the 5 ′ utr and beginning of orf from cd3299 gene of c . difficile 630 , accession number am180355 is as follows : seq id no : 1 : ttacagctttctgattttgataaatttaaaacttaccatctaatactaataacaggt taattttatctaattattatagattctcatactgtgccttattctatctataaatac aatttaagtgtccatattgaaatatttgtattgtaatacagctggatattacttaaa tccaattgtttccattataattttatgttaaaataatattacaaaatacatct gttt ttcttcataaac gggtg aaattccctatcggcggtaaaagcccgcgagccttatg gcataatttggtcatattccaaagccaacagtaaaatctggatggtagaagaaa ata gtatatgagtacctttatgtaattttacatgagtaatctatacaaatccttcaa ctaccgtatttattcatgaaattagacacattcaag tt tttttgttgtttattttacaattatatcgtacttataaaatctattaagattggagt gttatc aatggatagtattgattatcatctgtattggtgtatttatg tctactcttgatggaagtatactaaatatcgcaaa in the above depiction of the sequence , the riboswitch is highlighted in bold , and is the 0rf start site in the above sequence is downstram from the riboswitch and is depicted in italics and is : the hairpin can form a loop having a structure as depicted in formula 1 : a possible antiterminator has a structure as depicted in formula 2 : in one embodiment , compounds binding to and activating the cd3299 riboswitch are compounds of formula iv from pct application pct / us 09 / 04576 : ( i ) alk is c 1 - 8 alkyl ( e . g ., ethyl or n - butyl ); ( ii ) r a and r b are independently h , — c 1 - 4 alkyl ( e . g ., methyl ), —( ch 2 ) 3 c ( nh 2 )( cooh ) chf 2 , —( ch 2 ) 3 n ( h ) c (═ nh ) nh 2 , —( ch 2 ) 5 nh 2 , —( ch 2 ) 2 c ( h )( oh ) cooh , — c ( o )( ch 2 ) 2 cooh , — c 1 - 4 alkyl - c ( o ) or s ( e . g ., — ch 2 ch 2 ch 2 ch 2 c ( o ) or 9 , — ch 2 ch 2 ch 2 c ( o ) or 9 , — ch 2 ch 2 c ( o ) or 9 or — ch 2 c ( o ) or 9 , — c ( ch 3 )( ch 3 ) c ( o ) or 9 ), — c ( o ) ch 3 , aryl ( e . g ., phenyl ), — c ( o )- aryl , aryl - c 1 - 4 alkyl ( e . g ., benzyl , naphtha - 1 - ylmethyl , naphth - 2 - ylmethyl , phenylethyl , phenylpropyl , naphtha - 1 - ylethyl ), heteroaryl , heteroaryl - c 1 - 4 alkyl ( e . g ., pyrid - 2 - ylmethyl , pyrid - 3 - ylmethyl or quinoxalinyl ), wherein said aryl and heteroaryl groups are optionally substituted with one or more groups selected from — c ( o ) or 9 , — nh 2 , — s ( o ) 2 nh 2 , — ch 2 nh 2 , halo ( e . g ., chloro ), c 1 - 4 alkoxy ( e . g ., methoxy ), c 1 - 4 alkyl ( e . g ., methyl ); ( iii ) r 1 is h , c 1 - 8 alkyl ( e . g ., methyl ); ( iv ) r 2 is h , halo ( e . g ., chloro ), — o — c 3 - 7 cycloalkyl ( e . g ., — o - cyclopentyl ), — n ( r 4 )( r 5 ), c 3 - 7 cycloalkyl ( e . g ., cyclopropyl ), c 1 - 8 alkyl ( e . g ., methyl or ethyl ) or — o — c 1 - 8 alkyl wherein the alkyl group is optionally substituted with one or more halo or hydroxyl groups ( e . g ., trifluoromethyl , — o — ch 2 ch 2 oh ); ( v ) r 4 and r 5 are independently h , c 3 - 7 cycloalkyl ( e . g ., cyclopropyl or cyclopentyl ), c 1 - 8 alkyl ( e . g ., methyl ) wherein said alkyl is optionally substituted with one or more hydroxy groups ( e . g ., 2 , 3 - dihydroxypropyl , 2 , 3 , 4 , 5 , 6 - pentahydroxyhexyl ); ( vi ) r 9 is h or c 1 - 4 alkyl ( e . g ., t - butyl , isopropyl , methyl ); ( vii ) r 12 is c 1 - 8 alkyl ( e . g ., methyl , ethyl , t - butyl ) or — oc 1 - 8 alkyl ( e . g ., methoxy , ethoxy , t - butoxy ), the words “ treatment ” and “ treating ” are to be understood accordingly as embracing prophylaxis and treatment or amelioration of symptoms of disease as well as treatment of the cause of the disease . the compounds useful in the methods described herein may be administered orally , parentally ( e . g , intravenously ), topically , rectally or by other means depending on the nature and location of the infection . preferably the compounds are administered orally . dosages employed in practicing the present invention will vary depending , e . g . on the particular disease or condition to be treated , the age and size of the patient , the particular active compound used , the mode of administration , and the therapy desired . for example , in one embodiment , daily oral dosages for a 70 kg human suffering from diarrhea and colitis caused by c . difficile may be from 10 - 2000 mg . administration of a therapeutically active amount of the therapeutic compositions is defined as an amount effective , at dosages and for periods of time necessary to achieve the desired result . dosage regimens may be adjusted to provide the optimum therapeutic response . for example , several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation . the compound may administered as monotherapy or in combination with one or more antibiotics , for example in combination with metronidazole ( flagyl ®), vancomycin ( vancocin ®), linazolid , ramoplanin , and / or fidaxomicin , and / or in combination with one or more antibiotics selected from fluoroquinolones , cephalosporins , clindamycin and penicillins . the patient may receive anti - toxin therapy , for example monoclonal antibodies to c . difficile toxins , or anti - toxoid vaccine . the patient may receive probiotics , such as bacteria and yeast , which help restore a healthy balance to the intestinal tract , e . g ., saccharomyces boulardii ( florastor ®), and / or be undergoing fecal bacteriotherapy . pharmaceutical compositions comprising compounds as described herein may be prepared using conventional diluents or excipients and techniques known in the galenic art . thus oral dosage forms may include tablets , capsules , solutions , suspensions and the like . the term “ pharmaceutically acceptable carrier ” as used herein is intended to include diluents such as saline and aqueous buffer solutions , as well as solid carriers such as microcrystalline cellulose , hydroxypropylmethyl cellulose , or lactose . an in - line probing assay , as described in regulski and breaker , “ in - line probing analysis of riboswitches ”, ( 2008 ), methods in molecular biology , vol 419 , pp 53 - 67 , the contents of which are incorporated by reference , is used to estimate the dissociation binding constants for the interaction of each of the ligands described herein with a cd3299 riboswitch amplified from clostridium difficile . precursor mrna leader molecules are prepared by in vitro transcription from templates generated by pcr and [ 5 ′- 32 p ]- labeling using methods described previously ( regulski and breaker , in - line probing analysis of riboswitches ( 2008 ), methods in molecular biology vol 419 , pp 53 - 67 ). approximately 5 nm of labeled rna precursor is incubated for 41 hours at 25 ° c . in 20 mm mgcl 2 , 50 mm tris / hcl ( ph 8 . 3 at 25 ° c .) in the presence or absence of a fixed concentration of each ligand . binding to the cd3299 riboswitches are measured 100 m . in - line cleavage products are separated on 10 % polyacrylamide gel electrophoresis ( page ), and the resulting gel is visualized using a molecular dynamics phosphorimager . the location of products bands corresponding to cleavage are identified by comparison to a partial digest of the rna with rnase t1 ( g - specific cleavage ) or alkali ( nonspecific cleavage ). in - line probing exploits the natural ability of rna to self - cleave at elevated ph and metal ion concentrations ( ph ≈ 8 . 3 , 25 mm mgcl 2 ) in a conformation - dependent manner . for self - cleavage to occur , the 2 ′- hydroxyl of the ribose must be “ in - line ” with the phosphate - oxygen bond of the internucleotide linkage , facilitating a s n 2p nucleophilic transesterification and strand cleavage . typically , single - stranded regions of the riboswitch are dynamic in the absence of an active ligand , and the internucleotide linkages in these regions can frequently access the required in - line conformation . binding of an active ligand to the riboswitch generally reduces the dynamics of these regions , thereby reducing the accessibility to the in - line conformation , resulting in fewer in - line cleavage events within those regions . these ligand - dependent changes in rna cleavage can be readily detected by denaturing gel electrophoresis . the relative binding affinity of each ligand is expressed as i max , wherein i max represents the percent inhibition of in - line cleavage at selected internucleotide ligands in the presence of a fixed ligand concentration ( 100 m for the cd3299 riboswitch ) normalized to the percent inhibition in the absence of ligand and the percent inhibition in the presence of a saturation concentration of a control ligand . 100 m of compound a ( which is a compound identified as having high affinity to the cd3299 riboswitch ) is used as a control ligand for estimating binding to the cd3299 . the experiments show that compounds 1 and 2 have a binding affinity to the cd3299 switch with an i max value of 45 - 90 compared to the control at 100 m . the mic assays are carried out in a final volume of 100 μl in 96 - well clear round - bottom plates according to methods established by the clinical laboratory standards institute ( clsi ). briefly , test compound suspended in 100 % dmso ( or another suitable solubilizing buffer ) is added to an aliquot of media appropriate for a given pathogen to a total volume of 50 μl . this solution is serially diluted by 2 - fold into successive tubes of the same media to give a range of test compound concentrations appropriate to the assay . to each dilution of test compound in media is added 50 l of a bacterial suspension from an overnight culture growth in media appropriate to a given pathogen . final bacterial inoculum is approximately 10 5 - 10 6 cfu / well . after growth for 18 - 24 hours at 37 ° c ., the mic is defined as the lowest concentration of antimicrobial agent that completely inhibits growth of the organism as detected by the unaided eye , relative to control for bacterial growth in the absence of added antibiotic . ciprofloxacin is used as an antibiotic - positive control in each screening assay . each of the bacterial cultures that are available from the american type culture collection ( atcc , www . atcc . org ) is identified by its atcc number . the experiments show that compounds 1 and 2 have a minimum inhibitory concentration ( mic ) of 64 μg / ml or less against c . difficile strains atcc 700057 ( mmx 4381 ) and mmx3581 ( clinical ). all references indicated herein are incorporated by reference for any patent application in the united states .
the invention provides methods for treating or inhibiting infection by clostridium difficile in a subject in need of such treatment , comprising administering an effective amount of a compound binding to a cd3299 riboswitch , as well as assays for identifying compounds useful in such treatment , and the use of particular compounds in such treatment .