Patent Application: US-201213358512-A

Abstract:
a method for controlling yeast - to - filamentous growth transition in fungi comprising contacting a fungal cell with an anti - fungal small molecule in an amount effective to reduce or inhibit the yeast - to - filamentous growth transition , wherein the anti - fungal small molecule is 5 -- α - isopropyl - 4 - oxo - 2 - thioxo - 3 - thiozolidineacetic acid or an analog thereof . a method for treating fungal infections comprising contacting one or more fungal cells with an anti - fungal small molecule in an amount effective to control the fungal infection , wherein the anti - fungal small molecule is 5 -- α - isopropyl - 4 - oxo - 2 - thioxo - 3 - thiozolidineactic acid or an analog thereof .

Description:
the present invention is based on the discovery of novel anti - fungal properties of a small molecule that was previously shown to induce programmed cell death in mammalian cells . the invention provides a method for treating or controlling fungal infections ( such as those caused by c . albicans ) through inhibition of the yeast - to - filamentous growth transition , which is important for the pathogenicity of c . albicans . the invention also provides a method for controlling the yeast - to - filamentous growth transition in in vitro systems . the current generation of anti - fungal drugs works by inhibiting membrane biosynthesis and function and cell wall biosynthesis . bh3i - 1 represents a potentially new class of anti - fungal drugs in that it inhibits a process ( and presumably a molecular pathway ) not targeted by the current anti - fungal therapies . we have discovered that the small molecule bh3i - 1 is effective for inhibiting fungal morphogenesis . as used herein , the terms “ yeast - to - filamentous growth transition ” and “ fungal morphogenesis ” refer to all yeast - to - hypha growth transitions and all yeast - to - pseudohypha growth transitions . a simple in vitro assay system was designed to detect inhibition of the yeast - to - filamentous growth transition in c . albicans [ 8 ]. in this assay , c . albicans cells are first grown under conditions that maintain them in a yeast growth form . the yeast - form cells are then transferred to the wells of an optical microplate containing ( i ) a medium that induces either the yeast - to - hypha transition ( in wild type cells ) or the yeast - to - pseudohypha transition ( in a conditional pseudohyphal mutant strain ) and ( ii ) varying concentrations of bh3i - 1 ( typically 0 , 10 , 30 , 50 , 100 and 200 μm bh3i - 1 ). the plates are incubated for up to six hours at 37 ° c . ( to test for the hyphal transition ) or up to 24 hours 30 ° c . ( to test for the pseudohyphal transition ). the transition to filamentous growth ( or lack thereof ) is monitored in the optical microplates directly with a nikon te200 inverted microscope . c . albicans sc3514 was used as the “ wild type ” strain to monitor the yeast - to - hypha transition . a “ conditional pseudohyphal mutant ” strain was used to monitor the yeast - to - pseudohypha transition [ 9 ]. the conditional strain harbors deletions of both native grr1 alleles plus an introduced wild type copy of grr1 under the control of the regulatable met3 promoter [ 10 ]. deletion of grr1 was previously found to cause pseudohyphal growth [ 9 ]. standard yeast growth media were used for these experiments . to maintain wild type cells in the yeast - form , cells were grown in ypd medium ( 1 % yeast extract , 2 % peptone and 2 % dextrose ) at 30 ° c . to induce hyphal growth in the wild type strain , cells were transferred to spider medium ( 1 % nutrient broth , 1 % mannitol , 2 % k 2 hpo 4 ) at 37 ° c . or to lee &# 39 ; s medium ( 0 . 5 % ( nh 4 ) so 4 , 0 . 02 % mgso 4 , 0 . 5 % nacl , 1 . 25 % glucose , supplemented with alanine , leucine , lysine , ornithine , phenylalanine threonine and biotin ) at 37 ° c . to maintain the conditional pseudohyphal mutant strain in the yeast - form , cells were grown in ynb medium ( yeast nitrogen base , ammonium sulfate , succinic acid supplemented with the appropriate amino acids ) at 30 ° c . this growth condition allows expression of the met3p - driven grr1 gene ( and therefore yeast - form growth ) [ 9 ]. to induce pseudohyphal growth , the conditional strain was transferred to ynb medium plus 2 . 5 mm methionine and 2 . 5 mm cysteine at 30 ° c . methionine and cysteine in the growth medium represses the met3p - driven grr1 ( gene and therefore induces pseudohyphal growth [ 9 ]. bh3i - 1 was dissolved in dimethyl sulfoxide ( dmso ) as a 5 mm stock and diluted into 100 μl of the appropriate medium at 10 μm , 50 μm , 100 μm and 200 μm final concentration . the control condition ( no bh3i - 1 ) included medium plus dmso to a concentration equivalent to that of the bh3i - 1 - containing wells . blast searches of the ncbi , sod , and cgd databases using the amino acid sequences of various bh3 domains has not identified any significant bh3 domains in candida species . the closest match is from candida glabrata with an expect ( e ) value score of 13 . for comparison , an exact match from homo sapiens generates an e value score of 1 × 10 − 1 . the lower the e value score , the more likely that the match contains a true homologue of the bh3 domain . wild type sc3514 c . albicans cells treated with bh3i - 1 failed to undergo the yeast - to - hypha transition when transferred from ypd medium to either spider medium or lee &# 39 ; s medium . the bh3i - 1 - treated sc3514 cells continued to divide as yeast cells in the hyphal - inducing media . the minimum inhibitory concentration ( mic ) for the yeast - to - hypha transition was determined to be 50 - 100 μm bh3i - 1 in both hyphal - inducing media . in contrast , greater than 90 % of cells in the control condition ( no bh3i - 1 ) formed obvious hyphae in spider ( see fig1 , first panel in bottom row ) and lee &# 39 ; s media ( data not shown ; results similar to fig1 ). conditional pseudohyphal mutant cells treated with bh3i - 1 failed to undergo the yeast - to - pseudohypha transition when transferred from ynb medium to ynb plus methionine / cysteine medium . bh3i - 1 - treated conditional mutant cells continued to divide as yeast cells in the pseudohyphal - inducing medium ( see fig2 ). the mic for the yeast - to - pseudohypha transition was determined to be 100 μm bh3i - 1 in the pseudohyphal - inducing medium . conditional mutant cells in the control condition ( no bh3i - 1 ) formed abundant pseudohyphae in the pseudohyphal - inducing medium ( see fig2 , first panel in bottom row ). the anti - fungal morphogenesis small molecule bh3i - 1 has the chemical structure shown in fig3 . this basic structure may be modified such that the resulting analogs of bh3i - 1 also inhibit fungal morphogenesis . referring to fig4 , in one embodiment of the present invention , analogs of bh3i - 1 that inhibit fungal morphogenesis include , but are not limited to , modification the carboxylic acid group ( a ) to an ester group . the term “ ester ” is given its ordinary meaning as used in the field of organic chemistry . esters include a carbonyl group bonded to an oxygen atom , where the oxygen atom is also bonded to an alkyl group . in an alternate embodiment , analogs of bh3i - 1 that inhibit fungal morphogenesis include , but are not limited to , replacement of the methyl group ( b ) with another alkyl group and / or replacement of the methyl group ( c ) with another alkyl group . as used herein , the term “ alkyl ” is given its ordinary meaning as used in the field of organic chemistry . alkyl or aliphatic groups typically contains any number of carbon atoms , for example , between one and 20 carbon atoms . in some embodiments , the alkyl group will contain at least one carbon atom . typically , an alkyl group is a non - cyclic structure . in certain embodiments , the alkyl group is a methyl group or an ethyl group . the carbon atoms may be arranged in any configuration within the alkyl moiety , for example , as a straight chain ( i . e ., a n - alkyl such as methyl , ethyl , propyl , butyl , pentyl , hexyl , heptyl , octyl , nonyl , decyl , or undecyl ) or a branched chain ( e . g ., a t - butyl group or an isoalkyl group such as isopropyl , isobutyl , isopentanyl , or isohexanyl ). the alkyl moiety may contain none or any number of double or triple bonds within its structure , for example , as in an alkene , an alkyne , an alkadiene , an alkadiyne , an alkenyne , etc . the alkyl group may contain any number of substituents . for example , the alkyl group may contain a halogen , an alkoxy ( e . g ., a methoxy , an ethoxy , a propoxy , an isopropoxy , a butoxy , or a pentoxy ), an amine ( e . g ., a primary , secondary , or tertiary amine ), or a hydroxide as a substituent . as one example , if the alkyl group is a methyl group , then the methyl group may be substituted to form , for instance , a halogenated methyl group such as chloromethyl , bromomethyl , or iodomethyl . in some embodiments of the invention , more than one substituent may be present . for example , the alkyl group may have two or more halogen atoms ( for example , two chlorine atoms , or a chlorine and a bromine atom ), or it may have a halogen and an alkoxy group . as used herein the term “ halogen ,” or equivalently , “ halogen atom ,” is given its ordinary meaning as used in the field of chemistry . the halogens include fluorine , chlorine , bromine , iodine , and astatine . preferably , the halogen atoms used in the present invention include one or more of fluorine , chlorine , bromine , or iodine . in certain embodiments of the invention , the halogen atoms found within the structure are fluorine , chlorine , and bromine ; fluorine and chlorine ; chlorine and bromine , or a single type of halogen atom . as used herein the term “ alkoxy ” is given its ordinary meaning in the field of organic chemistry . an alkoxy group consists of an alkyl group covalently bound to an oxygen atom , where the oxygen atom is covalently bound to a carbon atom . in some embodiments of the invention , the alkyl group may also contain one or more heteroatoms substituted within the alkyl group , such as a nitrogen atom ( e . g ., as in an amine such as a primary , secondary , or tertiary amine ), an oxygen atom ( as in an ether moiety ) or a sulfur atom . in other embodiments of the invention , however , the main chain of the alkyl group is free of heteroatoms and includes carbon atoms . as used herein , the term “ heteroatoms ” refers to atoms that can replace carbon atoms within an alkyl group without affecting the connectivity of the alkyl group ; these typically include oxygen , nitrogen and sulfur atoms . halogen atoms and hydrogen atoms are not considered to be heteroatoms ; for example , a chlorine atom can replace a hydrogen atom within an alkyl group without affecting the connectivity of the alkyl group . in one embodiment of the invention , analogs of bh3i - 1 that inhibit fungal morphogenesis include , but are not limited to , replacement of the sulfur atom ( d ) with an oxygen atom . in another embodiment of the invention , analogs of bh3i - 1 that inhibit fungal morphogenesis include , but are not limited to , replacement of the sulfur heteroatom ( e ) with a nitrogen atom . in yet another embodiment of the invention , analogs of bh3i - 1 that inhibit fungal morphogenesis include reduction of the double bond ( f ) to a single bond . in yet another embodiment of the invention , analogs of bh3i - 1 that inhibit fungal morphogenesis include , but are not limited to , removal of the bromine atom ( h ), changing position of the bromine atom on the aromatic ring ( g ), inclusion of multiple bromine atoms at different positions on the aromatic ring ( g ), replacement of the bromine atom ( h ) with a different halogen atom , inclusion of non - bromine halogen atoms at different positions on the aromatic ring ( g ), replacement of the bromine atom ( h ) with a hydroxyl group , inclusion of hydroxyl groups at different positions on the aromatic ring ( g ), replacement of the bromine atom ( h ) with an alkyl group , inclusion of alkyl groups at different positions on the aromatic ring ( g ), replacement of the bromine atom ( h ) with an alkoxy group , inclusion of alkoxy groups at different positions on the aromatic ring ( g ), replacement of the bromine atom ( h ) with n ( ch 3 ) 2 , inclusion of n ( ch 3 ) 2 at different positions on the aromatic ring ( g ), or any chemically possible combination of the foregoing . as used herein , the term “ aromatic ring ” is given its ordinary meaning as used in the field of organic chemistry . an aromatic ring is a planar set of six carbon atoms that are connected by alternating single and double covalent bonds . as used herein , the term “ hydroxyl ” is given its ordinary meaning as used in the field of organic chemistry . a hydroxyl group consists of an oxygen atom covalently bound to a hydrogen atom , where the oxygen atom is also covalently bound to a carbon atom . according to some aspects of the invention , the bh3i - 1 used in the methods for inhibiting fungal morphogenesis or in the methods for treating fungal infection may exist in different isomeric forms . bh3i - 1 may be used in the methods of the invention as a substantially isomerically pure compound or as a mixture of isomers . as used herein the term “ isomerically pure ” means that one isomer will be present in an amount ranging from 51 to 100 % relative to other isomers but not with respect to other impurities or other compounds that may be present . as used herein , the term “ isomer ” refers to an e or z isomer , an r or s isomer , an enantiomer or a diastereomer . bh3i - 1 is useful for a variety of in vitro and in vivo uses . in one application of the present invention , a fungal cell is contacted with bh3i - 1 in an amount effective to reduce or inhibit fungal morphogenesis . it is intended that the fungal cell is contacted either in vitro or in situ , whereby in situ includes contacting a fungal cell in vivo or contacting a fungal cell on the surface of the skin . one of ordinary skill in the art would understand “ contacting ” to encompass putting a fungal cell into contact with bh3i - 1 , for example , in a culture plate or flask , whereby the fungal cell is placed into media containing bh3i - 1 . further “ contacting ” would be understood by one of ordinary skill in the art to mean adding bh3i - 1 to a fungal cell or population of fungal cells on the surface of the skin or parenterally or locally applying bh3i - 1 to a subject such that the fungus in the subject is exposed to bh3i - 1 . as used herein the term “ fungal cell ” is intended to encompass any cell originating from a fungal species or fungus . as used herein , the term “ fungus ” includes moulds , yeast and pathogenic yeast . a fungus includes , but is not limited to , candida ( for example , candida albicans , candida tropicalis , candida dubliniensis , candida parapsilosis , candida keyfyr , candida guilliermondii , candida inconspicua , candida famata , candida krusei , and candida lusitaniae ), aspergillus ( for example , aspergillus nidulans and aspergillus fumigatus ), saccharomyces ( for example , saccharomyces cerevisiae ), blastomyces ( for example , blastomyces dermatitidis ), coccidioides ( for example , coccidioides immitis ), histoplasma ( for example , histoplasma capsulatum ), and paracoccidioides ( for example , paracoccidioides brasiliensis ). as used herein , the terms “ treating ” and “ treat ” are intended to include preventing , ameliorating , curing , or reducing symptoms of fungal infections . as used herein , the term “ subject ” is any animal in need of treatment , including humans , primates and other mammals such as equines , cattle , swine , sheep , goats , primates , mice , rats , and pets in general , including , but not limited to , dogs , cats , guinea pigs , ferrets , and rabbits . the present invention can be used to treat fungal infections in subjects deemed medically of either having a fungal infection or being at significant risk of developing a fungal infection . a subject at risk of developing a fungal infection is a subject that has been exposed to a fungus or is susceptible to exposure to a fungus . for instance , subjects that are susceptible to exposure to a fungus includes those subjects who work in , live in or travel to areas with high fungal content or infectivity rates , as well as those subjects having particular susceptibility to fungal infection as a result of medical conditions or therapies . examples of subjects having particular susceptibility to fungal infections arising from medical conditions or therapies include , but are not limited to , an immunocompromised subject ( the compromised state of the subject &# 39 ; s immune system could be the result of an infectious disease such as aids , an inherited disorder , or a treatment protocol for , but not limited to , cancer , organ transplants , or infectious diseases ) or a subject having a central venous catheter . although the invention has been described in connection with specific embodiments and applications thereof the invention is capable of further modifications and / or applications , and this application is intended to cover any and all variations , uses , or adaptations of the invention that fall within the scope of the invention as described herein . the appended claims are therefore intended to cover all such variations , uses and adaptations as fall within the true spirit and scope of the invention . 1 . edmond , m . b ., s . e . wallace , d . k . mcclish , m . a . pfaller , r . n . jones and r . p . wenzel ( 1999 ). nosocomial bloodstream infections in united states hospitals : a three - 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