RNA as a target for small molecules

Proteins are folded to form a small binding site for catalysis or ligand recognition and this small binding site is traditionally the target for drug discovery. An alternative target for potential drug candidates is the translational process, which requires a precise reading of the entire mRNA sequence and, therefore, can be interrupted with small molecules that bind to mRNA sequence-specifically. RNA thus presents itself as a new upstream target for drug discovery because of the critical role it plays in the life of pathogens and in the progression of diseases. In this post-genomic era, RNA is becoming increasingly amenable to small-molecule therapy as greater structural and functional information accumulates with regard to important RNA functional domains. The study of aminoglycoside antibiotics and their binding to 16S ribosomal RNA has been a paradigm for our understanding of the ways in which small molecules can be developed to affect the function of RNA.     

Proteomic methods for drug target discovery

The field of drug target discovery is currently very popular with a great potential for advancing biomedical research and chemical genomics. Innovative strategies have been developed to aid the process of target identification, either by elucidating the primary mechanism-of-action of a drug, by understanding side effects involving unanticipated 'off-target' interactions, or by finding new potential therapeutic value for an established drug. Several promising proteomic methods have been introduced for directly isolating and identifying the protein targets of interest that are bound by active small molecules or for visualizing enzyme activities affected by drug treatment. Significant progress has been made in this rapidly advancing field, speeding the clinical validation of drug candidates and the discovery of the novel targets for lead compounds developed using cell-based phenotypic screens. Using these proteomic methods, further insight into drug activity and toxicity can be ascertained.     

Fast screening target sites for RNA interference using a cell-free system

A fast and simple procedure to screen target sites for RNA interference by using RNA in a cell-free system of Hela cells, and then evaluating the efficiency by Northern blotting, is described. This procedure produces results with an identical reliability compared to those described previously but which are more time-consuming than this present method.     

The characterization of Lucilia cuprina acetylcholinesterase as a drug target, and the identification of novel inhibitors by high throughput screening

Acetylcholinesterase (AChE, EC3.1.1.7.) is the molecular target for the carbamate and organophosphate pesticides that are used to combat parasitic arthropods. In this paper we report the functional heterologous expression of AChE from Lucilia cuprina (the sheep blowfly) in HEK293 cells. We show that the expressed enzyme is cell-surface-exposed and possesses a glycosyl-phosphatidylinositol membrane anchor. The substrates acetyl-, propionyl- and butyrylthiocholine (AcTC, PropTC, ButTC), and also 11 further thiocholine and homo-thiocholine derivatives were chemically synthesized to evaluate and compare their substrate properties in L. cuprina AChE and recombinant human AChE. The Michaelis-Menten constants K_M for AcTC, PropTC and ButTC were found to be 3-7-fold lower for the L. cuprina AChE than for the human AChE. Additionally, 2-methoxyacetyl-thiocholine and isobutyryl-thiocholine were better substrates for the insect enzyme than for the human AChE. The AcTC, PropTC and ButTC specificities and the Michaelis-Menten constants for recombinant L. cuprina AChE were similar to those determined for AChE extracted from L. cuprina heads, which are a particularly rich source of this enzyme. The median inhibition concentrations (IC₅₀ values) were determined for 21 organophosphates, 23 carbamates and also 9 known non-covalent AChE inhibitors. Interestingly, 11 compounds were 100- to >4000-fold more active on the insect enzyme than on the human enzyme. The substrate and inhibitor selectivity data collectively indicate that there are structural differences between L. cuprina and human AChE in or near the active sites, suggesting that it may be possible to identify novel, specific L. cuprina AChE inhibitors. To this end, a high throughput screen with 107,893 compounds was performed on the L. cuprina head AChE. This led to the identification of 195 non-carbamate, non-organophosphate inhibitors with IC₅₀ values below 10 ??M. Analysis of the most potent hit compounds identified 19 previously unknown inhibitors with IC₅₀ values below 200 nM, which were up to 335-fold more potent on the L. cuprina enzyme than on the human AChE. Some of these compounds may serve as leads for lead optimization programs to generate fly-specific pesticides.     

Virtual screening and evaluation of Ketol-Acid Reducto-Isomerase (KARI) as a putative drug target for Aspergillosis

Aspergillus is a leading causative agent for fungal morbidity and mortality in immuno-compromised patients. To identify a putative target to design or identify new antifungal drug, against Aspergillus is required. In our previous work, we have analyzed the various biochemical pathways, and we found Ketol Acid Reducto-Isomerase (KARI) an enzyme involves in the amino acid biosynthesis, could be a better target. This enzyme was found to be unique by comparing to host proteome through BLASTp analysis. A homology based model of KARI was generated by Swiss model server. The generated model had been validated by PROCHECK and WHAT IF programs. The Zinc library was generated within the limitation of the Lipinski rule of five, for docking study. Based on the dock-score six molecules have been studied for ADME/TOX analysis and subjected for pharmacophore model generation. The Zinc ID of the potential inhibitors is ZINC00720614, ZINC01068126, ZINC0923, ZINC02090678, ZINC00663057 and ZINC02284065 and found to be pharmacologically active agonist and antagonist of KARI. This study is an attempt to Insilco evaluation of the KARI as a drug target and the screened inhibitors could help in the development of the better drug against Aspergillus.     

Glutamate racemase as a target for drug discovery

The bacterial cell wall is a highly cross‐linked polymeric structure consisting of repeating peptidoglycan units, each of which contains a novel pentapeptide substitution which is cross‐linked through transpeptidation. The incorporation of d ‐glutamate as the second residue is strictly conserved across the bacterial kingdom. Glutamate racemase, a member of the cofactor‐independent, two‐thiol‐based family of amino acid racemases, has been implicated in the production and maintenance of sufficient d ‐glutamate pool levels required for growth. The subject of over four decades of research, it is now evident that the enzyme is conserved and essential for growth across the bacterial kingdom and has a conserved overall topology and active site architecture; however, several different mechanisms of regulation have been observed. These traits have recently been targeted in the discovery of both narrow and broad spectrum inhibitors. This review outlines the biological history of this enzyme, the recent biochemical and structural characterization of isozymes from a wide range of species and developments in the identification of inhibitors that target the enzyme as possible therapeutic agents.     

Human acidic mammalian chitinase as a novel target for anti-asthma drug design using in silico screening

Human acidic mammalian chitinase (hAMCase) was recently shown to be involved in the development of asthma, suggesting a possible application for hAMCase inhibitors as novel therapeutic agents for asthma. We therefore initiated drug discovery research into hAMCase using a combination of in silico methodologies and a hAMCase assay system. We first selected 23 candidate hAMCase inhibitors from a database of four million compounds using a multistep screening system combining Tripos Topomer Search technology, a docking calculation and two-dimensional molecular similarity analysis. We then measured hAMCase inhibitory activity of the selected compounds and identified seven compounds with IC₅₀ values ?100 μM. A model describing the binding modes of these hit compounds to hAMCase was constructed, and we discuss the structure–activity relationships of the compounds we identified, suggested by the model and the actual inhibitory activities of the compounds.     

Bacterial RNase P RNA is a drug target for aminoglycoside-arginine conjugates

The ribonuclease P (RNase P) holoenzymes are RNPs composed of RNase P RNA (PRNA) and a variable number of P protein subunits. Primary differences in structure and function between bacterial and eukaryotic RNase P and its indispensability for cell viability make the bacterial enzyme an attractive drug target. On the basis of our previous studies, aminoglycoside-arginine conjugates (AACs) bind to HIV-1 TAR and Rev responsive element (RRE) RNAs significantly more efficiently than neomycin B. Their specific inhibition of bacterial rRNA as well as the findings that the hexa-arginine neomycin derivative (NeoR6) is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, led us to explore the structure-function relationships of AACs in comparison to a new set of aminoglycoside-polyarginine conjugates (APACs). We here present predicted binding modes of AACs and APACs to PRNA. We used a multistep docking approach comprising rigid docking full scans and final refinement of the obtained complexes. Our docking results suggest three possible mechanisms of RNase P inhibition by AACs and APACs: competition with the P protein and pre-tRNA on binding to P1-P4 multihelix junction and to J19/4 region (probably including displacement of Mg2+ ions from the P4 helix) of PRNA; competition with Mg2+ ions near the P15 loop; and competition with the P protein and/or pre-tRNA near the P15 helix and interfering with interactions between the P protein and pre-tRNA at this region. The APACs revealed about 10-fold lower intermolecular energy than AACs, indicating stronger interactions of APACs than AACs with PRNA.     

RNA as a new target for toxic and protective agents

In contrast to damage of genomic DNA and despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome, such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human chronic diseases. We investigated whether RNA damage could be related to the exposure of particular xenobiotics by testing the RNA-damaging activity of a series of chemicals with different mechanisms of action. Cultured human T-lymphoblastoid cells were treated with ethyl methanesulfonate (EMS), H(2)O(2), doxorubicin, spermine, or S-nitroso-N-acetylpenicillamine (SNAP). Furthermore, we studied the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate can be mediated by alterations of the rates of toxic agent absorption and uptake, by trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA. We used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of pomegranate. We demonstrated that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H(2)O(2), spermine and SNAP. However, EMS, a well-known DNA-damaging agent, was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties, were able to damage RNA. Pomegranate reduced the RNA-damaging effect of doxorubicin, H(2)O(2), and spermine. Its inhibitory activity could be related with its ability to forms complexes with doxorubicin and H(2)O(2), or interacts with the intracellular formation of reactive species mediating their toxicity. For spermine, an alteration of the rates of spermine absorption and uptake can also be involved.     

The ribosome as a drug target

The elucidation of the crystal structure of the ribosome and its subunits has dramatically increased our understanding of this organelle and the molecular interactions that determine its functional capabilities. Two recent publications, one on the structure of the bacterial ribosome at 3.5A resolution and one on the identification of functionally relevant sites within the small subunit rRNA, illustrate the importance of interdisciplinary approaches in exploiting the ribosome as a drug target.     

Methionine recycling as a target for antiprotozoal drug development

The development of new and effective ontiprotozool drugs has been difficult because of the close metabolic relationship between protozoa and mammalian cells. In this article, Michael Riscoe, Al Ferro and john Fitchen present their hypothesis for chemotherapeutic exploitation of methylthioribose (MTR) kinase, an enzyme critical to methionine salvage in certain protozoa. They propose that analogues of MTR if properly designed, would be converted to toxic products in organisms that contain MTR kinase but not in mammalian cells, which lack this enzyme.     

The flavivirus protease as a target for drug discovery

Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures.     

RNA as a small molecule druggable target

Small molecule drugs have readily been developed against many proteins in the human proteome, but RNA has remained an elusive target for drug discovery. Increasingly, we see that RNA, and to a lesser extent DNA elements, show a persistent tertiary structure responsible for many diverse and complex cellular functions. In this digest, we have summarized recent advances in screening approaches for RNA targets and outlined the discovery of novel, drug-like small molecules against RNA targets from various classes and therapeutic areas. The link of structure, function, and small-molecule Druggability validates now for the first time that RNA can be the targets of therapeutic agents.     

Optogenetic methods in drug screening: technologies and applications

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Exploiting RNA as a new biomolecular target for synthetic polyamines

Anticancer chemotherapy is strongly hampered by the low therapeutic index of most anticancer drugs and the development of chemoresistance. Therefore, there is a continued need for the identification of new molecular targets in order to selectively hit cancer cells. RNA has been recently validated as a cancer target by the use of different specific ligands and/or by different agents able to destroy its diverse forms. The ability of synthetic polyamines to interact and to alter the RNA structure has been already reported. In the present paper the interaction and the ability to damage RNA structure by several synthetic polyamines were evaluated and quantified by microfluid capillary electrophoresis. This technique allowed us to visualize both the RNA impairment through different electropherograms and to assess the RNA integrity number. Finally, the ability to discriminate between RNA and DNA by these synthetic polyamines was also evaluated.     

In-cell NMR: From target structure and dynamics to drug screening

The cellular environment can affect the structure and function of pharmacological targets and the interaction with potential drugs. Such complexity is often overlooked in the first steps of drug design, where compounds are screened and optimized in vitro, leading to high failure rates in the pre-clinical and clinical tests. In-cell NMR spectroscopy has the potential to fill this gap, as it allows structural studies of proteins and nucleic acids directly in living cells, from bacteria to human-derived, providing a unique way to investigate the structure and dynamics of ligand–target interactions in the native cellular context. When applied to drug screening, in-cell NMR provides insights on binding kinetics and affinity toward a cellular target, offering a powerful tool for improving drug potency at an early stage of drug development.