Patent Application
20110237549
1. Field of Inventive Subject Matter
The inventive subject matter relates to a computer model generated from a data array, computer readable storage media encoded with the model, and computers comprising the model, wherein the model is derived from atomic structure coordinates of an FMN riboswitch or a lysine riboswitch. The inventive subject matter further relates to a pharmacophore having a spatial arrangement of atoms defined by the binding pocket identified in the model, along with a compound defined by the pharmacophore. Finally, the inventive subject matter additionally relates to methods for rational drug design based on the atomic structure data, and to compounds identified by the rational drug design process.
2. Background
Riboswitches regulate gene expression at the mRNA level, typically occurring when untranslated segments undergo structural rearrangement upon binding to a ligand such as, in a common example, a cognate ligand which is the expression product of the gene which is transcribed to produce the mRNA. Alternate riboswitch ligands include other naturally occurring or artificially-created ligands. However, identifying such alternate ligands has traditionally been a random, time-consuming, and expensive process, generally involving trial-and-error experimentation and a degree of luck.
As bacterial resistance becomes ever more prevalent, and as evolutionary pressures diminish or destroy the effectiveness of many common antibiotic compositions, there is an increasingly great need to develop new compounds and compositions which can target bacteria in new ways that will provide antibiotic drugs in the future. Riboswitch modulation is one target for drug development, and improving the process by which alternate ligands are identified will significantly increase the speed and effectiveness of drug development, while likely reducing costs as well.
There are a variety of known riboswitch classes:
Applicants herein have determined for the first time the structure of the FMN and lysine riboswitches, as well as the structure of each riboswitch bound to ligands of such riboswitches. It is expected that such structural data will permit rational drug design programs to identify alternate natural and/or synthetic ligands which will modulate the activity of riboswitches, and permit the synthesis of antibiotics of the future to target riboswitches of pathogenic bacteria.
The inventive subject matter relates to a computer model generated from a data array, computer readable storage media encoded with the model, and computers comprising the model, wherein the model is derived from atomic structure coordinates of an FMN riboswitch or a lysine riboswitch. The inventive subject matter further relates to a pharmacophore having a spatial arrangement of atoms defined by the binding pocket identified in the model, along with a compound defined by the pharmacophore. Finally, the inventive subject matter additionally relates to methods for rational drug design based on the atomic structure data, and to compounds identified by the rational drug design process.
In particular, the inventive subject matter relates to a computer model of an FMN riboswitch generated from a data array comprising the atomic structure coordinates of an FMN riboswitch as set forth in any one of Tables 6-14, or a composite thereof.
The inventive subject matter further relates to a pharmacophore having a spatial arrangement of atoms defined by the binding pocket identified in the computer model described above.
The inventive subject matter also relates to an isolated compound, or a salt or solvate thereof, defined by the pharmacophore described above.
The inventive subject matter additionally relates to a method for identifying a compound that interacts with an FMN riboswitch, utilizing a 3-D molecular model of an FMN riboswitch as shown in any one of Tables 6-14, or a composite thereof, comprising:
(a) using said model in a method of rational drug design to identify candidate compounds that can bind an FMN riboswitch; and
(b) assaying the binding of a candidate compound identified in step (a) using a purified FMN riboswitch to thereby determine a binding characteristic, or lack thereof, of said compound.
Further, the inventive subject matter relates to a method of killing bacteria, comprising contacting the bacteria with a compound identified by the method described above.
In yet another aspect of the inventive subject matter, a method for identifying a compound that interacts with an FMN riboswitch, utilizing the crystal structure of an FMN riboswitch, comprises the steps of:
(a) modeling the FMN riboswitch with a test compound; and
(b) determining if the test compound interacts with the FMN riboswitch.
In addition, the inventive subject matter relates to a computer model of a lysine riboswitch generated from a data array comprising the atomic structure coordinates of a lysine riboswitch as set forth in any one of Tables 15-26, or a composite thereof.
The inventive subject matter further relates to a pharmacophore having a spatial arrangement of atoms defined by the binding pocket identified in the computer model described above.
The inventive subject matter also relates to an isolated compound, or a salt or solvate thereof, defined by the pharmacophore described above.
The inventive subject matter additionally relates to a method for identifying a compound that interacts with a lysine riboswitch, utilizing a 3-D molecular model of a lysine riboswitch as shown in any one of Tables 15-26, or a composite thereof, comprising:
(a) using said model in a method of rational drug design to identify candidate compounds that can bind a lysine riboswitch; and
(b) assaying the binding of a candidate compound identified in step (a) using a purified lysine riboswitch to thereby determine a binding characteristic, or lack thereof, of said compound.
Further, the inventive subject matter relates to a method of killing bacteria, comprising contacting the bacteria with a compound identified by the method described above.
In yet another aspect of the inventive subject matter, a method for identifying a compound that interacts with a lysine riboswitch, utilizing the crystal structure of a lysine riboswitch, comprises the steps of:
(a) modeling the lysine riboswitch with a test compound; and
(b) determining if the test compound interacts with the lysine riboswitch.
θ is the fraction of RT pausing, No and Lo are RNA and ligand concentrations, respectively, and P50 is the apparent ligand concentration at which RT pausing is half-maximally attained. The P50 value for the lysine-induced RT pausing 5.50±0.53/−1M (mean±s.d.) (n=2) is similar to the Kd value 4.03±0.34/−1M (mean±s.d.) (n=2) determined by the equilibrium dialysis assay. The relative differences between the P50 values for lysine and its analogs are in overall agreement with the relative differences obtained from the Kd values for lysine and its analogs determined for the B. subtilis lysine riboswitch in ref. 5.
The term “riboswitch” refers to a part of an mRNA molecule that can directly bind a target molecule, and whose binding of the target affects the activity of the gene that produces the mRNA molecule. Thus, an mRNA molecule that contains a riboswitch is directly involved in regulating the activity of the DNA sequence from which it is transcribed.
The term “effecting” refers to the process of producing an effect on biological activity, function, health, or condition of an organism in which such biological activity, function, health, or condition is maintained, enhanced, diminished, or treated in a manner which is consistent with the general health and well-being of the organism.
The term “modulating” as used herein refers to the process of increasing or decreasing activity.
The term “enhancing” the biological activity, function, health, or condition of an organism refers to the process of augmenting, fortifying, strengthening, or improving.
The term “isomers” refer to different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. “Diastereoisomers” are stereoisomers which are not mirror images of each other. “Racemic mixture” means a mixture containing equal parts of individual enantiomers. “Non-racemic mixture” is a mixture containing unequal parts of individual enantiomers or stereoisomers.
The term “pharmaceutically acceptable salt, ester, or solvate” refers to a salt, ester, or solvate of a subject compound which possesses the desired pharmacological activity and which is neither biologically nor otherwise undesirable. A salt, ester, or solvate can be formed with inorganic acids such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, gluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, naphthylate, 2-naphthalenesulfonate, nicotinate, oxalate, sulfate, thiocyanate, tosylate and undecanoate. Examples of base salts, esters, or solvates include ammonium salts; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; salts with organic bases, such as dicyclohexylamine salts; N-methyl-D-glucamine; and salts with amino acids, such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups can be quarternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides, such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides, such as benzyl and phenethyl bromides; and others. Water or oil-soluble or dispersible products are thereby obtained.
Most clinical antibacterial compounds target one of a small number of cellular processes. Because bacteria have well-developed resistance mechanisms to protect these essential processes, it is very useful to discover and validate new targets. Riboswitches can be an effective target for controlling gene expression in natural organisms.
One potentially vulnerable bacterial process is the regulation of gene expression by riboswitches. Thus, an mRNA molecule that contains a riboswitch is directly involved in regulating its own activity, depending on the presence or absence of its target molecule. Typically found in the 5′ untranslated regions (5′ UTRs) of certain bacterial mRNAs, riboswitches form structured receptors (or aptamers) that bind fundamental metabolites. In most cases, ligand binding regulates the expression of genes involved in the synthesis and/or transport of the bound metabolite.
Because the biochemical pathways that are regulated by riboswitches may be essential for bacterial survival, modulation, in most cases down-regulation, of these pathways through riboswitch targeting is expected to be lethal.
Riboswitches are conceptually divided into two parts: an aptamer and an expression platform. The aptamer directly binds the small molecule, and the expression platform undergoes structural changes in response to the changes in the aptamer. The effect on the expression platform is what modulates or regulates gene expression.
Expression platforms typically turn off gene expression in response to the ligand, but some turn it on. Exemplary expression platforms may include:
The FMN riboswitch (also known as the RFN-element) binds flavin mononucleotide (FMN) to regulate riboflavin biosynthesis and transport. The FMN riboswitch is a highly conserved RNA element that is found frequently in the 5′-untranslated regions of prokaryotic mRNAs that encode for flavin mononucleotide (FMN) biosynthesis and transport proteins. This element is a metabolite-dependent riboswitch that directly binds FMN in the absence of proteins. In Bacillus subtilis, the riboswitch most likely controls gene expression by causing premature transcription termination within the 5′ untranslated region of the rib DEAHT operon and precluding access to the ribosome-binding site of ypaA mRNA.
The lysine riboswitch (also known as the L-box) binds lysine to regulate lysine biosynthesis, catabolism and transport. The Lysine riboswitch is a metabolite binding RNA element found within certain messenger RNAs that serve as a precision sensor for the amino acid lysine. Allosteric rearrangement of mRNA structure is mediated by ligand binding, and this results in modulation of gene expression. This riboswitches is found in a number of genes involved in lysine metabolism, including lysC. The lysine riboswitch has also been identified independently.
Riboswitches as antibiotic targets. Riboswitches are expected to be targets for novel antibiotics. Indeed, some antibiotics whose mechanism of action was unknown for decades have been shown to operate by targeting riboswitches. For example, when the antibiotic pyrithiamine enters the cell, it is metabolized into pyrithiamine pyrophosphate. Pyrithiamine pyrophosphate has been shown to bind and activate the TPP riboswitch, causing the cell to cease the synthesis and import of TPP. Because pyrithiamine pyrophosphate does not substitute for TPP as a coenzyme, the cell dies.
One potential advantage that riboswitches have as an antibiotic target is that many of the riboswitches have multiple instances per genome, where each instance controls an operon containing many genes, many of which are essential. Therefore, in order for bacteria to evolve resistance to the antibiotic by mutations in the riboswitch, all riboswitches must be mutated.
Disclosed are the crystalline atomic structures of riboswitches. These structures are useful in modeling and assessing the interaction of a riboswitch with a binding ligand. They are also useful in methods of identifying compounds that interact with the riboswitch.
Also disclosed are compositions and methods for selecting and identifying compounds that can activate, deactivate, or block a riboswitch. Activation of a riboswitch refers to the change in state of the riboswitch upon binding of a trigger molecule. A riboswitch can be activated by compounds other than the trigger molecule and in ways other than binding of a trigger molecule. The term trigger molecule is used herein to refer to molecules and compounds that can activate a riboswitch. This includes the natural or normal trigger molecule for the riboswitch and other compounds that can activate the riboswitch. Natural or normal trigger molecules are the trigger molecule for a given riboswitch in nature or, in the case of some non-natural riboswitches, the trigger molecule for which the riboswitch was designed or with which the riboswitch was selected (as in, for example, in vitro selection or in vitro evolution techniques). Non-natural trigger molecules can be referred to as non-natural trigger molecules.
Deactivation of a riboswitch refers to the change in state of the riboswitch when the trigger molecule is not bound. A riboswitch can be deactivated by binding of compounds other than the trigger molecule and in ways other than removal of the trigger molecule. Blocking of a riboswitch refers to a condition or state of the riboswitch where the presence of the trigger molecule does not activate the riboswitch. Also disclosed are methods of identifying a compound that interacts with a riboswitch comprising modeling the atomic structure of the riboswitch with a test compound and determining if the test compound interacts with the riboswitch. This can be done by determining the atomic contacts of the riboswitch and test compound. Furthermore, analogs of a compound known to interact with a riboswitch can be generated by analyzing the atomic contacts, then optimizing the atomic structure of the analog to maximize interaction. These methods can be used with a high throughput screen.
Also disclosed are methods for activating, deactivating, or blocking a riboswitch. Also disclosed are compositions for activating, deactivating or blocking a riboswitch. Riboswitches function to control gene expression through the binding or removal of a trigger molecule. Compounds can be used to activate, deactivate or block a riboswitch. The trigger molecule for a riboswitch (as well as other activating compounds) can be used to activate a riboswitch. Compounds other than the trigger molecule generally can be used to deactivate or block a riboswitch. Riboswitches can also be deactivated by, for example, removing trigger molecules from the presence of the riboswitch. A riboswitch can be blocked by, for example, binding of an analog of the trigger molecule that does not activate the riboswitch. The nature of the inaction between a riboswitch and a ligand other than a natural ligand can be as an agonist or an antagonist.
Also disclosed are methods for identifying compounds which are capable of altering expression of an RNA molecule, or of a gene encoding an RNA molecule, where the RNA molecule includes a riboswitch, by bringing a compound into contact with the RNA molecule. Riboswitches function to control gene expression through the binding or removal of a trigger molecule.
Thus, subjecting an RNA molecule of interest that includes a riboswitch to conditions that activate, deactivate or block the riboswitch can be used to alter expression of the RNA. Expression can be altered as a result of, for example, termination of transcription or blocking of ribosome binding to the RNA. Binding of a trigger molecule can, depending on the nature of the riboswitch, reduce or prevent expression of the RNA molecule or promote or increase expression of the RNA molecule.
Also disclosed are compositions and methods for regulating expression of an RNA molecule, or of a gene encoding an RNA molecule, by operably linking a riboswitch to the RNA molecule. A riboswitch can be operably linked to an RNA molecule in any suitable manner, including, for example, by physically joining the riboswitch to the RNA molecule or by engineering nucleic acid encoding the RNA molecule to include and encode the riboswitch such that the RNA produced from the engineered nucleic acid has the riboswitch operably linked to the RNA molecule. Subjecting a riboswitch operably linked to an RNA molecule of interest to conditions that activate, deactivate or block the riboswitch can be used to alter expression of the RNA.
Also disclosed are compositions and methods for regulating expression of a naturally occurring gene or RNA that contains a riboswitch by activating, deactivating or blocking the riboswitch. If the gene is essential for survival of a cell or organism that harbors it, activating, deactivating or blocking the riboswitch can result in death, stasis or debilitation of the cell or organism. For example, activating a naturally occurring riboswitch in a naturally occurring gene that is essential to survival of a microorganism can result in death of the microorganism (if activation of the riboswitch turns off or represses expression). This is one basis for the use of the disclosed compounds and methods for antimicrobial and antibiotic effects.
Also disclosed are compositions and methods for regulating expression of an isolated, engineered or recombinant gene or RNA that contains a riboswitch by activating, deactivating or blocking the riboswitch. The gene or RNA can be engineered or can be recombinant in any manner. For example, the riboswitch and coding region of the RNA can be heterologous, the riboswitch can be recombinant or chimeric, or both. If the gene encodes a desired expression product, activating or deactivating the riboswitch can be used to induce expression of the gene and thus result in production of the expression product. If the gene encodes an inducer or repressor of gene expression or of another cellular process, activation, deactivation or blocking of the riboswitch can result in induction, repression, or de-repression of other, regulated genes or cellular processes. Many such secondary regulatory effects are known and can be adapted for use with riboswitches. An advantage of riboswitches as the primary control for such regulation is that riboswitch trigger molecules can be small, non-antigenic molecules.
Also disclosed are compositions and methods for altering the regulation of a riboswitch by operably linking an aptamer domain to the expression platform domain of the riboswitch (which is a chimeric riboswitch). The aptamer domain can then mediate regulation of the riboswitch through the action of, for example, a trigger molecule for the aptamer domain. Aptamer domains can be operably linked to expression platform domains of riboswitches in any suitable manner, including, for example, by replacing the normal or natural aptamer domain of the riboswitch with the new aptamer domain. Generally, any compound or condition that can activate, deactivate or block the riboswitch from which the aptamer domain is derived can be used to activate, deactivate or block the chimeric riboswitch.
Also disclosed are compositions and methods for inactivating a riboswitch by covalently altering the riboswitch (by, for example, crosslinking parts of the riboswitch or coupling a compound to the riboswitch). Inactivation of a riboswitch in this manner can result from, for example, an alteration that prevents the trigger molecule for the riboswitch from binding, that prevents the change in state of the riboswitch upon binding of the trigger molecule, or that prevents the expression platform domain of the riboswitch from affecting expression upon binding of the trigger molecule.
Also disclosed are methods of identifying compounds that activate, deactivate or block a riboswitch. For examples, compounds that activate a riboswitch can be identified by bringing into contact a test compound and a riboswitch and assessing activation of the riboswitch. If the riboswitch is activated, the test compound is identified as a compound that activates the riboswitch. Activation of a riboswitch can be assessed in any suitable manner. For example, the riboswitch can be linked to a reporter RNA and expression, expression level, or change in expression level of the reporter RNA can be measured in the presence and absence of the test compound. As another example, the riboswitch can include a conformation dependent label, the signal from which changes depending on the activation state of the riboswitch. Such a riboswitch preferably uses an aptamer domain from or derived from a naturally occurring riboswitch. As can be seen, assessment of activation of a riboswitch can be performed with the use of a control assay or measurement or without the use of a control assay or measurement. Methods for identifying compounds that deactivate a riboswitch can be performed in analogous ways.
Identification of compounds that block a riboswitch can be accomplished in any suitable manner. For example, an assay can be performed for assessing activation or deactivation of a riboswitch in the presence of a compound known to activate or deactivate the riboswitch and in the presence of a test compound. If activation or deactivation is not observed as would be observed in the absence of the test compound, then the test compound is identified as a compound that blocks activation or deactivation of the riboswitch.
Also disclosed are compounds made by identifying a compound that activates, deactivates or blocks a riboswitch and manufacturing the identified compound. This can be accomplished by, for example, combining compound identification methods as disclosed elsewhere herein with methods for manufacturing the identified compounds. For example, compounds can be made by bringing into contact a test compound and a riboswitch, assessing activation of the riboswitch, and, if the riboswitch is activated by the test compound, manufacturing the test compound that activates the riboswitch as the compound.
Also disclosed are compounds made by checking activation, deactivation or blocking of a riboswitch by a compound and manufacturing the checked compound. This can be accomplished by, for example, combining compound activation, deactivation or blocking assessment methods as disclosed elsewhere herein with methods for manufacturing the checked compounds. For example, compounds can be made by bringing into contact a test compound and a riboswitch, assessing activation of the riboswitch, and, if the riboswitch is activated by the test compound, manufacturing the test compound that activates the riboswitch as the compound. Checking compounds for their ability to activate, deactivate or block a riboswitch refers to both identification of compounds previously unknown to activate, deactivate or block a riboswitch and to assessing the ability of a compound to activate, deactivate or block a riboswitch where the compound was already known to activate, deactivate or block the riboswitch.
Also contemplated are the development of isolated and recombinant riboswitches, recombinant constructs containing such riboswitches, heterologous sequences operably linked to such riboswitches, and cells and transgenic organisms harboring such riboswitches, riboswitch recombinant constructs, and riboswitches operably linked to heterologous sequences. The heterologous sequences can be, for example, sequences encoding proteins or peptides of interest, including reporter proteins or peptides. Preferred riboswitches are, or are derived from, naturally occurring riboswitches.
The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches, also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. Applicants have determined for the first time the crystal structures of the Fusobacterium nucleatum riboswitch, as bound to FMN, riboflavin, and antibiotic roseoflavin. Applicants' structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds.
The determination of the FMN riboswitch structure is especially interesting and timely, given the predicted complexity of its riboswitch fold, the abundance of this riboswitch in pathogenic species, and its involvement in riboflavin overproduction in biotechnologically relevant bacterial strains.
The inventive subject matter thus relates to a computer model generated from a data array, computer readable storage media encoded with the model, and computers comprising the model, wherein the model is derived from atomic structure coordinates of an FMN riboswitch or a lysine riboswitch. The inventive subject matter further relates to a pharmacophore having a spatial arrangement of atoms defined by the binding pocket identified in the model, along with a compound defined by the pharmacophore. Finally, the inventive subject matter additionally relates to methods for rational drug design based on the atomic structure data, and to compounds identified by the rational drug design process.
In particular, the inventive subject matter relates to a computer model of an FMN riboswitch generated from a data array comprising the atomic structure coordinates of an FMN riboswitch as set forth in any one of Tables 6-14, or a composite thereof. The data found in Tables 6-14 is X-ray diffraction data depicting the spatial coordinates of the atoms of an exemplary, consensus FMN riboswitch in crystal form and interacting with several different FMN riboswitch ligands in the presence of several possible metal cations are facilitate the interactions between the riboswitch and said ligands.
In one aspect of the inventive subject matter, said model is encoded onto a computer-readable storage medium.
In another aspect of the inventive subject matter, said model is stored in the memory of a computer. With the model and supporting data stored in memory, said computer becomes a special-purpose computer which is capable of performing molecular modeling functions, of which a general-purpose is incapable. In a preferred embodiment, the computer described immediately above additionally comprises executable code for:
(a) displaying the data array as a 3-dimensional model;
(b) analyzing the binding site of the model of an FMN riboswitch;
(c) screening in silico a library for small molecules that fit into said binding site; and (d) controlling a unit for assaying the small molecules determined in step (c) in a an FMN riboswitch binding assay.
In a further aspect of the inventive subject matter, the computer model described above is based upon a data array comprising atomic structure coordinates of an FMN riboswitch obtained in the presence of a sufficient amount of magnesium to saturate all sites for binding interactions between said FMN riboswitch and said magnesium. As discussed in the Examples below, the absence of magnesium or the substitution of another metal cation appears to substantially impair the binding interaction between an FMN riboswitch and its natural and artificial ligands.
The inventive subject matter further relates to a pharmacophore having a spatial arrangement of atoms defined by the binding pocket identified in the computer model described above.
In an alternate aspect, said spatial arrangement of atoms is determined in the presence of a sufficient amount of magnesium to saturate all sites for binding interactions between said FMN riboswitch and said magnesium.
The inventive subject matter also relates to an isolated compound, or a salt or solvate thereof, defined by the pharmacophore described above.
The inventive subject matter additionally relates to a method for identifying a compound that interacts with an FMN riboswitch, utilizing a 3-D molecular model of an FMN riboswitch as shown in any one of Tables 6-14, or a composite thereof, comprising:
(a) using said model in a method of rational drug design to identify candidate compounds that can bind an FMN riboswitch; and
(b) assaying the binding of a candidate compound identified in step (a) using a purified FMN riboswitch to thereby determine a binding characteristic, or lack thereof, of said compound.
In one aspect of the inventive subject matter, determining the binding characteristics of said compound in interaction with the riboswitch comprises determining a predicted minimum interaction energy, a predicted binding constant, a predicted dissociation constant, or a combination thereof, for the test compound in the model of the riboswitch.
In another aspect, determining if the test compound interacts with the riboswitch comprises determining one or more predicted bonds, one or more predicted other interactions, or a combination thereof, for the test compound in the model of the riboswitch.
As above, said 3-D molecular model of an FMN riboswitch is preferably determined from data obtained in the presence of a sufficient amount of magnesium to saturate all sites for binding interactions between said FMN riboswitch and said magnesium.
Further, the inventive subject matter relates to a method of killing bacteria, comprising contacting the bacteria with a compound identified by the method described above.
In yet another aspect of the inventive subject matter, a method for identifying a compound that interacts with an FMN riboswitch, utilizing the crystal structure of an FMN riboswitch, comprises the steps of:
(a) modeling the FMN riboswitch with a test compound; and
(b) determining if the test compound interacts with the FMN riboswitch.
In addition, the inventive subject matter relates to a computer model of a lysine riboswitch generated from a data array comprising the atomic structure coordinates of a lysine riboswitch as set forth in any one of Tables 15-26, or a composite thereof. The data found in Tables 15-26 is X-ray diffraction data depicting the spatial coordinates of the atoms of an exemplary, consensus lysine riboswitch in crystal form and interacting with several different lysine riboswitch ligands in the presence of several possible metal cations are facilitate the interactions between the riboswitch and said ligands.
In one aspect of the inventive subject matter, said model is encoded onto a computer-readable storage medium.
In another aspect of the inventive subject matter, said model is stored in the memory of a computer. With the model and supporting data stored in memory, said computer becomes a special-purpose computer which is capable of performing molecular modeling functions, of which a general-purpose is incapable. In a preferred embodiment, the computer described immediately above additionally comprises executable code for:
(a) displaying the data array as a 3-dimensional model;
(b) analyzing the binding site of the model of a lysine riboswitch;
(c) screening in silico a library for small molecules that fit into said binding site; and
(d) controlling a unit for assaying the small molecules determined in step (c) in a lysine riboswitch binding assay.
In a further aspect of the inventive subject matter, the computer model described above is based upon a data array comprising atomic structure coordinates of a lysine riboswitch obtained in the presence of a sufficient amount of magnesium to saturate all sites for binding interactions between said lysine riboswitch and said magnesium. As discussed in the Examples below, the absence of magnesium or the substitution of another metal cation appears to substantially impair the binding interaction between a lysine riboswitch and its natural and artificial ligands.
The inventive subject matter further relates to a pharmacophore having a spatial arrangement of atoms defined by the binding pocket identified in the computer model described above.
In an alternate aspect, said spatial arrangement of atoms is determined in the presence of a sufficient amount of magnesium to saturate all sites for binding interactions between said lysine riboswitch and said magnesium.
The inventive subject matter also relates to an isolated compound, or a salt or solvate thereof, defined by the pharmacophore described above.
The inventive subject matter additionally relates to a method for identifying a compound that interacts with a lysine riboswitch, utilizing a 3-D molecular model of a lysine riboswitch as shown in any one of Tables 15-26, or a composite thereof, comprising:
(a) using said model in a method of rational drug design to identify candidate compounds that can bind a lysine riboswitch; and
(b) assaying the binding of a candidate compound identified in step (a) using a purified lysine riboswitch to thereby determine a binding characteristic, or lack thereof, of said compound.
In one aspect of the inventive subject matter, determining the binding characteristics of said compound in interaction with the riboswitch comprises determining a predicted minimum interaction energy, a predicted binding constant, a predicted dissociation constant, or a combination thereof, for the test compound in the model of the riboswitch.
In another aspect, determining if the test compound interacts with the riboswitch comprises determining one or more predicted bonds, one or more predicted other interactions, or a combination thereof, for the test compound in the model of the riboswitch.
As above, said 3-D molecular model of a lysine riboswitch is preferably determined from data obtained in the presence of a sufficient amount of magnesium to saturate all sites for binding interactions between said lysine riboswitch and said magnesium.
Further, the inventive subject matter relates to a method of killing bacteria, comprising contacting the bacteria with a compound identified by the method described above.
In yet another aspect of the inventive subject matter, a method for identifying a compound that interacts with a lysine riboswitch, utilizing the crystal structure of a lysine riboswitch, comprises the steps of:
(a) modeling the lysine riboswitch with a test compound; and
(b) determining if the test compound interacts with the lysine riboswitch.
FMN riboswitches. Fusobacterium nucleatum plays a role in periodontal disease and other human infections and is considered one of the most pathogenic bacteria of the genus. The intracellular concentration of FMN in F. nucleatum is apparently controlled by a transcription attenuation mechanism involving two riboswitches, positioned before the riboflavin synthetic genes of the ribHDE(B/A) operon and the candidate riboflavin transporter impX gene (
In
Applicants have determined the 2.95-A° structure of the FMN-bound 112-nucleotide F. nucleatum impX RFN element (SEQ ID NO: 1), which conforms well with the consensus sequence and the six-helical junctional secondary structure characteristic of this riboswitch family (
In
In
In
The pseudo-symmetrical FMN does not take advantage of the two-fold symmetry between riboswitch elements, because its isoalloxazine ring and phosphate are oriented towards different riboswitch domains. The peripheral domains contain small RNA motifs, such as a T-loop (
In
In
Comparison of the loop-helix interactions uncovers a small difference that may contribute to gene expression regulation. In the P2-P6 domain, invariant G12 from the G12N(G93-C30) triple (
In contrast to other multi-stem junctional riboswitches, the junctional region of the FMN riboswitch is not constructed on the basis of collinear stacking of adjacent helices, but instead is composed of several non-paired segments, which provide a smooth transition between adjacent helices (
In
In support of the structural data, the binding affinity of riboflavin, the FMN precursor which lacks the phosphate and does not control gene expression, decreases by about 1,000-fold, compared with binding of FMN to both F. nucleatum and Bacillus subtilis riboswitches (
In
In
The identity of metal M1 as Mg+2 has been confirmed by substitution with Mn+2, a mimic of Mg+2 (
In
In
In
In
In
In
In
In
In
The identity of the divalent cation appears not to be crucial for FMN binding, because Mg+2 can be substituted by Ca+2, as well as and Mn+2, even in the absence of monovalent cations (Supplementary
To gain further insights into the ligand discrimination by FMN riboswitches, Applicants have determined crystal structures of the riboswitch in complex with riboflavin and roseoflavin 7 (
Applicants' structure-based modeling of a FAD-riboswitch complex supports the possibility of FAD-mediated control of the FMN riboswitch because FAD can be accommodated within the binding pocket after minor conformational adjustments (
Because the bound FMN is enveloped by the RNA, the FMN riboswitch is anticipated to re-arrange its conformation on complex formation. To access these potential conformational changes, Applicants have performed footprinting experiments using nucleases V1 (paired and stacked regions) and T2 (single-stranded regions) (
The presence of V1 and the absence of T2 cleavages indicate that nucleotides of the junctional segments are most likely involved in stacking interactions in the unbound state. Most of these nucleotides, which are clustered around the phosphate (nucleotides 10, 29-33) and isoalloxazine ring system (nucleotides 46-50, 97-102) of FMN and are adjacent to the P1 helix (nucleotides 9, 104), become protected on FMN binding owing to the mobility restrictions and shielding by neighboring RNA segments. Applicants' structural and footprinting data, corroborated by the in-line probing results on the B. subtilis riboswitch (
Because riboflavin biosynthetic capability is lacking in higher animals, riboflavin is traditionally used for food and feed fortification. Riboflavin can be produced in bacterial strains selected as roseoflavin resistant mutants with deregulated riboflavin biosynthesis. The FMN riboswitch structure (
In
Recent studies have demonstrated slow kinetics of association and dissociation for the FMN-riboswitch complex, supportive of a kinetically driven riboswitch mechanism 30. These kinetic characteristics are consistent with the recognition principles identified in Applicants' three-dimensional structure. Indeed, riboswitch folding requires formation of multiple non-canonical and tertiary interactions and other conformational adjustments on FMN binding, which together may account for the slow association rate. Subsequent FMN release is likely to be slowed down due to envelopment of the ligand by the RNA. The propensity of other large riboswitches to function as kinetically driven genetic switches remains a challenging area for future exploration.
Lysine Riboswitches. To understand the molecular basis of amino acid recognition by riboswitches, here Applicants present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the 1.9 angstrom lysine-bound and 3.1 angstrom free states.
The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+1-mediated hydrogen bonds to its charged ends. Applicants' structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. Applicants have also determined several structures of the riboswitch bound to different lysine analogues 5, including antibiotics, in an effort to understand the ligand binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Applicants' results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.
RNA sensors play a crucial part in many regulatory loops, owing to their capacity for directing gene expression in response to various stimuli in the absence of protein participation 6-8. Recent three-dimensional structures of a thermosensor 9, a metallosensor 10, a metabolite bound ribozyme 11,12 and riboswitches specific for purine nucleobases 13,14, and for co-enzymes thiamine pyrophosphate 15-17 and S-adenosylmethionine 18,19 have highlighted how each ribosensor uses unique structural features to sense its cognate stimulus. However, the molecular details of the organization of amino-acid-specific riboswitches, such as the lysine riboswitch, which efficiently discriminates against other free amino acids, their precursors and amino acids within a peptide context 1,2,5, remain obscure. The determination of the lysine riboswitch structure presents a considerable challenge, because of its large metabolite-sensing domain, predicted to form a five-way junction 1-3.
The T. maritima riboswitch is a typical lysine riboswitch 1-3,5 (Supplementary
The P2-P2a-L2 stem-loop reverses its orientation through two turns important for riboswitch function 5,21: one of them adjacent to a loop E motif and the other centered on a turn that replaces the kink-turn motif found in other lysine riboswitches (
The five-helical junction contains three layers of nucleotides, each composed of two interacting base pairs, organized around the centrally positioned lysine which fits into a tight pocket and is specifically recognized by its charged ends (
Both the carboxylate and ammonium groups of the lysine ‘main chain’ segment are hydrogen-bonded to the minor groove edges of purine bases and sugar 29-OH groups (
A notable feature of the lysine-binding pocket is a K+1 cation (
In
In
In
The importance of K+1 for lysine binding has been demonstrated in primer extension experiments. In the presence of lysine and at physiological concentration of K+1, reverse transcriptase pauses before the junction at A169, reflecting the formation of a stable junctional conformation (
RNA. b, Lysine binding affinity measured by equilibrium dialysis for riboswitches from T. maritima (top) and B. subtilis (bottom). Mg+2, K+1 and Na+1 concentrations are 20, 100 and 100 mM, respectively. The dissociation constants (mean6s.d., mM; n52-4) are: T. maritima, 0.1060.03 (Mg+2K+1), 4.1460.67 (Mg+2Na+1), 15.9360.09 (Mg+2); B. subtilis, 2.9560.30 (Mg+2K+1). c, The apparent ligand concentration at which reverse transcriptase pausing is half-maximally attained (P50; n52) in the primer extension experiments. HArg, L-homoarginine; IEL, iminoethyl-L-lysine; Lys, L-lysine; Oxa, L-4-oxalysine. d, In-line probing of 59 32P-labelled T. maritima 174-nucleotide RNA (1.6 nM) in the absence and presence of lysine (1.1 mM). T1 and 2OH designate RNase T1 and alkaline ladders, respectively. NR, no reaction. Strong and weak cleavage reductions are shown in red and pink colors, respectively. e, f, Probing of 174-nucleotide RNA (1 mM) by V1 and T2 nucleases with or without a tenfold excess of lysine. Weak and strong cleavage enhancements are shown in light and dark green, respectively, in e. g, Strong lysine-induced cleavage reductions are color coded in the riboswitch structure. Light orange, green and red are reductions identified in ref. 1 using B. subtilis RNA with a short P1 helix, overlapping reductions of the present study and in ref. 1, and extra reductions found in the present study, respectively.
The preference of K+1 for binding to the negatively charged carboxylate group contrasts with Mg+2-mediated phosphate recognition in other ribosensors 11,12,15,16 and might also be a characteristic for other amino-acid-specific riboswitches. Because more than 20 lysine-binding proteins (listed in the Protein Data Bank) do not use cations to mediate lysine recognition, this feature is probably unique to RNA, given that it lacks the positively-charged side chains found in proteins.
Folding of most RNAs, including the B. subtilis lysine riboswitch 21, requires Mg+2. However, the crystals of the T. maritima lysine riboswitch can be grown in the absence of Mg+2 (
The identification of antibiotic-resistance mutations 27,28 in the lysine riboswitch 1,2, together with the demonstration of a direct interaction between the riboswitch and lysine-like antibacterial compounds 1,2,5, suggest that riboswitch targeting, along with other processes 29, is an important component of the antibiotic activity.
To understand the molecular basis of antibiotic resistance and explore the pharmaceutical potential of the lysine riboswitch, Applicants have determined structures of the riboswitch bound to antibacterial compounds S-(2-aminoethyl)-L-cysteine (AEC) and L-4-oxalysine-5, which contain sulphur and oxygen at position C4, respectively (
In
θ is the fraction of RT pausing, No and Lo are RNA and ligand concentrations, respectively, and P50 is the apparent ligand concentration at which RT pausing is half-maximally attained. The P50 value for the lysine-induced RT pausing 5.50±0.53/−1M (mean±s.d.) (n=2) is similar to the Kd value 4.03±0.34/−1M (mean±s.d.) (n=2) determined by the equilibrium dialysis assay. The relative differences between the P50 values for lysine and its analogs are in overall agreement with the relative differences obtained from the Kd values for lysine and its analogs determined for the B. subtilis lysine riboswitch in ref. 5.
Next, Applicants determined the structures with lysine analogues L-homoarginine and N6-1-iminoethyl-L-lysine, where the ammonium group of lysine is replaced by a guanidinium group and its methyl-substituted variant, respectively (
The lysine-binding pocket has two openings, which could be exploited for the design of next generation lysine-like analogues (
To gain insights into lysine-induced conformational rearrangements of the riboswitch in solution, Applicants performed footprinting experiments using in-line probing, specific for flexible RNA regions, and cleavage by the nucleases T2 (single-stranded RNA) and V1 (paired and stacked regions). As in B. subtilis 1,5, the transition from the free to the lysine-bound states of the T. maritima riboswitch is accompanied by conformational changes within the junctional core, detected as strong cleavage reductions in both in-line and V1 probing (
In
The P2-L4 tertiary contact seems to be more dynamic in character as reflected in the rather strong T2 (nucleotides 126-127,
Prompted by pre-formation of tertiary riboswitch elements in solution, Applicants have crystallized the riboswitch in the absence of lysine. This 3.1 A ° structure (
In
The L box structure readily explains mutations that deregulate gene expression and confer resistance to AEC 27,28 (
The G12A, G12C and G81A mutations disrupt the lysine-binding pocket, whereas the G11A, G11U, G9C and C166U substitutions prevent pairing of the P1 helix. Therefore, intracellular lysine and AEC cannot bind the mutated riboswitches, and the segment downstream of G161 engages in formation of an anti-terminator stem (
The unusual architecture and high ligand specificity, achieved through a combination of shape complementarity and K+1-assisted recognition of the bound lysine, distinguishes the lysine riboswitch from other riboswitches. Given the importance of lysine riboswitch controlled gene expression for bacterial viability and the absence of the diaminopimelate pathway in mammals, the structure provides critical details towards facilitating the design of lysine-like analogues targeting riboswitches and other cellular sites.
The following examples are illustrative of the inventive subject matter and are not intended to be limitations thereon. When used and unless otherwise indicated, all percentages are based upon 100% by weight of the final composition.
The complexes of the riboswitch with FMN and its analogues were prepared by mixing 0.4m mRNA with 0.7 mM ligand in a buffer containing 100 mM potassium acetate, pH 6.8, and 4 mM MgCl2. FMN-riboswitch crystals were grown by hanging-drop vapor diffusion after mixing the complex with the reservoir solution (0.1M MES-sodium, pH 6.5, 100 mM MgCl2 and 10% (w/v) PEG 4000) at equimolar ratio. For soaking, crystals were incubated for 10 h in the reservoir solution supplemented with 25% glycerol and the following heavy atom salts: 5 mM [Ir(NH3)6]Cl3, 15 mM MnCl2, 10 mM [Co(NH3)6]Cl3, 30 mM BaCl2 and 30 mM CsCl. Crystals were flash frozen in liquid nitrogen and data were collected at 100 K. The structure was determined using 3.0-A° multiwavelength anomalous dispersion (MAD) iridium data (See Table 1) and refined to Rwork/Rfree 20.0/24.3 with a native data set. FMN and cations were added to the model based on the analysis of 2Fo2Fc, Fo2Fc and anomalous electron density maps. Analogue-bound and metal-soaked riboswitch structures were refined using the native riboswitch model (See Table 2).
)
]Cl3
)
)
)
)
)
(
)
)
)
)
(790)
(
)
)
]
(
)
/R
(%)
)
aValues for the highest-resolution shell are in parentheses.
bEstimated coordinate error based on maximum likelihood was calculated with REFMAC31.
indicates data missing or illegible when filed
Values for the highest-resolution shell are in parentheses.
Estimated coordinate error based on maximum likelihood was calculated with REFMAC
indicates data missing or illegible when filed
A 0.4 mM lysine riboswitch complex was prepared by mixing in vitro transcribed RNA and lysine in a buffer containing 100 mM potassium acetate, pH 6.8, and 4 mM MgCl2. Crystals were grown by hanging-drop vapor diffusion after mixing the complex and the reservoir (18% (w/v) PEG4000, 100 mM sodium citrate, pH 5.7, and 20% isopropanol (v/v)) solutions at 1:1 ratio. For soaking, crystals were placed in the reservoir solution with PEG4000 replaced by 20% PEG400, and then incubated in the presence of either 3 mM [Ir(NH3)6]31 or 10-50 mM of Cs+1, Ti+1 and Mn+2 salts for 7 h. Crystals were flash frozen in liquid nitrogen and data were collected at 100 K. The structure was determined using 2.4 A° multiwavelength anomalous dispersion iridium data and SHARP30. The RNA model was built using TURBO-FRODO (http://www.afmb.univ-mrs.fr/-TURBO-), and refined using the 1.9 A° native data set to Rwork/Rfree 19.2/22.9 (See Table 3 and
)
]
+
sites
/R
indicates data missing or illegible when filed
)
/R
indicates data missing or illegible when filed
/R
(%)
indicates data missing or illegible when filed
The 112-nucleotide sensing domain of the F. nucleatum FMN riboswitch followed by a hammerhead ribozyme was cloned into the pUT7 vector and transcribed in vitro using T7 RNA polymerase. The RNA was purified by denaturing polyacrylamide gel electrophoresis and anion-exchange chromatography. Alternatively, the riboswitch was formed by the annealing of two chemically synthesized RNAs (0.4 mM each):
designed to engineer crystal contacts, in the presence of FMN or its analogues in the binding buffer at 37° C. for 30 min followed by incubation on ice. The 143-nucleotide sensing domain of the B. subtilis FMN riboswitch, the 220-nucleotide full-length F. nucleatum FMN riboswitch and the 172-nucleotide fragment of the B. subtilis lysine riboswitch were transcribed and purified as above. Ligand concentrations were estimated spectrophotometrically using the extinction coefficients ε450=12,500 M−1 cm−1 for FMN, ε505=31,000 M−1 cm−1 for roseoflavin, and ε445=12,500 M−1 cm−1 for riboflavin and lumiflavin.
Crystals were grown at 20° C. using hanging-drop vapor diffusion by mixing 1 ml complex with 1 ml reservoir solution. The FMN-bound complex produces crystals of the saturated yellow color, characteristic for the oxidized form of FMN, under several conditions. The best crystals were obtained in the solution containing 0.1M MES-sodium, pH 6.5, 100-200 mM MgCl2 and 6-13% (w/v) PEG 4000 for 1-4 weeks. The crystals of the analogue-bound complexes grew in solution containing 0.1M Tris-HCl, pH 8.4, 200 mM MgCl2 and 6-10% (w/v) PEG 4000 for about 1 week. The FMN-bound native and heavy-atom-soaked crystals were grown using the transcribed RNA. Although the analogue-bound crystals could grow using the transcribed RNA, resolution of the crystals was improved with the annealed riboswitch.
X-ray diffraction data were reduced using HKL2000 (HKL Research). The structure was determined using 3.0-A° MAD iridium data and autoSHARP (see, e.g., de La Fortelle, E. & Bricogne, G. in Methods in Enzymology 472-494, Academic Press, 1997). The RNA model was built using TURBO-FRODO (see http://www.afmb.univmrs.fr/-TURBO-) and refined with REFMAC (see, e.g., Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240-255 (1997)). The final 2.95-A° riboswitch model contained 109 nucleotides (nucleotides 54-56 were cleaved off during crystallization), one FMN molecule, two potassium and 14 magnesium cations. Mn2+, Cs+, Ba2+ and [Co(NH2)6]3+ cations were positioned based on the anomalous electron density maps (Supplementary
Fluorescent assays were performed based on intrinsic fluorescence of FMN and its analogues, which become quenched after specific interaction of the ligands with the riboswitch fragment. In all assays, intensity of fluorescence emission was measured at 530 nM with excitation at 450 nM. Each experiment was performed about two to four times at room temperature using a Tecan M1000 fluorimeter.
For the binding affinity measurements, the fragments of the F. nucleatum and B. subtilis FMN riboswitches were titrated against 6×10−8 M FMN or 10−8 M analogues. RNA and ligands were premixed in 50 mM Tris-HCl, pH 7.4, 100 mM KCl and 2 mM MgCl2 in the 96 half-area black flat plates for 10-60 min. The B. subtilis lysine riboswitch 27 was used as a negative control. After subtraction of the buffer fluorescence and normalization to the free ligand fluorescence, the data were fitted to equation (1):
where F is normalized fluorescence intensity, L0 and R0 are the concentrations of ligand and RNA, Kd is the apparent dissociation constant, and f is a residual fluorescence intensity at the saturated concentration of ligand, determined by plotting F versus R0.
For cation-dependence studies, the 112-nucleotide F. nucleatum FMN riboswitch was titrated against 6×10−8M of FMN in 50 mM Tris-HCl, pH 7.4, supplemented with 2 mM different cations (MgCl2, MnCl2, BaCl2, CaCl2, [Ir(NH3)6]Cl3 and [Co(NH3)6]Cl3) in the presence and absence of 100 mM KCl or in the presence of other monovalent cations (NaCl or CsCl). Binding affinities were determined using equation (1). The cation concentration required for the FMN binding was estimated by titration of different cations against a mixture of RNA (2×10−7 M) and ligand (6×10−8 M) in 50 mM Tris-HCl, pH 7.4. After background subtraction of the fluorescent quenching at each point in the absence of RNA, data were fitted to the Hill equation (2),
θ=[M]n/(Kd+[M]n) (2)
where h is the normalized FMN-bound fraction, n is Hill coefficient, [M] is the concentration of cation and Kd is the apparent dissociation constant. As the parameters Kd and n covary, the cation binding was roughly estimated as the ion concentration [M]1/2=(Kd)1/n at which approximately 50% of FMN was bound to RNA.
For footprinting experiments (see, e.g., Serganov, A., Polonskaia, A., Ehresmann, B., Ehresmann, C. & Patel, D. J. Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA. EMBO J. 8, 1898-1908, 2003), the 112-nucleotide F. nucleatum riboswitch was radioactively labelled at the 59 end by the kinase reaction. Samples (20 μl) of the radiolabelled RNA (100,000 c.p.m.) with a final RNA concentration 0.5 μM were preheated at 37° C. for 10 min in 50 mM Na-HEPES, pH 7.9, 50 mM KCl and 2 mM MgCl2. Sixfold excess of FMN was added to the RNA and the mixtures were additionally incubated at 37° C. for 15 min. Cleavage reactions were performed with 0.003 U RNase V1 (Pierce) or 0.25 U RNase T2 (Sigma) at 37° C. for 10 min. Reactions were quenched by the addition of 80 μl cold buffer and were immediately extracted with phenol-chloroform and precipitated by ethanol. Radiolabelled pellets were dissolved and analysed by polyacrylamide gel electrophoresis.
The lysine riboswitch, followed by the hammerhead ribozyme, was transcribed in vitro using T7 RNA polymerase. RNA was purified by denaturing polyacrylamide gel electrophoresis (PAGE) and anion-exchange chromatography. Lysine analogues were added to RNA at a 2.5-2.75 to 1 molar ratio. To form a complex without Mg+2, the RNA was mixed with 100 mM potassium-acetate, 1.0 mM EDTA and lysine. To prepare RNA for crystallization without lysine, 0.2 mm RNA was supplemented with 50 mM potassium acetate, pH 6.8, 50 mM sodium acetate, pH 6.9, and 2 mM MgCl2, and concentrated two fold by Speedvac. Before crystallization, sodium-citrate, pH 5.7, was added to the mixture up to 100 mM, and the RNA sample was heated at 55° C. for 5 min and cooled on ice for 15 min.
Hanging drops were prepared by mixing 1 ml of the complex with 1 ml of the reservoir solution. The drops were equilibrated against 1 ml of reservoir solution at 20° C. for 1-2 weeks. The riboswitch in the free state was crystallized in the solution containing 21% (w/v) PEG4000, 100 mM Bis-Tris, pH 5.5, and 25% isopropanol (v/v). For cryoprotection, crystals were quickly passed through the stabilizing solution, which was the reservoir solution with PEG4000 replaced by 20% PEG400. For soaking, crystals were passed through several 5 ml drops of the stabilizing solution, and then incubated in 5 ml of stabilizing solution supplemented with 3 mM [Ir(NH3)6]Cl3, 10 mM CsCl, 10 mM thalium acetate, or 50 mM MnCl2 salts for 7-8 h.
Data were reduced using HKL2000 (HKL Research). The structure was determined using the autoSHARP option of SHARP and 2.4 A° MAD iridium data. The resulting experimental map was of excellent quality (
Water molecules were added using ARP/wARP32. K+1 cations were added on the basis of the 1.9 A° 2Fo2Fc and 2.9 A° anomalous (
Primer extension experiments were performed using 265-nucleotide full-length riboswitch. The 32P-labelled 13-mer DNA oligonucleotide (100,000 c.p.m.), complementary to nucleotides 253-265 of RNA, was annealed to the 39 end of RNA (final RNA concentration 1 mM in the assay). Primer extension was conducted in 15 ml volume with 40 U of moloney murine leukaemia virus reverse transcriptase in 50 mM Na-HEPES, pH 7.9, 2 mM MgCl2, and variable concentrations of NaCl and KCl (
For footprinting experiments, the 174-nucleotide metabolite-sensing domain of the riboswitch was radioactively labelled at the 59 end by the kinase reaction. For in-line probing, 30,000-300,000 c.p.m. of 174-nucleotide RNA (1.6-16 nM) was incubated in 10-30 ml solution containing 50 mM Tris-HCl, pH 8.3, 100 mM KCl and 20 mM MgCl2 in the absence or presence of ,6-600-fold excess of lysine (
For nuclease footprinting experiments, 20 ml samples of 174-nucleotide RNA (100,000 c.p.m.) with a final RNA concentration 1 mM were preheated at 37° C. for 10 min in 50 mM Na-HEPES, pH 7.9, 50 mM KCl and 2 mM MgCl2. Mixtures were incubated with a tenfold excess of lysine over RNA at 37° C. for 15 min. Cleavage reactions were performed with 0.0025 U RNase V1 (Pierce) or 0.2 U RNase T2 (Sigma) at 37° C. for 10 min. Reactions were quenched by the addition of 80 ml cold buffer and were immediately extracted with phenolchloroform and precipitated with ethanol. Radiolabelled RNA products were dissolved and analysed by PAGE.
The assay was performed as described in Sudarsan, et al., An mRNA structure in bacteria that controls gene expression by binding lysine, Genes Dev. 17:2688-2697 (2003) using 5 kDa DispoEquilibrium DIALYZERS (Harvard Apparatus). In brief, 30 ml RNA (from 0.001 to 20 mM) in the in-line probing buffer was placed in chamber A of the dialyser and equilibrated for 16 h at room temperature against chamber B containing 30 ml 3H-labelled lysine (1 nM; 6,000 c.p.m.) in the same buffer. The amount of bound lysine was calculated by subtracting the radioactivity counts of chamber B from chamber A. The data were fitted using a bimolecular equilibrium equation, assuming that the free lysine concentration is negligible.
Synthesis of Compounds of the Inventive Subject Matter
The compounds of the inventive subject matter may be readily prepared by standard techniques of organic chemistry and molecular biology.
In the preparation of the compounds of the inventive subject matter, one skilled in the art will understand that one may need to protect or block various reactive functionalities on the starting compounds or intermediates while a desired reaction is carried out on other portions of the molecule. After the desired reactions are complete, or at any desired time, normally such protecting groups will be removed by, for example, hydrolytic or hydrogenolytic means. Such protection and deprotection steps are conventional in organic chemistry. One skilled in the art is referred to “Protective Groups in Organic Chemistry,” McOmie, ed., Plenum Press, New York, N.Y.; and “Protective Groups in Organic Synthesis,” Greene, ed., John Wiley & Sons, New York, N.Y. (1981) for the teaching of protective groups which may be useful in the preparation of compounds of the inventive subject matter.
The product and intermediates may be isolated or purified using one or more standard purification techniques, including, for example, one or more of simple solvent evaporation, recrystallization, distillation, sublimation, filtration, chromatography, including thin-layer chromatography, HPLC (e.g. reverse phase HPLC), column chromatography, flash chromatography, radial chromatography, trituration, and the like.
Tables 6-26 comprise a large data set, were consolidated into a separate part of the description, and were submitted in text format attached to this application on compact disk (CD) under PCT Rule 5.2(a). In Tables 6-26, the following abbreviations apply: FMN=flavin mononucleotide; IR6=iridium hexamine; BA2=barium cation; CS=cesium cation; MN2=manganese cation; CO6=cobalt hexamine; B2=riboflavin; RFN=roseoflavin; S2L=S-(2-aminoethyl)-L-cysteine; LYS=lysine; HOM=homoarginine; N61=N6-(1-iminoethyl)-L-lysine; KAD=potassium cation (anomalous data); MG2=magnesium cation; L4O=L-4-oxalysine; and TI=titanium cation.
The following literature references are believed to useful to an understanding of the inventive subject matter in the context of its place in the relevant art. Citation here is not to be construed as an assertion or admission that any reference cited is material to patentability of the inventive subject matter. Applicants will properly disclose information material to patentability in an Information Disclosure Statement. Each of the following documents is hereby incorporated by reference in its entirety, in this application.
The inventive subject matter being thus described, it will be obvious that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the inventive subject matter and all such modifications and variations are intended to be included within the scope of the following claims.
This application claims the benefit of U.S. Provisional Patent Application No. 61/064,817, filed Mar. 27, 2008, and U.S. Provisional Patent Application No. 61/129,005, filed May 30, 2008, the contents of which are hereby incorporated by reference in their entirety.
The present invention was made in part with support from a grant from the National Institutes of Health (NIGMS), grant R01 GM073618-18. Therefore, the government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2009/001883 | 3/27/2009 | WO | 00 | 6/14/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61064817 | Mar 2008 | US | |
| 61129005 | May 2008 | US |
