The overall goal of this project is to develop promising anti-AIDS drug leads using a new drug design paradigm. Using essential RNA elements as targets for developing small therapeutic drugs is a little-used strategy, despite the precedent that many antibiotics work by binding specific sites on rRNA. Our lab and others have determined 3D structures of some crucial HIV-1 RNA genome elements that can serve as targets for computational screening of databases of available compounds for binding. For RNA targeting, we continue to develop two docking programs, DOCK and MORDOR, the latter quite novel in that it permits flexibility in both putative ligand and target for a true induced fit;such flexible fitting is useful for virtual screening of protein targets but is very important for RNA targets, as they are typically quite malleable. Candidate compounds are water-soluble, nonpeptide, nonnucleotide organic compounds generally with molecular weight <500 daltons and a charge of no more than +2 (generally 0 or +1). Computational """"""""hits"""""""" will be tested experimentally using NMR and surface plasmon resonance for binding to the RNA target but not off-target RNA controls. Those that bind will serve as models for similarity searches to identify other potential binders. NMR will be used to map the binding site on the RNA and to measure Kd values for the best hits using two control RNAs to determine selectivity. NMR will be used to model the structure of the most promising binders;such structures will define scaffolds for subsequent organic synthesis (not explicitly proposed here) to enhance affinity and selectivity. In this short research project, we propose a continuation of needed methodology development. Application to a novel target will illustrate the utility of the methodology with the prospect that promising hits will result. The primary target is the G-rich internal loop (GRIL) of stem-loop 1 (SL1) in the packaging sequence in the 5'-UTR of the HIV-1 RNA genome, which we discovered to be essentially 100% conserved (suggesting the possibility to avert drug resistance). More importantly, human rRNA and tRNAs do not have any structural motifs similar to GRIL. GRIL is critical for packaging of the genome into new infectious virions but, considering the lack of sequence conservation in other parts of the 5'UTR, it may play another unknown crucial role as well. We have determined the structure of SL1, which will be used for targeting. As we have identified another ligand that binds to the packaging site, studies focused on our knowledge of that ligand will attempt to identify inhibitors of HIV-1 replication.
Screening a large database of drug-like compounds by computer for binding to a 3D RNA or protein structure important for HIV-1 activity followed by experimental verification of the top predicted compounds should be a fast, cost effective way to find compounds that might become drug leads. We have developed software for screening that is a significant improvement over existing software, especially for targeting crucial sites on RNA, which has largely been ignored compared with the typical protein targets. We propose to improve the software and to use it to find promising compounds that bind to an important conserved portion of the RNA genome of HIV-1, where the sequence conservation suggests evolution of drug resistance will be minimized, and ultimately to inhibit HIV-1 replication.
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|Guilbert, Christophe; James, Thomas L (2008) Docking to RNA via root-mean-square-deviation-driven energy minimization with flexible ligands and flexible targets. J Chem Inf Model 48:1257-68|
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|Chung, Janet; Mujeeb, Anwer; Jiang, Yongying et al. (2008) 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. Biochemistry 47:8148-56|
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