversion from kissing dimer to extended dimer. Here we report the structure-activity relationship for KA-AMC for promoting dimer maturation. Guanidino groups and increasing positive charge on the side chain enhance activity. For activity, the charged side chain is preferred on the benzene ring, and O 1 in the coumarin scaffold is essential. NMR studies show that the coumarin derivatives stack with aromatic bases of the RNA. The coumarin derivatives may aid in the investigation of some aspects of dimer maturation and serve as a scaffold for design of maturation inhibitors or of activators of premature maturation, either of which can lead to a potential HIV therapeutic.

Docking to RNA via root-mean-square-deviation-driven energy minimization with flexible ligands and flexible targets.

Structure-based drug design is now well-established for proteins as a key first step in the lengthy process of developing new drugs. In many ways, RNA may be a better target to treat disease than a protein because it is upstream in the translation pathway, so inhibiting a single mRNA molecule could prevent the production of thousands of protein gene products. Virtual screening is often the starting point for structure-based drug design. However, computational docking of a small molecule to RNA seems to be more challenging than that to protein due to the higher intrinsic flexibility and highly charged structure of RNA. Previous attempts at docking to RNA showed the need for a new approach. We present here a novel algorithm using molecular simulation techniques to account for both nucleic acid and ligand flexibility. In this approach, with both the ligand and the receptor permitted some flexibility, they can bind one another via an induced fit, as the flexible ligand probes the surface of the receptor. A possible ligand can explore a low-energy path at the surface of the receptor by carrying out energy minimization with root-mean-square-distance constraints. Our procedure was tested on 57 RNA complexes (33 crystal and 24 NMR structures); this is the largest data set to date to reproduce experimental RNA binding poses. With our procedure, the lowest-energy conformations reproduced the experimental binding poses within an atomic root-mean-square deviation of 2.5 A for 74% of tested complexes.

New insights into the allosteric mechanism of human hemoglobin from molecular dynamics simulations.

It is still difficult to obtain a precise structural description of the transition between the deoxy T-state and oxy R-state conformations of human hemoglobin, despite a large number of experimental studies. We used molecular dynamics with the Path Exploration with Distance Constraints (PEDC) method to provide new insights into the allosteric mechanism at the atomic level, by simulating the T-to-R transition. The T-state molecule in the absence of ligands was seen to have a natural propensity for dimer rotation, which nevertheless would be hampered by steric hindrance in the "joint" region. The binding of a ligand to the alpha subunit would prevent such hindrance due to the coupling between this region and the alpha proximal histidine, and thus facilitate completion of the dimer rotation. Near the end of this quaternary transition, the "switch" region adopts the R conformation, resulting in a shift of the beta proximal histidine. This leads to a sliding of the beta-heme, the effect of which is to open the beta-heme's distal side, increasing the accessibility of the Fe atom and thereby the affinity of the protein. Our simulations are globally consistent with the Perutz strereochemical mechanism.