Patent Application
20090203754
The present invention relates generally to the field of screening for novel antimicrobial compounds.
Antibiotic resistant strains of bacteria as well as emerging bacterial pathogens pose a growing threat to world health, and the development of antibiotics necessary to counter these threats has dramatically slowed over the previous decades, leaving the population more vulnerable to these risks (Katz et al., 2006, Nat. Biotech. 24: 1529-1531; Payne et al., 2007, Nat. Rev. Drug Discovery 6: 29-40). Most newly developed antibiotics merely target one of the proven vulnerable pathways previously exploited by prior generations of antibiotics. While these drugs are effective, resistance to them is rising at an alarming rate, suggesting the need for antibiotics that target additional bacterial molecules or cellular processes.
Single-stranded DNA-binding proteins (SSBs) in bacteria form essential intermolecular complexes with at least a dozen other DNA replication, DNA recombination, and DNA repair proteins (Molineux and Gefter, 1975, J. Mol. Biol. 98: 811-825; Butland et al., 2005, Nature 433: 531-537). In addition, SSB proteins play important organizational roles through their interactions with many different genome maintenance proteins. Several, if not all, of these interactions are known to be mediated by the carboxy-terminal-most 8-10 residues from SSB, which form a peptide sequence that is highly conserved among bacterial SSBs but is not found in eukaryotic SSBs (Sandigursky et al., 1996, Radiation Res. 145: 619-623; Curth et al., 1996, Nucleic Acids Res. 24: 2706-2711; Genschel et al., 2000, Biol. Chem. 381: 183-192). Previous studies have shown that mutations within, or deletions of, this interaction sequence from SSB, have drastic effects on bacterial viability, indicating that formation of proper protein interactions with this site is critical for bacterial growth.
SSBs are a conserved protein family found throughout all forms of life, playing a variety of essential roles in nearly all aspects of DNA metabolism. SSBs bind and protect sensitive single-stranded DNA (ssDNA) intermediates that occur during DNA replication, recombination, and repair. SSBs recruit genome maintenance proteins to ssDNA, and play instrumental regulatory roles in ssDNA degradation, replication initiation, initiation of homologous recombination, relaxation of supercoiled DNA, and numerous other genome maintenance processes. Deletions of SSBs have been shown to be lethal and since all known SSBs play similar roles in cells, finding compounds that inhibit bacterial SSBs could have similar effects on eukaryotic cells.
It has been discovered that interfaces involving SSBs may be used as targets to generate promising new classes of antimicrobial compounds. Methods are provided that include reacting polypeptides comprising prokaryotic single-stranded DNA binding proteins, polypeptides that bind to the prokaryotic single-stranded DNA binding proteins, and candidate compounds, and assaying for binding of the polypeptides comprising the prokaryotic single-stranded DNA binding proteins to the polypeptides that bind to the prokaryotic single-stranded DNA binding proteins, where a decrease in binding, relative to the binding of the polypeptides comprising the prokaryotic single-stranded DNA binding proteins to the polypeptides that bind to the prokaryotic single-stranded DNA binding proteins in absence of the candidate compounds, identifies the candidate compounds as antimicrobial compounds. In the practice of the methods, the polypeptides comprising the prokaryotic single-stranded DNA binding proteins may include carboxy-tails that are at least 90% identical to the carboxy-tails of the polypeptides with an amino acid sequence of SEQ ID NO:2, or to the carboxy-tails of the polypeptides with an amino acid sequence of SEQ ID NO:2 having 1 to 4 conservative amino acid substitutions. The polypeptides comprising the prokaryotic single-stranded DNA binding proteins polypeptides may include carboxy-tails comprising the amino acid sequence Asp-Ile-Pro-Phe (SEQ ID NO:5), or the amino acid sequence Asp-Ile-Pro-Phe (SEQ ID NO:5) having 1 to 2 conservative amino acid substitutions.
In the practice of the methods, the polypeptides that bind to the prokaryotic single-stranded DNA binding proteins may include exonucleases, DNA polymerase subunits, primases, helicases, topoisomerases, DNA repair enzymes, etc. In one example, the polypeptides that bind to the prokaryotic single-stranded DNA binding proteins include Exonuclease I. The polypeptides that bind to the prokaryotic single-stranded DNA binding proteins may be at least 90% identical to the amino acid sequence of SEQ ID NO:4. The methods may include assaying that comprises measuring fluorescence polarization.
Methods of inhibiting the binding of SSB-binding proteins to the C-terminal tails of prokaryotic SSBs are provided. The methods include contacting the antimicrobial compounds identified as described above with the SSB-binding proteins and with the C-terminal tails of prokaryotic SSBs, where the antimicrobial compounds inhibit the binding of the prokaryotic SSBs to the SSB-binding proteins. The SSB-binding proteins may be exonucleases, and in some embodiments the SSB-binding proteins may be prokaryotic Exonuclease I.
Methods of inhibiting the growth of microorganisms are provided, which include contacting the antimicrobial compounds identified as described above with the microorganisms, thereby inhibiting the growth of the microorganisms.
Three-dimensional models of crystal structures of prokaryotic Exonucleases I bound to the C-terminal tails of prokaryotic SSBs are provided. In one embodiment, the models substantially represent the atomic coordinates specified in the Protein Data Bank under the accession code 3C94. Methods are provided, which include: a) identifying candidate compounds using three-dimensional models of crystal structures of Exonuclease I bound to C-terminal tails of prokaryotic single-stranded DNA binding proteins, where the models substantially represent the atomic coordinates specified in the Protein Data Bank under the accession code 3C94; b) contacting the candidate compounds with polypeptides comprising the prokaryotic single-stranded DNA binding proteins and with polypeptides that bind to the prokaryotic single-stranded DNA binding proteins; and c) assaying for binding of the polypeptides comprising the prokaryotic single-stranded DNA binding proteins to the polypeptides that bind to the prokaryotic single-stranded DNA binding proteins; where a decrease in binding, relative to the binding of the polypeptides comprising the prokaryotic single-stranded DNA binding proteins to the polypeptides that bind to the prokaryotic single-stranded DNA binding proteins in absence of the candidate compounds, identifies the candidate compounds as antimicrobial compounds. The contacting may be performed in solution. Alternatively, the contacting may be simulated in silico. The methods may also include the step (d) of administering the identified antimicrobial compounds to subjects to determine if the compounds reduce growth of microorganisms in the subjects. Compounds identified by these methods are provided. Antimicrobial pharmaceutical compositions that include as active agents the compounds identified by these methods are also provided.
Methods of inhibiting the binding of prokaryotic Exonucleases I to the C-terminal tails of prokaryotic SSBs are provided, which include: a) designing candidate compounds using three-dimensional models of crystal structures of the prokaryotic Exonucleases I bound to the C-terminal tails of the prokaryotic SSBs, where the models substantially represent the atomic coordinates specified in the Protein Data Bank under the accession code 3C94; and b) contacting the candidate compounds with the Exonucleases I and with the prokaryotic SSBs, where the candidate compounds inhibit the binding of the prokaryotic Exonucleases I to the prokaryotic SSBs. The contacting may be performed in solution. Alternatively, the contacting may be simulated in silico.
Three-dimensional models of crystal structures of prokaryotic Exonuclease I bound to compounds identified according to the present invention are provided. For example, the three-dimensional model of crystal structure of prokaryotic Exonuclease I bound to compound 9 is provided, where the models substantially represent the atomic coordinates specified in Table 6. As well, the three-dimensional models of crystal structure of prokaryotic Exonuclease I bound to compound 10 is provided, where the models substantially represent the atomic coordinates specified in Table 7. Methods are provided, which include: a) identifying candidate compounds using three-dimensional models of crystal structures of Exonuclease I bound to compound 9 or compound 10, where the models substantially represent the atomic coordinates specified in Tables 7 and 8, respectively; b) contacting the candidate compounds with polypeptides comprising the prokaryotic single-stranded DNA binding proteins and with polypeptides that bind to the prokaryotic single-stranded DNA binding proteins; and c) assaying for binding of the polypeptides comprising the prokaryotic single-stranded DNA binding proteins to the polypeptides that bind to the prokaryotic single-stranded DNA binding proteins; where a decrease in binding, relative to the binding of the polypeptides comprising the prokaryotic single-stranded DNA binding proteins to the polypeptides that bind to the prokaryotic single-stranded DNA binding proteins in absence of the candidate compounds, identifies the candidate compounds as antimicrobial compounds. The contacting may be performed in solution. Alternatively, the contacting may be simulated in silico. The methods may also include the step (d) of administering the identified antimicrobial compounds to subjects to determine if the compounds reduce growth of microorganisms in the subjects. Compounds identified by these methods are provided. Antimicrobial pharmaceutical compositions that include as active agents the compounds identified by these methods are also provided.
Methods of inhibiting the growth of microorganisms are provided, which include contacting the antimicrobial compounds identified as described herein with the microorganisms, thereby inhibiting the growth of the microorganisms.
Antimicrobial pharmaceutical compositions comprising as the active agent the antimicrobial compounds identified as described herein are provided. Also provided are methods of treating subjects that have microbial infections, which include the step of administering to the subjects therapeutically effective amounts of these novel pharmaceutical compositions.
Novel and selective targets for antibiotic development are provided. The compounds and assays of the present invention can be used for identification of molecules that selectively inhibit interaction between SSB and its target proteins. Such molecules may be used as novel, broad-spectrum antibiotics. Alternatively, such molecules may be chemically modified, i.e. they may be used as starting points for the development of novel, broad-spectrum antibiotics. The compositions of the present invention find use in methods to inhibit the growth of microorganisms. They find use as disinfectants. They find use as active agents that can be used in pharmaceutical compositions, and they also find use in methods of treating subjects with microbial infections. The term “subject” is intended to include patients, normal volunteers, non-human mammals such as primates, and also other animals.
In one aspect, the present invention relates to novel antimicrobial compounds and to methods of identifying same. An “antimicrobial” is a substance that kills or inhibits the growth of microbes such as bacteria (antibacterial activity), fungi (antifungal activity), viruses (antiviral activity), or parasites (anti-parasitic activity). “Antibiotic” is an antimicrobial substance that is generally used to treat bacterial infections.
The invention is particularly well-suited for the identification of antimicrobial compounds in the form of small molecules that can inhibit the growth of bacteria. In some examples, the bacteria whose growth is inhibited using the compounds of the present invention are Gram-negative bacteria, e.g. Escherichia coli. In other examples, the bacteria whose growth is inhibited using the compounds of the present invention are Gram-positive bacteria, e.g. Staphylococcus aureus. The present invention may be practiced with bacteria that are not necessarily classified as Gram-positive or Gram-negative, e.g. Deinococcus radiodurans and Mycobactedia such as Mycobacterium tuberculosis, and also with any other bacteria that have conserved C-terminal residues as described herein.
In one embodiment, the present invention provides for the identification of compounds that inhibit protein complex formation mediated by the single-stranded DNA binding protein's highly conserved C-terminal tail, which could lead to a novel class of chemotherapeutics. The term “SSB” refers to a single-stranded DNA-binding protein. In some embodiments of the present invention, the E. coli SSB has the nucleotide sequence of SEQ ID NO:1, and has the amino acid sequence of SEQ ID NO:2.
Prokaryotic SSBs of the present invention include polypeptides that are polymorphic variants, mutants, and interspecies homologs of SEQ ID NO:2. Prokaryotic SSBs of the present invention also include functional equivalents or fragments of SEQ ID NO:2. A “functional fragment” or “functional equivalent” or “functional homolog” of a polypeptide of the present invention is a polypeptide that is homologous to the specified polypeptide but has one or more amino acid differences from the specified polypeptide. A functional fragment or equivalent of a polypeptide retains at least some, if not all, of the activity of the specified polypeptide.
The term “genome maintenance” as used herein refers to the maintenance of the structure and integrity of a genome. Proteins such as the single-stranded DNA-binding protein (SSB) may be involved in genome maintenance.
“Carboxy tail” (also known as the carboxy-tail, carboxyl-terminus, carboxy-terminus, C-terminal end, C-terminus, or COOH-terminus) of a protein or polypeptide is the end of the amino acid chain terminated by a free carboxyl group (—COOH). For purposes of the present invention, the C-terminus of a protein encompasses approximately 10-20 amino acids of the C-terminus of a protein. The SSB's C-terminus is herein also referred to as SSB-Ct.
The canonical prokaryotic SSBs are homotetramers composed of four identical subunits, with each monomeric unit consisting of an oligonucleotide/oligosaccharide binding (OB)-fold followed by a disordered C-terminal tail. The final ten amino acids of the C-terminal tail are very highly conserved among bacteria, and are essential for viability in E. coli, yet they are notably absent from both eukaryotic and mitochondrial SSBs. The C-terminal tail of E. coli SSB is the primary interaction site between SSB and many, if not all, of its functional partners including Topoisomerase III, PriA DNA helicase, Chi subunit of DNA polymerase III, RecQ DNA helicase, and Exonuclease I. Removal of the C-terminal tail of SSB has little effect on SSB's DNA binding capabilities, but it is nonetheless lethal to E. coli.
Methods are provided for the identification of compounds that inhibit protein-protein interactions mediated by the C-terminal tail of SSB. The present invention also provides compounds that are identified using these methods, which compounds can inhibit protein-protein interactions mediated by the C-terminal tail of SSB. These compounds can serve as broad-spectrum antibiotics by disrupting the formation of genome maintenance protein complexes in prokaryotes, and in particular in bacteria. These compounds are not detrimental to eukaryotic genome maintenance complexes since eukaryotic RPA (Replication Protein A) does not contain a similar C-terminal tail, and likely uses a distinct method to recruit binding partners.
Alignment of SSB Tails from Different Prokaryotic Organisms
The alignment of the carboxy-tails of approximately 280 prokaryotic SSBs is shown in Table 1, which shows the conservation of the approximately ten amino acid residues that comprise the carboxy-tails of prokaryotic SSBs. In particular, the alignment of the C-terminal 20 residues of approximately 280 prokaryotic SSBs shows that the four extreme C-terminal residues (DIPF, i.e. Asp-Ile-Pro-Phe; also shown in SEQ ID NO:5) are the most highly conserved residues in the carboxy-tail of these prokaryotic SSBs (Table 1). These final C-terminal residues from the carboxy-tail of prokaryotic SSBs appear to be the most important for binding of proteins.
The present invention contemplates the use of polypeptides with carboxy-tails as shown in Table 1. The present invention contemplates the use of polypeptides with carboxy-tails homologous to the carboxy-tails shown in Table 1. For example, other useful polypeptides may have carboxy-tails as indicated in Table 1 with amino acid sequences comprising one or more conservative amino acid substitutions.
Desulfovibrio_desulfuricans--------FDDLGPAFPSEVSGMDDVPF
Desulfovibrio_vulgaris-------------DDDLGPAFPSEASGMDDVPF
Lawsonia_intracellularis----------GFEDISP-FPSEASGMDDVPF
Deinococcus_geothermalis-----------GLDIDQGLDDFPPEEEDLPF
Deinococcus_radiodurans------------GLDIDQGLDDFPPEEDDLPF
Thermus_thermophilus--------------GGVDIDEGLEDFPPEE-DLPF
Streptococcus_pneumoniae-----------ENNAGQDLADLVLEEEELPF
Caldicellulosiruptor_saccharol-----DIGTSSKLDLDENPEDDLPF
Streptococcus_pyogenes-------------NTSSLADSMPDYGPEPDLPF
Bacteroides_thetaiotaomicron-------QAQPSQAQPIQDNPADDLPF
Bacteroides_fragilis---------------SQQPQQPVSSQDNSADDLPF
Gramella_forsetii------------------NFANKNEFYSQDEEEDDLPF
Escherichia_coli_K12------------SAPAAPSNEPPMD---FDDDIPF
Shigella_dysenteriae_Sd197------SAPAAPSNEPPMD---FDDDIPF
Shigella_flexneri---------------SAPAAPSNEPPMD---FDDDIPF
Shigella_sonnei_Ss046-----------SAPAAPSNEPPMD---FDDDIPF
Shigella_boydii_Sb227-----------SAPAAPSNEPPMD---FDDDIPF
Haemophilus_influenzae-----------QQAAPQAEPPMDG--FDDDIPF
Pseudoalteromonas_atlantica-----QNKPAPMAEPDFD---FDDDIPF
Mannheimia_succiniciproducens----TRPAPAAEPAMDN--FDDDIPF
Pasteurella_mult-------------------PAPQNEPPMDMGFEBDNIPF
Salmonella_enterica-------------QSAPAPSNEPPMD---FDDDIPF
Salmonella_typhimurium----------QSAPAPSNEPPMD---FDDDIPF
Sodalis_glossinidius------------NSAPAPSNEPPMD---FDDDIPF
Erwinia_carotovora--------------NNAPAQSNEPPMD---FDDDIPF
Aeromonas_hydrophila------------QSAPPVYNEPPMD---FDDDIPF
Aeromonas_salmonicida-----------QSAPPVYNEPPMD---FDDDIPF
Vibrio_parahaemolyticus---------QQPQQQYNEPPMD---FDDDIPF
Photobacterium_profundum--------QQPQQQYNEPPMD---FDDDIPF
Vibrio_cholerae-----------------QYSQPQYNEPPMD---FDDDIPF
Vibrio_vulnificus---------------MQSQPQYNEPPMD---FDDDIPF
Vibrio_fischeri-----------------QAAQPQYNEPPMD---FDDDIPF
Psychromonas_ingrahamii---------QPTQTQYNEPSMD---FDDDIPF
Yersinia_enterocolitica---------AAQPQGGNEPPMD---FDDDIPF
Shewanella_oneidensis-------------QPQQNFTPDLDDG-WDDDIPF
Shewanella_loihica----------------QPQQNFTPDLDDG-WDDDIPF
Shewanella_putrefaciens-----------QPQQNFTPDLDDG-WDDDIPF
Shewanella_baltica----------------QPQQNFTPDLDDG-WDDDIPF
Shewanella_amazonensis------------PQQQNYTPDLDDG-WDDDIPF
Xanthomonas_campestris------------PAQQQSAPPMDDF-ADDDIPF
Xanthomonas_axonopodis------------PAQQQSAPPMDDF-ADDDIPF
Xanthomonas_oryzae----------------PAQQQSVPPMDDF-ADDDIPF
Xylella_fastidiosa----------------QSPQSSPPPMDDF-ADDDIPF
Hahella_chejuensis----------------QQPKPPMPEPMDD-FDDDIPF
Marinobacter_aquaeolei------------QQQGGGMPEPIDD-FDDDIPF
Alcanivorax_borkumensis---------TNQGGGFSGPADD---FDDDIPF
Magnetococcus_MC-1-------------FSSPADTFN-EGPD---FDDDIPF
Geobacter_metallireducens-------FGGGPAYDEPAFN---PDDDIPF
Geobacter_sulfurreducens-------GFGG-PSYDEPAFN---PDDDIPF
Pelobacter_carbinolicus---------QPQQNQYEEPPFN---PDDDIPF
Syntrophobacter_fumaroxidans----SRADELPPHPGGG---PDDDIPF
Leptospira_borgpetersenii-------SSSPESYNPPAPD---GDDDIPF
Buchnera_aphidicola----------------PKKIEKIDSSEIDFDDEIPF
Coxiella_burnetii------------------QTPTAGDDSSVADFDDDIPF
Photorhabdus_luminescens-----------SSVPPRGSEPPIDFDELIPF
Nitrosococcus_oceani-------------PRPSAPPSSSND--DFEDDIPF
Colwellia_psychrerythraea----------QAPKVNPQEPSIDFDDDIPF
Pseudoalteromonas_haloplanktis-----QGGASNPMEPTIDFDDDIPF
Wigglesworthia_glossinidia---------KESKKNKIEEEINFDDDIPF
Pseudomonas_entomophila------------QQPAPQPAADFDSFDDDIPF
Pseudomonas_putida-----------------QQPAPQPAADFDSFDDDIPF
Pseudomonas_syringae---------------QQQAPQPAADFDSFDDDIPF
Pseudomonas_fluorescens------------QQAAPQPAPDFDSFDDDIPF
Pseudomonas_aeruginosa-------------QQPAPQPAQDYDSFDDDIPF
Pseudomonas_stutzeri---------------PAARQQPAPDYDSEDDDIPF
Pseudomonas_mendocina--------------APQQAQPAPDYDSFDDDIPF
Saccharophagus_degradans-----------PAPAAPPAPDMDSFDDDIPF
Methylibium_petroleiphilum---------RQQARQPATAGDGFDDEIPF
Thiomicrospira_crunogena-----------PAQQVPAYTANDFDDDDVPF
Solibacter_usitatus---------------APAQHNDDFNQG-ITDDDVPF
Idiomarina_loihiensis-------------KPAEPAPFSPDNDF-DDDIPF
Burkholderia_383-------------------SRPSAPAGGGFDEMDDDIPF
Burkholderia_cepacia---------------SRPSAPAGGGFDEMDDDIPF
Burkholderia_pseudomallei----------SRPSAPAGGGFDEMDDDIPF
Burkholderia_mallei----------------SRPSAPAGGGFDEMDDDIPF
Burkholderia_cenocepacia-----------SRPSAPAGGGFDEMDDDIPF
Burkhoideria_xenovorans------------SRPSAPAGGGFDEMDDDIPF
Burkholderia_vietnamiensis---------SRPSAPAGGGFDEMDDDIPF
Acidovorax_avenae------------------AAQAPRAASGFDDMDDDIPF
Verminephrobacter_eiseniae---------AAPGPRAASGFDDMDDDIPF
Polaromonas_JS666------------------APAPTKAASGFDDMDDDIPF
Rhodoferax_ferrireducens-----------APAQAKPSSGFDDMDDDIPF
Thiobacillus_denitrificans---------AGSQRPASSGFDDMDDDIPF
Polaromonas_naphthalenivorans------PAASRASPSGFDDMDDDIPF
Nitrosomonas_eutropha--------------KTGTTGSSTGFDDMEDDIPF
Nitrosomonas_europaea--------------STPPAKSNTGFDDMEDDIPF
Nitrosospira_multiformis-----------GRAPARSSTGFDDMDDDIPF
Azoarcus_EbN1----------------------KAPTKSSGAGFGDFDDDIPF
Methylobacillus_flagellatus--------AASKPAGGSNFDDFEDDIPF
Rhodospirillum_rubrum-------------TAPASGPAGGP-VDMDDDIPF
Ralstonia_eutropha----------------RRQQQAPSNGF-EDMDDDIPF
Ralstonia_solanacearum------------ARRQQAPSNGF-EDMDDDIPF
Agrobacterium_tumefaciens----------RGGGQPSGGFSNDMDDDIPF
Neisseria_gonorrhoeae--------------RRQPVPAAAPVEDIDDDIPF
Neisseria_meningitidis-------------RRQPVPAAAPVEDIDDDIPF
Herminiimonas_arsenicoxydans-------RPAAKPAASNFNDMDDDIPF
Nitrobacter_hamburgensis-----------PRRTVAAGARRSDMDDDIPF
Nitrobacter_winogradskyi-----------PRRAAPASSHRGDMDDDIPF
Bordetella_parapertussis-----------PAPQAAPAANLADMDDDIPF
Bordetella_pertussis---------------PAPQAAPAANLADMDDDIPF
Bordetelia_bronchiseptica----------PAPQAAPAANLADMDDDIPF
Polynucleobacter_QLW-P1DMWA-1------SAPSASNAASLGAMDDDIPF
Acidobacteria_bacterium------------NDFDSAPAASTGITDDDIPF
Candidatus_pelagibacter-----------ANNFEDSPQTSN-DMDDEIPF
Psychrobacter_arcticus-------------PAQSKPTAMLDGPVDDDIPF
Psychrobacter_cryohalolentis-------PAQSKPTAMPDGPVDDDIPF
Haemophilus_ducreyi----------------NKSSKKSTTQQPEVDDDIPF
Dechloromonas_aromatica------------APPKNKPKPSFDDLGDDIPF
Neorickettsia_sennetsu-----------DLGTPTNHVNDTLDD--DDIPF
Baumannia_cicadellinicola---------NKILQDMGNEQPIEFDDEIPF
Bdellovibrio_bacteriovorus---------FNFQDFGPEPSFNSNDEIPF
Chromohalobacter_salexigens--------DNYGAPNPGNFDDFDDEIPF
Dichelobacter_nodosus------------SSPDYGP--DGAFDDPDDEIPF
Aquifex_aeolicus-------------------EKLGKEEEKPFTDEEDEIPF
Hyphomonas_neptunium--------------QQMSGPKESFS-QDLDDEIPF
Caulobacter_crescentus------------SQPSGPRESES-ADLDDEIPF
Maricaulis_mans--------------------SMDGPKEDFRNADLDDEIPF
Rickettsia_felis-------------------HPEAKNHSFDHSDLDDEIPF
Rickettsia_conorii-----------------HPETKNHSFDHSDLDDEIPF
Rickettsia_typhi-------------------YPEIKNHSFDHSDLDDEIPF
Rickettsia_prowazekii--------------YPETKNHSFDHSDLDDEIPF
Rickettsia_bellii------------------EYKHSKPSFDHSDLDDEIPF
Halorhodospira_halophila-----------GSGGGGMQEAPADFDDDIPF
Paracoccus_denitrificans-----------SGGGGGQSQSRPDFDDDIPF
Rhodopseudomonas_palustris---------RPMPASSGGGRSDMDDDIPF
Bradyrhizobium_japonicum-----------RAVAAGGGGRNSDMDDDIPF
Granulibacter_bethesdensis--------SGGSGWEPSHGG-DLDDEIPF
Rhodobacter_sphaeroides-----------RGNAPSGCGRRS-DLDDEIPF
Silicibacter_pomeroyi-------------GGGRGRGPASGG-IDDDEIPF
Roseobacter_denitrificans---------GGGNAPSPAPSR-DLDDEIPF
Magnetospirillum_magneticum-------GGGCGQSWEPPA-DLDDEIPF
Mesorhizobium_BNC1----------------PAESGGGGGHSR-DLDDEIPF
Mesorhizobium_loti----------------APRGGGGGGSSR-ELDDEIPF
Brucella_melitensis---------------GPSSGSSGGFSR-DLDDEIPF
Brucella_suis---------------------GPSSGSSGGFSR-DLDDEIPF
Brucella_abortus------------------GPSSGSSGGFSR-DLDDEIPF
Jannaschia_CCS1--------------------SGGGYGGGGGSSDLDDEIPF
Silicibacter_TM1040---------------NRGGGYGSGSQSID-DDEIPF
Novosphingobium_aromaticivoran----NQGGGSGGGFGD-DLDDDIPF
Myxococcus_xanthus-------------PPDDMGGGHGGGNGD----DDIPF
Erythrobacter_litoralis-----------GGSGGGGGSNYD-DLDDDIPF
Anaeromyxobacter_dehalogenans-----GPGFGSGGGAGG-GGPDDIPF
Desulfotomaculum_reducens--------SGSGSGFGSEISF-NG-DDIPF
Sphingopyxis_alaskensis-----------GAPGGGR-PPFDDDLDDDVPF
Pelobacter_propionicus----------AGTSGGGYEPPPFQD---DDIPF
Gluconobacter_oxydans--------------GSNGGWDAPPDNDLDDEIPF
Bartonella_quintana----------------QNNSQSEESFSHKLDDDVPF
Bartonella_henselae----------------SNNSQLGESFSHKLDDDVPF
Bartonella_bacilliformis-----------QNNNQSGGSFSHQLDDDVPF
Rhizobium_etli---------------------SSNRGGGGNFSRDLDDDIPF
Rhizobium_leguminosarum------------SRGGGGGGNFSRDLDDDIPF
Sinorhizobium_meliloti-------------SNQPNQGGNFSRDLDDDIPF
Desulfitobacteriurn_hafniense------HTASGEAYGHEMSLDDDIPF
Shewanella_denitrificans-----------KSHLVDSTSKIDFDDEDIPF
Candidatus_blochmannia------------NNHELNSESILVNFNEDDIPF
Wolbachia_endosyinbiont------------QYENFDSEVKEELIDDEIPF
Tropheryma_whipplei----------------KVLVGDNVSYEDFDSDEVPF
Ehrlichia_chaffeensis--------------KENSLNSSCDDIIIDDEIPF
Ehrlichia_canis--------------------KENFQDSSCDDIIIDDEIPF
Ehrlichia_ruminantium--------------NKMPFQNSCEDVIIDDEIPF
Helicobacter_acinonychis-----------PSKYQNSVPEINIDEEEIPF
Helicobacter_pylori----------------PSKYQNSVPEINIDEEEIPF
Thiomicrospira_denitrificans-------QMPSNSSIPEIDIDEDEIPF
Wolinella_succinogenes-------------APYKEPQIPEINIDDDEIPF
Helicobacter_hepaticus-------------TGNYPQNIPEINIDDEDIPF
Campylobacter_fetus----------------RQNKPKQNIDVNIDDEEIPF
Dehalococcoides_ethenogenes-----MDARDDDNGGGELEP---EDIPF
Dehalococcoides_CBDB1-----------IDAREDDNGGGELEP---EDIPF
Symbiobacterium_thermophilum-------RREDGMGSELTLGDDEDVPF
Clostridium_novyi------------------IFDQGYDEEITPIDDGDIPF
Clostridium_acetobutylicum---------DFGVPVQEDITPVDNSDIPF
Clostridium_perfringens------------DSSFNSNDDMTPIDDGDIPF
Clostridium_tetani-----------------NNEDNYNDDITPVDEGEVPF
Borrelia_afzelii-------------------EDVIKDIDIVDDKFNEDIPF
Borrelia_burgdorferi---------------EDVVKDIDIVDDKFSEDIPF
Borrelia_garinii-------------------EDAIKNIDIVDDKFNEDIPF
Syntrophomonas_wolfei--------------EVNMDNIDLVDQHEDEDIPF
Anapiasma_marginale----------------ENAIVEEVSFADEDMDEIPF
Anaplasma_phagocytophilum----------AGSFGGGVDFLDPDVDEIPF
Treponema_pallidum-----------------ATSSLDEADFSSSDLDTVPF
Rubrobacter_xylanophilus-----------GRGAGDEVDINESDFDDIPF
Treponema_denticola----------------PSYDDYQPDMGNSDLDNIPF
Mycoplasma_penetrans---------------DDEDPDQVVSNLDWLDEFKF
Candidatus_ruthia------------------PVLDPIAPVDNSEFDDDIPF
Acidovorax_JS42--------------------PVLDPIAPVDNSEFDDDIPF
Francisella_tularensis-------------DNMPDFAEINSSNFDDDIPF
Porphyromonas_gingivalis-----------SSVRDTAKEESSEPPSDLPF
Acinetobacter_ADP1---------------YVPKAPQQP--APADLDDDLPF
Acinetobacter_baumannii------------PKAPQQPASAPADLDDDLPF
Synechococcus_elongates------------GSRRDQEGGMAPRDPDSDLF
Clostridium_thermocellum----------EPENTDGEGFFPAE-DDELPF
Clostridium_difficile-------------EPQGLDPQGFQAID-DDDIPF
Thermoanaerobacter_tengcongens-----DIPDDFDGFTPIESEDDLPF
Streptococcus_sanguinis----------DESPFGNSN--PMDISDDDLPF
Streptococcus_thermophilus------DESPFGNSN--PMDISDDDLPF
Streptococcus_agalactiae---------DESPFGNSN--PMDISDDDLPF
Streptococcus_mutans-------------DESPFGDSN--PMDISDDDLPF
Streptococcus_suis---------------EESPFGASN--PMDISDDDLPF
Lactococcus_lactis--------------QNNDSFG-SD--PMEISDDDLPF
Zymomonas_inobilis---------------SSNTNHDPF--GMDDLDDDVPF
Oenococcus_oeni------------------PFNTDTGND--SLDISDDDLPF
Carboxydothermus_hydrogenoform----DFDPSDFG-TEIEISDEDIPF
Moorella_therinoacetica-----------NQDFSDLG-TEVEIGEDDLPF
Geobacillus_kaustophilus----------DDPFANDG-QPIDISDDDLPF
Geobacillus_thermodenitrifican----EDPFANDG-QPIDISDDDLPF
Bacillus_licheniformis------------DDPFANDG-KPIDISDDDLPF
Bacillus_subtilis-----------------DDPFANDG-KPIDISDDDLPF
Bacillus_halodurans---------------EDPFANDG-KPIDISDDDLPF
Listeria_welshimeri---------------NDPFASDG-KPIDISDDDLPF
Lactobacillus_casei---------------PDPFANNG-KPIDISDDDLPF
Oceanobacillus_iheyensis----------EDPFKNNG-EPIDISDDDLPF
Bacillus_thuringiensis------------DDPFSNVG-QPIDISDDDLPF
Bacillus_anthracis----------------DDPFSNVG-QPIDISDDDLPF
Bacillus_cereus-------------------DDPFSNVG-QPIDISDDDLPF
Bacillus_clausii-----------------DNDPFSNDG--SIDISDDDLPF
Pediococcus_pentosaceus-----------NDPFANSG-QSIDISDDDLPF
Lactobacillus_plantarum-----------ADPFANNG-DQIDISDDDLPF
Lactobacillus_brevis--------------ADPFANSG-DSIDISDDDLPF
Lactobacillus_sakei---------------ADPFANNG-QAIDISDDDLPF
Lactobacillus_salivarius----------ADPFADNG-QSIDISDDDLPF
Leuconostoc_mesenteroides----------NPFAASGNTEIDISDDDLPF
Lactobacillus_gasseri-------------QDPFADSG-STIDISDDDLPF
Enterococcus_faecalis-------------SDPFGGSG-SSIDISDDDLPF
Staphylococcus_haemolyticus-------DNPFANAN-GPIDISDDDLPF
Staphylococcus_saprophyticus------DNPFANAN-GPIDISDDDLPF
Staphylococcus_epidermidis--------DNPFANAN-GPIDISDDDLPF
Listeria_innocua------------------SDSFANEG-KPIDINPDDLPF
Listeria_monocytogenes------------SDSFASEG-KPIDINEDDLPF
Prosthecochloris_vibrioformis------TSQPPSGPMIENNDKDDLPF
Pelodictyon_luteolum--------------PPQTAPSAPMIEN-DKDDLPF
Chlorobium_phaeobacteroides-------DYPQQSSGPMIES-EKDDLPF
Chlorobium_tepidum-----------------YGASPSSGGAQEFEKDDLPF
Chlorobium_chlorochromatii---------PPATPTVPTMIDTDKDDLPF
Staphylococcus_aureus--------------TQTGNNPFDNTEEDFSDLPF
Lactobacillus_delbrueckii----------TNPFDSSDDAINVSNDDLPF
Salinibacter_rubber----------------GGDGQPGSDETFEPDDDLPF
Cytophaga_hutchinsonii-------------AMESAGSFEPQTSGADDLPF
Methylococcus_capsulatus-----------GGSGAGSSQFDEGFDDDVPF
Alkalilimnicola_ehrlichei----------GGRQDNMGDDAGAFEDDIPF
Rhodopirellula_baltica-------------SSDSQPTGDGPGYDEPDIPF
Synechococcus_WH_8102-------------DNQEAAGSFG-GQASDEEIPF
Synechococcus_CC9902--------------DNQESGGNFG-GQASDEDIPF
Synechococcus_CC9311---------------SEAGSGGFGGGSPSDEEVPF
Prochlorococcus_marinus-----------ASNFGGGGFGDG-PSBEEVPF
Arthrobacter_aurescens-----------NPSANAGSSWGNS-P-DSEPPF
Arthrobacter_FB24----------------PGVSNAGGGWGNG-P-DSEPPF
Mycobacterium_tuberculosis------PWGSAPASGSFGGG---DDEPPF
Mycobacterium_avium-------------PWGSAPASGSFGGG---DDEPPF
Mycobacterium_bovis-------------PWGSAPASGSFGGG---DDEPPF
Mycobacterium_vanbaalenii-------PWGSAPASGSFGGA---DDEPPF
Mycobacterium_gilvum------------PWGSAPASGSFGGA---DDEPPF
Mycobacterium_smegmatis---------PWGSAPASGSFSGA---DDEPPF
Nocardia_farcinica----------------GSAPAAGSFGGGRM-DDEPPF
Mycobacterium_ulcerans---------DPWGSAPASGSFG-G---DDEPPF
Mycobacterium_leprae------------PWGSAPTSGSEGVG---DEEPPF
Corynebacterium_jeikeium-------DPWNSAPQSG-FGDG---DDEPPF
Corynebacterium_diphtheriae-----PWSSAPQAGGFGGA---EQDPPF
Salinispora_tropica---------------APAPSRGGSGGGNF-DEEPPF
Corynebacterium_efficiens---------NSAPPAGSGGFGGA-DDEPPF
Corynebacterium_glutamicum--------NSAPPAGSGGFGGA-DDEPPF
Rhodococcus_RHA1----------------APQASGSFGGSGGG---SDEPPF
Streptomyces_avermitilis---------GSSGGSSGGSGGG-Y-SDEPPF
Thermobifida_fusca--------------WATGGGGFGG-GGG-Y-SDEPPF
Frankia_CcI3---------------------APIDDPWSQPAGG--YSDEPPF
Nocardioides_JS614--------------SAPANDPWGAPGVG---SDEPPF
Acidothermus_cellulolyticus--------AEDPWAAGSATGNFSDEPPF
Propionibacterium_acnes-------ANRGGGVDPWASAQT-----DEPPF
Leifsonia_xyli------------------PSAGTDVWNTPGAYN---DSTPF
Anabaena_variabilis----------------EESTSTSLPNETQAVANANF
Nostoc_PCC_7120--------------------EESTSTSAPNETQAVANANF
Synechocystis_---------------------LLGSKRDNAEATMNNYPEEF
Trichodesinium_erythraeum----------ELLGSKRDSEQAALASYNEF
Flavobacterium_johnsoniae----------AKNTNFDAPSEGLPINDLPF
Thermotoga_maritime----------------LEIPEEDFSSDTFSEDEPPF
Syntrophus_aciditrophicus----------EGHFSPFNDLPPLPEDDVPF
Leptospira_interrogans-------------VVGQMIRFDGLPGKKEREVA
Desulfotalea_psychrophila----------SFPEPTGPDAYGGTGNDVPF
Campylobacter_jejuni---------------EKLKEIDIDAYDSDDTNLPF
Thermosynechococcus_elongates-------LSSKRDTDPNAVPAGYVPEI
Shewanella_W3-18-1-----------------PADDASSQANWAQTYPEPDF
Gloeobacter_violaceus--------------KVDQLELLGRAARPDEPESF
Shewanella_ANA-3---------------------AQPQGGHQQNTQQQAYNYHR
In one aspect, the present invention relates to the determination of the previously unpublished high-resolution structure of the E. coli SSB bound to a target protein, Exonuclease I (Exo I) from Escherichia coli. The E. coli Exonuclease I was described by Lehman, 1960, J. Biol. Chem. 235: 1479-1487. Exonucleases, which can be found as individual enzymes, or as parts of larger enzyme complexes, are enzymes that cleave nucleotides one at a time from an end of a polynucleotide chain. The E. coli Exonuclease I has the nucleotide sequence of SEQ ID NO:3, and it has the amino acid sequence of SEQ ID NO:4. The atomic coordinates of the crystal structure of the E. coli Exonuclease I have been deposited in the Protein Data Bank under the accession code 3C95. The present invention contemplates the use of other prokaryotic exonucleases, and in particular the use of other prokaryotic homologs of the E. coli Exonuclease I.
Prokaryotic exonucleases of the present invention include polypeptides that are polymorphic variants, mutants, and interspecies homologs of SEQ ID NO:4. Prokaryotic exonucleases of the present invention also include functional equivalents or fragments of SEQ ID NO:4.
Methods are provided for assaying binding of a prokaryotic SSB to a “SSB-binding protein” (also referred to herein as a “target protein”). An SSB-binding protein is a protein that binds to SSB. The SSB may be a prokaryotic SSB, for example the SSB may be an E. coli SSB. The SSB-binding protein may be a prokaryotic SSB-binding protein, for example the SSB-binding protein may be Exonuclease. The Exonuclease may be prokaryotic Exonuclease, and in particular it may be Exonuclease I. The Exonuclease I may be an E. coli Exonuclease I.
Also provided are rapid fluorescence polarization methods for measuring the binding of SSB to Exonuclease I in solution. Fluorescence polarization (FP) is a technique that can measure the binding of a tagged molecule to a target molecule using polarized light and a fluorescent tracer. When excited with polarized light, tracers attached to molecules emit high levels of polarized fluorescence. Tracers attached to larger molecules are slower in rotations compared to tracers attached to smaller molecules. For example, fluorescein-labeled peptides can be used to detect specific binding of the peptides to a variety of molecules, since the fluorescein-labeled peptides are depolarized more slowly when bound to targets due to their slower tumbling rates relative to their free tumbling rates. In chemistry, fluorescence anisotropy assays the rotational diffusion of a molecule from the decorrelation of polarization in fluorescence, i.e., between the exciting and emitted (fluorescent) photons. From the rotational diffusion constants, one can estimate the shape of a macromolecule and use that information to design other molecules that may be drug candidates.
Given (1) the importance of protein interactions with SSBs, (2) the broad conservation of SSB protein-binding sequences across bacterial species, and (3) the apparent absence of SSB protein-binding sequences from eukaryotic (human) systems, these assays can be specific for the identification of compounds that selectively inhibit prokaryotic growth. Small molecules that selectively inhibit interaction between SSB and its target proteins are thus attractive, novel, broad-spectrum antibiotics. The present invention contemplates the use of the methods described herein for a high-throughput screen for small-molecule inhibitors of this interaction. Such inhibitors can serve as lead compounds for future biochemical and anti-microbial studies.
The methods of the present invention may be practiced in solution, i.e., in vitro. Thus, by conducting in vitro assays, it is possible to identify novel antimicrobial compounds according to the present invention. In another example, the methods of the present invention may be practiced in silico, i.e. through simulations using computer programs. Thus, using information on the crystal structures of the present invention, it is possible to design and/or to identify structures of novel antimicrobial compounds that can then be chemically synthesized using methods known in the art.
Examples of libraries of compounds that can be screened used in the practice of this invention include a variety of screening libraries that can be obtained from Maybridge (e.g., the HitFinder collection of 14,400 compounds); the 16,000 compound DIVERSet library from ChemBridge; the Chemdiversity library, Chemical Diversity Labs, Inc., etc. Any other candidate compounds can be used in the assays according to this invention. Candidate compounds do not have to be obtained from libraries in order to be screened. Candidate compounds can be synthesized using methods known in the art, and can then be used in the assays of the present invention. For example, candidate compounds can be designed in silico, using data on the crystal structures of the interacting proteins (e.g. SSB and Exonuclease). Once candidate compounds are designed in silico, these candidate compounds can be synthesized using known methods of synthetic chemistry, and can then be tested for their efficacy to inhibit or influence the interaction between the interacting proteins (e.g. SSB and Exonuclease).
In one example, the identification of compounds that inhibit SSB C-terminal tail mediated protein/protein interactions is assayed using a high throughput fluorescence polarization (FP) based screen. The inventors used an FP screen to identify compounds that inhibit binding of ExoI to the C-terminal tail of E. coli SSB. The use of ExoI-SSB C-terminal tail binding as a reporter for the high throughput screen has several advantages. First, binding between ExoI and SSB's C-terminal tail is quite strong in comparison to other known binding events allowing determination of an accurate Kd using FP and allowing screening of significant populations of ExoI-SSB-Ct complexes since complex formation can be saturated. Second, FP is very amenable to a high throughput platform, thus allowing for screening large numbers of compounds quickly. Third, the inventors' recent discovery of the crystal structures of Exonuclease I bound to the C-terminal tail of SSB has provided the first known high-resolution picture of the SSB C-terminal tail bound to a functional partner. The crystal structure suggests that the final amino acids of the C-terminal tail play vital roles in binding. These findings are supported by FP data indicating that mutation of the penultimate Pro residue of SSB to Ser eliminates the ability of the ExoI to bind the C-terminal tail of SSB. Not wanting to be bound by the following theory, it is possible that, given the extremely high conservation between the final four amino acids of SSB among disparate bacterial SSB homologs, this mode of protein binding may be a conserved mechanism of recruitment for proteins vital for genome maintenance.
By finding small molecules that inhibit these binding events, it is possible to disrupt the SSB-Exonuclease I interactions thus slowing bacterial growth or killing the bacteria. Furthermore, small molecules that inhibit binding may also inhibit binding between SSB C-terminal tail and other binding partners.
In one example, high throughput screens could be done looking for molecules that disrupt the interaction between SSB's C-terminal tail and other known SSB binding partners. For example, over a dozen SSB-interacting proteins have been already identified in E. coli (Butland et al., 2005, Nature 433: 531-537). Many of these interactions are mediated by contacts between the C-terminal tail of SSB and its heterotypic binding partners, e.g. ExoI (Sandigursky et al., 1996, Radiation Research 145: 619-623; Genschel et al., 2000, Biol. Chem. 381: 183-192), the χψ subunit of DNA polymerase III (Yuzhakov et al., 1999, Cell 96: 153-163; Witte et al., 2003, Nucleic Acids Res. 31: 4434-4440), uracil DNA glycosylase (Hanada et al., 2001, J. Biol. Chem. 276: 16992-16997), PriA DNA helicase (Cadman and McGlynn, 2004, Nucleic Acids Res. 32:6378-6387), and RecQ DNA helicase (Shereda et al., 2007, J. Biol. Chem. 282: 19247-19258). Since bacterial SSB proteins are predominantly homotetrameric (with four C-termini that can act as protein binding sites) and multiple SSB tetramers can bind to a single extended ssDNA region (Lohman and Ferrari, 1994, Annu. Rev. Biochem. 66: 527-570), utilization of SSB as a common target for many genome maintenance enzymes fosters efficient targeting and coordination of several DNA metabolic activities.
Order of Addition of Assay Components when Performing the Screen for Antimicrobial Compounds
The present invention contemplates variations of the screens for antimicrobial compounds described herein. As shown below, it is possible to screen libraries of compounds, to identify a number of hits that as candidate antimicrobial compounds. These candidate compounds can be mapped to a peptide-binding site in a crystal structure that represents the C-terminus of SSB bound to a SSB-binding protein (e.g. ExoI). In one example, it is possible to make a screen using a solution of fluorescently labeled C-terminal SSB peptide prebound to ExoI and titrate in compounds. Alternatively, it would be possible to conduct a screen by adding Exonuclease I (e.g. to 384 well plates), and then adding a single (e.g. different) candidate compound to each well. This would allow the compound some time to bind to the binding pocket is of ExoI. Fluorescently labeled peptide of the present invention can then be added. This fluorescently labeled peptide would compete for any compounds that were bound to the pocket/s. Not wanting to be bound by the following theory, it is possible that the latter would be a more physiologically relevant screen, since a drug in vivo would likely see its target before the target saw the SSB tail. While in the examples below the screen was conducted using ExoI and the peptide tail, there are numerous variations on this theme that could potentially be useful. The screen can also be performed using other targets from E. coli, e.g. Topoisomerase III, the Chi subunit of DNA polymerase, Gyrase, mixtures of these or other targets, etc. Furthermore, this screen could be done using proteins from another bacterium, including but not limited to Staphylococcus, Bacillus, Enterococcus, etc. These assays can be done using the entire protein or the C-terminal tail from the species that is being tested.
Computer modeling and docking procedures can be used to examine and improve the hits from small molecule screening libraries by analyzing the docking modes of known small molecules in the binding pocket. Docking starts with a crystal structure of the small molecule in the receptor pocket. It may happen that an initial hit from a database may not be an optimal structure for a drug. This can be tested by modeling the hit in the active site, then docking other molecules. A binding energy is calculated and compared to the known hits. Conformational fit is also an important part of the docking results and is usually examined by modeling.
After the initial crystal structure is determined for a bound small molecule, computational screening and docking programs such as AutoDock (Scripps research Institute, La Jolla, Calif.), FlexX (BioSolveIT GmbH, Sankt Augustin, Germany), and SLIDE (Michigan State University, East Lansing, Mich.) can be used to screen thousands of compounds from known databases. In this example, each molecule is allowed conformational flexibility to fit in the receptor site and scored on binding ability. In most cases, the receptor is also allowed flexibility. The top compounds from the databases are scored and ranked and the binding modes and binding energies are compared to actual hits from the small molecule assays. This ranks the reliability of both the chemical assay and the docking modes. This procedure leads to a group of molecules that can serve as starting molecules for optimizing the binding pocket with atom by atom de novo or by similar drug binding computer programs.
The methods described herein are amenable to this type of procedure. Indeed, the inventors of the present application have solved a number of crystal structures in which inhibitors are bound to the binding pocket. Using these tools, it is possible to search in silico (i.e., using computer programs) for molecules that bind as well or perhaps even better than the ones that have already been identified herein. In essence, appropriate computer software might be used to help identify the structure of a compound that will optimally inhibit peptide binding. The advantage of using the in silico techniques is the rapidity with which the potential candidate compounds can be screened, since this is a much faster way than doing thousands of binding assays in solution, i.e. in vitro.
Pharmaceutical Compositions that Include Identified Compounds
Pharmaceutical compositions are also provided, which include as active agents the antimicrobial compounds that are identified according to the present invention. The pharmaceutical compositions may be liquid or solid. The liquid composition may, for example, be an aqueous solution comprising one or more antimicrobial compounds. The solid composition may, for example, be a solid preparation obtainable by freeze-drying or spray-drying the above aqueous solution. In one embodiment the solid preparation is a lyophilizate. The aqueous preparation mentioned above includes an aqueous solution of such solid preparation. Still, the above solid composition may be in the form of a kit wherein the solid preparation and an infusion are formulated into separate preparations.
An example of the technology for producing the pharmaceutical compositions of this invention is described below. An aqueous solution can be produced by dissolving the antimicrobial compound in a conventional manner, in a solvent of choice. This aqueous solution may be alkaline, neutral, or basic; it is just sufficient that the antimicrobial compound is dissolved in the aqueous solution. The concentration of the antimicrobial compound in such an aqueous solution may for example be at concentrations consistent with measured minimal inhibitory concentrations (Table 3). In some embodiments, the concentration is chosen so that it permits successful lyophilization in a subsequent procedure. Referring to the manufacture of a solid pharmaceutical composition, a lyophilizate, for instance, can be produced by freeze-drying an aqueous solution that includes one or more antimicrobial compounds of the present invention. An exemplary procedure comprises freezing the aqueous solution at about −25° C. and, with the internal negative pressure of the freeze-dryer being maintained at about 0.1 Torr or less, increasing the plate temperature at a rate of about 5° C. to 20° C./hour to an ultimate temperature of about 25° C. to 40° C. Where lyophilization is carried out, a form regulator may be added to an aqueous solution of the antimicrobial compound for the purpose of improving the morphology of the lyophilizate. The form regulator may include various sugars (e.g. sugar alcohols such as mannitol, xylitol, inositol, sorbitol, etc., hexose-based disaccharides such as maltose, sucrose, lactose, etc., and monosaccharides such as glucose), neutral amino acids (e.g. glycine, alanine, proline, valine, methionine, etc.) and alkali metal salts of succinic acid (e.g. sodium succinate, etc.). Where a spray-dried preparation is the desired product, the aqueous solution described above is spray-dried by a per se known technique. An exemplary procedure comprises ejecting the aqueous solution in mist form from a spray dryer nozzle (e.g. a twin nozzle, a pressure nozzle, etc.) or rotary disk into its drying chamber at a flow rate of about 5-20 ml/minute (e.g. drying chamber inlet and out temperatures: about 80° C. to 120° C. and about 30° C. to 50° C., respectively; air flow rate about 70-100 kg/hour).
For insuring a further increase in drug absorption, a surfactant can be used concomitantly in the pharmaceutical compositions of this invention. Examples of suitable surfactants include nonionic surfactants such as sorbitan fatty acid esters (e.g. sorbitan monopalmitate, sorbitan sesquistearate, etc.), glycerin fatty acid esters (e.g. glyceryl monostearate, etc.), propylene glycol fatty acid esters (e.g. propylene glycol monostearate), polyoxyethylene glycerin fatty acid esters (e.g. polyoxyethylene glyceryl monostearate, etc.), polyethylene glycol fatty acid esters (e.g. polyoxyethylene monostearate, PEG distearate, etc.), polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, etc.), polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitol beeswax derivatives, polyoxyethylene lanolin alcohol, polyoxyethylene sorbitol fatty acid esters, Pluronic series surfactants, anionic surfactants such as alkali metal dodecyl sulfates, alkali metal stearates, alkali metal palmitates, and liquid surfactants such as Tween 20 and Tween 80, among others. These surfactants can be used singly or plurally in a suitable ratio.
For improving the solubility or stability of the antimicrobial compound, a variety of salts (e.g. salts of organic acids such as sodium citrate, sodium tartrate, sodium benzoate, etc.) and/or stabilizers (e.g. basic inorganic salts such as magnesium carbonate, calcium carbonate, magnesium hydrogen carbonate, calcium hydrogen carbonate, etc.) may be incorporated or added to the compositions of this invention. If necessary, an isotonizing agent (e.g. sodium chloride) for osmotic pressure adjustment and/or a soothing or local anesthetic agent (e.g. glucose, sorbitol, mannitol, benzyl alcohol, mepivacaine hydrochloride, xylocaine hydrochloride, etc.) can also be employed.
A preservative and a pH control agent can be added in small amounts as required. The preservative includes parabens such as methyl p-hydroxybenzoate, propyl p-hydroxybenzoates, etc., alcohols such as chlorobutanol, quaternary ammonium salts such as benzalkonium chloride, benzethonium chloride, cetrimide, etc., sorbic acid, chlorhexidines, thimerosal and so on. The pH control agent includes various acids, e.g. inorganic acids such as hydrochloric acid, boric acid, phosphoric acid, carbonic acid, hydrogen carbonic acid, etc., organic acids such as mono- or polycarboxylic acids, amino acids, etc., and various bases, e.g. alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, etc. and alkali metal (hydrogen) carbonates such as sodium hydrogen carbonate, sodium carbonate and so on. These additives can be used alone or in combination and can be added in a proportion of about 0.001-10 mg, preferably about 0.01-5 mg, per milligram of the antimicrobial compound.
The pharmaceutical compositions of this invention are generally administered orally or parenterally in a dosage form manufactured by formulating such an active ingredient with a pharmacologically acceptable carrier or excipient. The pharmaceutical composition of this invention can be put to use in the following manner. Taking a solid composition as an example, it can be extemporaneously dissolved in sterile distilled water or an infusion fluid (e.g. physiological saline, glucose infusion, etc.) and used as, for example, an intravenous, subcutaneous, intramuscular or intravenous drip injection or as an ophthalmic solution. Preparation of such an injection is preferably carried out by known aseptic procedures.
The dosage of the pharmaceutical composition of this invention is dependent on dosage form, therapeutic regimen, species of active compound and other factors. In some embodiments, the pharmaceutical composition may be administered once or divided into 2 to 3 times a day.
It is to be understood that this invention is not limited to the particular methodology, protocols, subjects, or reagents described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the claims. The following examples are offered to illustrate, but not to limit the claimed invention.
Shown below are examples of compounds that were identified in the screens for compounds that inhibit SSB-Exonuclease I binding, according to this invention. For each of the compounds, the name of the compound, the chemical formula of the compound, the molecular weight of the compound (MW), and the chemical structure of the compound are shown. Also shown are the name of the company that provided the library from which the compound was identified, and the product code for the specific compound in the library. The Ki value is dissociation constant used to describe the affinity between a ligand (such as a drug, in the present case the tested inhibitory compound) and a peptide or a protein, i.e. how tightly a ligand (i.e., the tested inhibitory compound) binds to a particular peptide or protein. The IC50 value refers to the half maximal inhibitory concentration, i.e. the concentration of an inhibitory compound that is required for 50% inhibition of its target in vitro. It measures how much of a particular substance/molecule is needed to inhibit some biological process (e.g., bacterial growth in LB) by 50%.
Name: 2-[5-(3-bromobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-phenylpropanoic acid. Formula: C19H14BrNO3S2. MW: 448.36. Company: ChemBridge. Product Code: 6044448. Ki=˜2.5 μM. IC50 E. coli 4213=5.5 μM. IC50 Staphylococcus aureus=10 μM.
Name: [5-(2-methyl-3-phenyl-2-propen-1-ylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl](phenyl)acetic acid. Formula: C21H17NO3S2. MW: 395.5. Company: ChemBridge. Product Code: 5767720. Ki=˜4 μM. IC50 E. coli 4213=11 μM. IC50 Staphylococcus aureus=29 μM.
Name: 3-(tert-butyl)-1-(6-chloro-1,3-benzothiazol-2-yl)-4,5-dihydro-1H-pyrazol-5-one. Formula: C14H14ClN3OS. MW: 307.79766. Company: Maybridge. Product Code: SEW01297. Ki=˜773 nM. IC50 E. coli 4213=6 μM. IC50 Staphylococcus aureus=5.5 μM.
Name: 2-[2-chloro-5-(trifluoromethyl)anilino]-5-methoxybenzoic acid. ACD Code: MFCD00175807. Formula: C15H11ClF3NO3. MW: 345.705449. Company: Maybridge. Product Code: S07197. Ki=˜124.7 nM. IC50 E. coli 4213=30 μM. IC50 Staphylococcus aureus=18 μM.
Name: 1-(1,3-benzothiazol-2-yl)-3,4-dimethyl-1H-pyrazol-5-ol. Formula: C12H11N3OS. MW: 245.3. Company: Chembridge. Product Code: 9040944.
Name: 1-(1,3-benzothiazol-2-yl)-3-isopropyl-1H-pyrazol-5-ol. Formula: C13H13N3OS. MW: 259.33. Company: ChemBridge. Product Code: 9036389.
Name: 2-(1,3-benzothiazol-2-yl)-5-methyl-2,4-dihydro-3H-pyrazol-3-one. Formula: C11H9N3OS. MW: 231.27. Company: Chembridge. Product Code: 5108305.
Name: 2-[3-(trifluoromethyl)anilino]benzoic acid. Formula: C14H10F3NO2. MW: 281.234109. Company: Maybridge. Product Code: RJC02179.
Name: 5-[2-chloro-5-(trifluoromethyl)phenoxy]-2-nitrophenol. Formula: C13H7ClF3NO4. MW: 333.64. Company: ChemBridge. Product Code: 5524827.
Name: N-(6-chloro-1,3-benzothiazol-2-yl)-3,5-dimethyl-4-isoxazolecarboxamide. Formula: C13H10ClN3OS. MW: 307.7543. Company: Maybridge. Product Code: HTS08909.
Name: 1-(6-chloro-1,3-benzothiazol-2-yl)-3-methyl-4,5-dihydro-1H-pyrazol-5-one. Formula: C11H8ClN3OS. MW: 265.71702. Company: Maybridge. Product Code: SEW01296.
Peptides corresponding to the C-terminal tail of SSB or variants thereof were synthesized and purified by the University of Wisconsin-Madison Biotechnology Center. “WT” peptide comprises an added N-terminal Trp (W) residue for quantification followed by the 9 C-terminal-most residues from E. coli SSB: Trp-Met-Asp-Phe-Asp-Asp-Asp-Ile-Pro-Phe (SEQ ID NO:6). Thus, the synthesized peptide was fluorescein-WMDFDDDIPF (i.e., fluorescein-SEQ ID NO:6). The Trp residue absorbs at 280 nm thus allowing accurate quantification of the peptide concentration. Alternatively, other residues or tags could be used for practicing the invention, so long as they can be used to accurately quantify the peptide concentration. In competitive binding experiments, the peptide WMDFDDDIPF (SEQ ID NO:6) was used without a fluorescent moiety.
A second peptide that was used, “F-WT”, comprises the same sequence but adds an N-terminal fluorescein moiety. Two additional peptides that alter the F-WT sequence were also synthesized: the first, “F-P176S”, substitutes a Ser residue for the penultimate Pro in the WT sequence, and the second, “F-mixed”, is a randomly-chosen sequence of the SSB-derived portion of the F-WT peptide sequence Trp-Asp-Phe-Met-Asp-Asp-Pro-Phe-Ile-Asp (SEQ ID NO:8). Other peptides that were used in the experiments described below were WDDIPF (SEQ ID NO:7) and WMDFDDDIPF (SEQ ID NO:6). Neither of these peptides was fluorescently labeled.
Peptide binding assays were performed in solution, using the peptides from above, and Exonuclease I, in the absence or presence of various compounds. Different amounts of Exonuclease I (ExoI) were used in the peptide binding assays. Typically, 0.1-10,000 nM E. coli ExoI (or a variant) was incubated with 10 nM F-WT, F-P176S, or F-mixed peptide in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 4% (v/v) glycerol, 1 mM 2-mercaptoethanol, and 0.1 mg/ml bovine serum albumin (BSA) at room temperature for 10 minutes. Fluorescence polarization (FP) was measured at 25° C. in triplicate; the average FP value was plotted with standard deviation of the mean shown as error.
The data in Table 2 summarize the values of the dissociation constants (Ki) for several candidate compounds tested. Graphs showing peptide binding data for individual compounds (i.e., inhibitors) are shown in
In one example, it was noteworthy that the only difference between compounds (inhibitors) 46 and 31 was the presence of a Cl atom in the former. To examine the significance of one atom substitution, the peptide binding inhibition values of inhibitor 46 were plotted against the peptide binding inhibition values of inhibitor 31 (
The compounds 28, 29, and 31 did not inhibit binding (Table 3).
High Throughput Screen to Find Molecules that Inhibit SSB/Exonuclease I Interaction
Concentrated Exonuclease I was dialyzed overnight into 2 liters dilution buffer (DB) (20 mM Tris pH 8.0 HCl, 100 mM NaCl, 1 mM MgCl2, and 1 mM Beta-Mercaptoethanol). After dialysis, Exonuclease I was mixed with fluorescently labeled SSB C-terminal peptide, amino acid sequence (N-terminus) Fluorescein-WMDFDDDIPF, i.e. fluorescein-SEQ ID NO:6 (C-terminus), also previously diluted in DB. The final concentrations of the components of the Exonuclease I/SSB C-terminal peptide mixture after-adjustment with DB were 1 μM Exonuclease I, 10 nM fluorescently labeled SSB C-terminal peptide with 20 mM Tris pH 8.0 HCl, 100 mM NaCl, 1 mM MgCl2, and 1 mM Beta-Mercaptoethanol. After sitting at room temperature for 5 min, a liquid handling system was used to aliquot this solution into 384 well plates providing 30 microliters/well. One microliter of 1 mM compound was then added. The screened compounds were obtained from the ChemBridge DIVERSet library (ChemBridge Corporation, San Diego, Calif.), from the Maybridge HitFinder library (Thermo Fisher Scientific, Waltham, Mass.), from the ChemDiv library (Chemical Diversity Labs, San Diego, Calif.), or from the Known Bioactive Library—KBA01.
The FP (fluorescence polarization) values for each well were read using a (plate reader) 5 minutes after compound addition. Compounds that lowered the FP values were identified. This was typically done using three standard deviations. Compounds that were fluorescent or caused excessive precipitation were eliminated from consideration. Roughly 20 compounds that met these criteria were isolated and more stringently tested using a PanVera Beacon 2000 FP (PanVera Corp., Madison, Wis.). From these experiments 5 compounds were identified that were able to lower the FP values to a degree that suggested a complete inhibition of peptide binding thus allowing the determination of Ki.
To determine the Ki, a master mix was prepared similar to what was previously described above, using 1 μM Exonuclease I, 10 nM fluorescently labeled SSB C-terminal peptide with 20 mM Tris pH 8.0 HCl, 100 mM NaCl, 1 mM MgCl2, and 1 mM Beta-Mercaptoethanol. Increasing amounts of compound were titrated into peptide protein mixture until FP values were lowered to values given by only fluorescently labeled peptide alone. Ki values were determined using the Ki calculator for fluorescence-based competitive binding assays of Prof. Shaomeng Wang, University of Michigan (Nikolovska-Coleska et al., 2004, Anal. Biochem. 332: 261-273).
The candidate compounds were tested for their ability to inhibit bacterial growth, as shown in
100 μL of LB containing E. coli 1655, E. coli 4213, or S. aureus at A ˜0.05 OD 600 was aliquoted in 96 well plates. One μL of compound in DMSO was added to final concentrations given. Plates were incubated at 37° C. with shaking at 250 RPM. Rate of growth was calculated by determining the average change in OD/hour that took place between hours 3 and 7 (log growth phase). The graphs shown herein are plots of this bacterial growth rate (change in OD/h) against inhibitor concentration in μM.
The data in Table 3 summarize the concentrations of selected compounds according to the present invention, which are necessary to reduce bacterial growth by 50% in liquid media.
E. coli
S.
aureus
b(μM)
NDd
aKi is the amount of compound needed to dissociate 50% of Exonuclease I/SSB-Ct complexes in vitro.
bHemolysis is the minimum concentration of compounded necessary to lyse red blood cells.
cMIC is the minimal concentration necessary to inhibit growth of E. coli (imp4213) or B. subtilis cells on solid media. Data are the same for either bacterial strain.
dNot detected; no dissociation of the Exonuclease I/SSB-Ct complex was observed even in the highest concentrations tested.
eTo be determined.
Data from the Escherichia coli inhibition experiments are summarized in Table 4.
Data from the Staphylococcus aureus inhibition experiments are summarized in Table 5.
A number of the tested candidate compounds had profound inhibitory effects on bacterial growth. In addition, it was discovered that the identified drug compounds can slow the growth of a number of other Gram-positive bacterial species. For example, the growth of Bacillus subtilis and Enterococcus faecalis was inhibited when these bacteria were grown on agar plates containing 100 μM of either compound 9 or compound 10. Growth of Deinococcus radiodurans was also inhibited when tested in a similar manner.
Exonuclease I crystals were transferred to a soak solution containing 12% PEG 4000, 1 mM MgCl2, 20 mM Tris pH 8.0, and 5 mM compound. These solutions were allowed to soak in watch glasses for 5 days at which point the crystals were transferred to a cryo solution containing 12% PEG 4000, 1 mM MgCl2, 20 mM Tris pH 8.0, 5 mM compound, and 25% glycerol. Crystals were then flash frozen in liquid Nitrogen.
In spite of the noted importance of SSB interactions with heterologous proteins in cells, a structural and mechanistic understanding of interactions between SSB and its partner proteins has remained unclear. In one aspect, the present invention provides elucidation of the high-resolution structure of E. coli ExoI bound to the C-terminus of its cognate SSB. The crystal structures of apo and SSB peptide-bound forms of E. coli ExoI were determined, as were candidate compounds that bind in one of the binding pockets previously occupied by the C-terminal peptide tail of SSB in the crystal structure (Lu and Keck, 2008, Proc. Natl. Acad. Sci. USA, in press).
The crystal structure of E. coli ExoI bound to a peptide comprising the SSB-Ct element was determined. ExoI/SSB-Ct protein crystals diffracted to 2.7-Å resolution and the structure of the complex was determined by molecular replacement using the apo ExoI structure (Breyer and Matthews, 2000, Nat. Struct Biol. 7: 1125-1128) as a search model (Table 9). In addition, the 1.7-Å-resolution structure of E. coli ExoI crystallized in the absence of the SSB-Ct peptide (but otherwise under the same crystallization conditions as the peptide-bound form) was determined, for comparative structural analysis. As was described for the initial apo-ExoI structure, ExoI in both crystal forms is comprised of exonuclease (residues 1-201), SH3-like (residues 202-352), and helical (residues 360-476) domains (
Inspection of difference electron-density maps from the SSB-Ct-bound ExoI crystals revealed features corresponding to the C-termini from two SSB-Ct peptides associated with the ExoI surface, as shown in
Electron density for the second peptide, “peptide B”, includes SSB residues 175-177, which are bound entirely by the ExoI SH3-like domain (
To examine ExoI/SSB-Ct complex formation in solution, an equilibrium fluorescence-anisotropy assay that measures ExoI binding to a fluorescein-labeled SSB-Ct peptide (F-SSB-Ct) was developed. ExoI binds F-SSB-Ct with an apparent equilibrium dissociation constant (Kd, app) of 136+/−11 nM in this assay (
A panel of ExoI variants was created in which surface-exposed residues forming the peptide-A (Arg148, Tyr207, Gln311 and Arg316) or peptide-B (Arg327 and Leu331) binding sites were individually mutated to alanine to assess the contributions of the two sites to SSB-Ct binding. Additional Ala variants were made to test whether the prominent “basic ridge” on ExoI (Lys227 and Arg338) and ExoI residues that bind Mg2+ in the presence of the SSB-Ct peptide (Glu150, Glu318 and Asp319) have roles in F-SSB-Ct binding. Finally, Ala variants altering residues from the helical domain (Gln448 and Gln452) that mediate inter-protein contacts with peptide A in the crystal structure were also created. Each ExoI variant was purified and tested for binding to the F-SSB-Ct peptide. Far UV circular dichroic spectra of the variants were indistinguishable from wild type ExoI, indicating that the mutations did not alter the secondary structure of the variants significantly.
Three of the peptide-A-site variants displayed dramatically reduced F-SSB-Ct binding (
Additional variants were used to test the roles of basic ridge, Mg2+-binding and helical domain surfaces in binding the F-SSB-Ct peptide in solution (
ExoI/SSB Complex Formation is Essential for SSB stimulation of ExoI Activity
Early studies showed that E. coli SSB stimulates ExoI nuclease activity and that the two proteins physically interact. However, whether ExoI/SSB complex formation is required for this stimulation has not been tested. To examine this, a nuclease assay in which hydrolysis of a radiolabeled ssDNA substrate is catalyzed by ExoI in a reaction that can be stimulated ˜4-fold with the addition of SSB was developed (
To assess whether association with SSB is necessary for ExoI stimulation, two SSB variants wee substituted into the assay. The first, SSB113, is a well-characterized E. coli SSB variant with dramatically reduced ExoI binding affinity but wild type ssDNA binding attributes, and the second, SSB-mixed, is a variant with a mixed C-terminal sequence that matches the sequence used in the F-mixed peptide. SSB113 provided greatly reduced stimulation (<2-fold) compared to wild type SSB, whereas SSB-mixed entirely failed to stimulate ExoI activity (
The effects of Ala substitutions on SSB-stimulated ExoI nuclease activity were also tested. If SSB stimulates ExoI by recruiting the enzyme to its substrate, then ExoI variants that are defective for binding the SSB-Ct peptide would have parallel defects in SSB-stimulated activity. The peptide-A- and peptide-B-site variants matched this prediction remarkably well. Two of the peptide-A-site variants (Arg148 and Tyr207) appeared to be unaffected by the addition of SSB, maintaining wild-type levels of nuclease activity at all SSB concentrations tested (
The basic ridge and Mg2+-binding ExoI variants were also tested for SSB-dependent nuclease activities. The basic ridge variants (Lys227 and Arg338), which had defects in F-SSB-Ct binding, similarly had reduced stimulation by SSB (
The atomic coordinates of the crystal structure of Escherichia coli Exonuclease I bound to the C-terminal peptide from E. coli SSB have been deposited in the Protein Data Bank under the accession code 3C94. Prokaryotic exonucleases that bind to SSBs and that upon binding have crystal structures whose models substantially represent the atomic coordinates specified in the model deposited in the Protein Data Bank under the accession code 3C94, can be used for practicing the present invention. As well, SSBs that bind to prokaryotic exonculeases and that upon binding have crystal structures whose models substantially represent the atomic coordinates specified in the model deposited in the Protein Data Bank under the accession code 3C94, can be used for practicing the present invention.
Table 6 shows the coordinates of the crystal structure of E. coli ExoI bound to compound 9 that was identified in a screen according to the present invention. Compounds that bind prokaryotic exonucleases and that have crystal structures whose models substantially represent the atomic coordinates specified in Table 6, can be used for practicing the present invention.
Table 7 shows the coordinates of the crystal structure of E. coli ExoI bound to compound 10 that was identified in a screen according to the present invention. Compounds that bind prokaryotic exonucleases and that have crystal structures whose models substantially represent the atomic coordinates specified in Table 7, can be used for practicing the present invention.
Table 8 illustrates the number and identity of known protein interactions with bacterial SSBs.
Table 9 illustrates the crystallographic data collection and structure refinement statistics.
Table 10 shows Kd, app values for F-SSB-Ct binding by ExoI and variants.
Topoisomerase III binding assays were conducted (
The data indicate that peptide binding is much weaker in the case of TopoIII/peptide, making it difficult to accurately determine a binding constant (Kd). In order to determine if the compounds had an effect on peptide binding to TopoIII, a similar set of experiments as stated above was set up, except for the omission of DMSO from the buffer solution. Either unlabeled peptide or various compounds were then added to a final concentration of 100 μM. These compounds and unlabeled peptide were dissolved in DMSO and their addition brought the final concentration of DMSO to 1%.
Some of the candidate compounds were able to inhibit binding of the peptide to TopoIII, lowering the observed anisotropy value. As shown in
The toxicity of the identified antimicrobial candidate compounds was tested using human colorectal adenocarcinoma cells in vitro. The tested compounds are significantly less toxic to HT-29 (human colorectal adenocarcinoma) than to Gram-positive bacterial cells or the cell wall compromised E. coli cells. The mammalian cells were tested using a CellTiter-Glo cell viability assay (Promega, Madison, Wis.).
According to the data obtained with this assay compound 3 had an IC50>100 μM, compound 8 had an IC50>100 μM, compound 9 had an IC50=38 μM, compound 10 had an IC50=60 μM, compounds 28, 29, 32, and 37 all had an IC50>100 μM, compound 42 had an IC50=51 μM, and compound 46 had an IC50=55 μM. Comparing these data to the IC50 from the bacterial growth curves, the conclusion is that the compounds are much more effective at killing bacteria than mammalian cells.
In vitro hemolysis experiments were carried out to determine if the identified drug candidates merely inhibited bacterial growth by causing cell lysis. The identified small molecules were incubated with red blood cells to see what percentage of cells would lyse after 1 hour. The results from these experiments are shown in Table 11. Estimated hemolysis data are due to problem with reading absorbance, when the compound absorbs at the same wavelength as heme or if the compound precipitates. In such instances it is difficult to quantitate the absorbance readings; however, visual determination of how many cells have lysed can then be performed, which provides an estimate of cell lysis.
It is to be understood that this invention is not limited to the particular devices, methodology, protocols, subjects, or reagents described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the claims. Other suitable modifications and adaptations of a variety of conditions and parameters, obvious to those skilled in the art of biochemistry and pharmaceutical chemistry, are within the scope of this invention. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.
This invention claims priority to U.S. Provisional Patent Application Ser. No. 60/931,411, filed May 22, 2007, which is herein incorporated by reference.
This invention was made with United States government support awarded by the National Institutes of Health, grant No. GM068061. The United States government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60931411 | May 2007 | US |
