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WO2024148237A1 - Procédés de criblage pour identifier des inhibiteurs spécifiques de mycobactéries, y compris m. tuberculosis - Google Patents

Procédés de criblage pour identifier des inhibiteurs spécifiques de mycobactéries, y compris m. tuberculosis Download PDF

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WO2024148237A1
WO2024148237A1 PCT/US2024/010438 US2024010438W WO2024148237A1 WO 2024148237 A1 WO2024148237 A1 WO 2024148237A1 US 2024010438 W US2024010438 W US 2024010438W WO 2024148237 A1 WO2024148237 A1 WO 2024148237A1
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compound
ribosome
asl
trna
binding
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Dick GUENTHER
Steve Peterson
Sam YENNE
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Trana Discovery Inc
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Trana Discovery Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • a representative S-D sequence for tuberculosis is AGAAAGGAGG, and a representative “box” sequence following the S-D sequence is AUAAUAA.
  • Minimal mRNA sequences that include the S-D sequence, the “box” sequence, the AUG sequence encoding methionine, and the triplet codons used by mycobacteria (or streptomyces and mycobacteria), including M. tuberculosis, to encode various amino acids, are provided above.
  • sequences can include additional bases to the left and right of the listed sequences.
  • the mRNA consensus sequence 5' AGGAGGU 3' is between 5 and 8 bases upstream from the AUG translation initiation codon (i.e., the codon for methionine).
  • the S-D sequence forms complementary base pairs with a consensus sequence found at the 3’ end of the 16S rRNA molecule (q.v.) in the 30S subunit of the ribosome.
  • the S-D sequence thus serves as the binding site for bacterial mRNA molecules on ribosomes.
  • the S-D sequence for tuberculosis is different than that in E. coli, the mechanism is the same.
  • the screening assay involves the further step of screening active compounds for their ability to inhibit the propagation of bacteria other than mycobacteria (or, as specified above, streptomyces and mycobacteria), including M. tuberculosis, and which use a different ASL for the various amino acids than mycobacteria, including M. tuberculosis.
  • This is indicative of compounds that inhibit or stabilize the formation of a complex between the ribosome and the Shine-Delgamo sequence.
  • the compounds have broad-spectrum antimicrobial activity, and inhibit the formation of a complex between the ribosome and the Shine-Delgarno sequence, the compounds demonstrate a heretofore unknown mechanism of action.
  • Such compounds, and a method of treating bacterial infections using such compounds are intended to be within the scope of the invention described herein.
  • the present invention relates to compositions and methods for identifying compounds useful for specifically inhibiting mycobacteria (or, as specified above, streptomyces and mycobacteria), including M. tuberculosis propagation, as well as pharmaceutical compositions and methods for treating mycobacteria (or, as specified above, streptomyces and mycobacteria), including M. tuberculosis infections by inhibiting bacterial propagation.
  • Propagation can be inhibited by inhibiting translation of mycobacteria (or, as specified above, streptomyces and mycobacteria), including M. tuberculosis RNA into proteins.
  • the inhibition can be achieved by either inhibiting or stabilizing the complex formed between the bacterial ribosome and the codon specific for Arg, Gin, Glu, Leu or Lys, as described herein. Either approach inhibits the ability of the bacteria to produce proteins.
  • an “inhibitor” refers to any compound capable of preventing, reducing, or restricting S. aureus propagation.
  • An inhibitor may inhibit propagation of Mycobacteria, such as M. tuberculosis (and, as discussed herein, in some embodiments, also streptomyces), for example, by preventing, reducing or restricting protein formation in these bacteria, specifically by inhibiting incorporation or one or more of Arg, Gin, Glu, Leu or Lys into a growing protein strand, and, more specifically, by inhibiting incorporation of these amino acids by focusing on a) disrupting or b) stabilizing a complex formed by a tRNA specific for these bacteria’s incorporation of Arg, Gin, Glu, Leu and/or Lys into a growing protein strand.
  • the inhibition is at least 20% (e.g., at least 50%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%) of the propagation as compared to the propagation in the absence of the inhibitor.
  • an inhibitor prevents, reduces, or restricts the binding of a tRNA, or fragment thereof, to a ribosome, preferably a ribosome associated with protein and peptide synthesis.
  • a stabilizer stabilizes the binding of a tRNA, or fragment thereof, to a ribosome, preferably a ribosome associated with protein and peptide synthesis.
  • the binding in the foregoing two aspects is related to the incorporation or arginine into a growing peptide or protein, and, most particularly, the binding is specific for the incorporation of Arg, Gin, Glu, Leu or Lys into a protein or peptide encoded by Mycobacteria, such as M. tuberculosis (and in some cases, streptomyces), and the tRNA is not useful for the incorporation of these amino acids into proteins or peptides of other bacteria.
  • a “label” or “detectable label” is any composition that is detectable, either directly or indirectly, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • Useful labels include, but are not limited to, radioactive isotopes (for example, 32 P, 35 S, and 3 H), dyes, fluorescent dyes (for example, Cy5 and Cy3), fluorophores (for example, fluorescein), electron-dense reagents, enzymes and their substrates (for example, as commonly used in enzyme-linked immunoassays, such as, alkaline phosphatase and horse radish peroxidase), biotin-streptavidin, digoxigenin, or hapten; and proteins for which antisera or monoclonal antibodies are available.
  • radioactive isotopes for example, 32 P, 35 S, and 3 H
  • dyes for example, fluorescent dyes (for example, Cy5 and Cy3), fluorophores (for example, fluorescein), electron-dense reagents, enzymes and their substrates (for example, as commonly used in enzyme-linked immunoassays, such as, alkaline phosphatase and horse radish
  • host refers to human or animal cells or tissues in vitro and human or animal subjects (e.g., avian or mammalian cells, tissues and subjects such as chickens, turkeys, mouse, rat, cats, dogs, cows, pigs, horses, etc.).
  • avian or mammalian cells, tissues and subjects such as chickens, turkeys, mouse, rat, cats, dogs, cows, pigs, horses, etc.
  • the complementary sequence is called the anti-Shine-Dalgarno sequence and is located at the 3' end of the 16S rRNA in the ribosome.
  • the eukaryotic equivalent of the Shine-Dalgarno sequence is called the Kozak sequence.
  • Shine-Dalgarno sequence can reduce translation. This reduction is due to a reduced mRNA-ribosome pairing efficiency, as evidenced by the fact that complementary mutations in the anti-Shine-Dalgarno sequence can restore translation. Accordingly, it is preferred that the Shine-Dalgarno sequence be appropriately matched with the ribosome that is used. That is, if a AT. tuberculosis ribosome is used, then the Shine-Dalgarno sequence should be the sequence used by M. tuberculosis. If an E. coli ribosome is used, then the Shine-Dalgarno sequence should be the sequence used by E. coli.
  • the translation initiation factors IF2-GTP, IF 1 , IF3, as well as the initiator tRNA fMet-tRNA(fmet) are recruited to the ribosome. Once the tRNA(fmet) is recruited to the ribosome, the protein translation will begin (starting with methionine).
  • an “inhibitor” refers to any compound capable of preventing, reducing, or restricting Mycobacteria (and in some embodiments, Steptomyces), such as M. tuberculosis propagation.
  • An inhibitor may inhibit propagation, for example, by preventing, reducing or restricting protein (or peptide) translation in a specific manner, i.e., by inhibiting the use of the codon specific for the amino acids mentioned above used by these bacteria but not most other bacteria.
  • the mRNA including the codon for these amino acids is present in a ribosome, and the bacterial tRNA (ASL Arg) forms a complex with the mRNA and the ribosome.
  • ASL Arg bacterial tRNA
  • tRNA is preferably a unique or unusual tRNA: that is, one that contains one or more modified bases other than adenine, guanine, cytosine, or uracil in the anticodon binding region (including both the stem and loop thereof), and/or preferably a tRNA that is only found in these bacteria for binding to a corresponding amino acid (e.g., Arg, Gin, Glu, Leu and Lys, as discussed above) during protein translation.
  • a corresponding amino acid e.g., Arg, Gin, Glu, Leu and Lys, as discussed above
  • the tRNA is specific for these amino acids, and is only found in these bacteria, or at least is not found in the vast majority (i.e., greater than 90%, preferably greater than 95%, and, ideally, greater than 99% of other bacteria).
  • the modified base or bases is/are a nucleotide(s) that is/are at a binding site as described below (e.g., nucleotides 27 through 43) and participates in the binding event.
  • the tRNA for the corresponding amino acid bound by the Mycobacterial (and in some cases, Streptomyces) tRNA preferably does not have the same modified base at the binding site or corresponding nucleotide in the host organism (i.e., pathogen specific modification). Many of these exist in the human host and in agronomically important animal hosts as set forth above). Examples of modified bases are set forth below.
  • tRNA fragments for use in the screening methods described herein are tRNA fragments from Mycobacterium (and in some cases, Streptomyces), such as M. tuberculosis.
  • Streptomyces and Mycobacterium specifically use the anticodon stem loop (ASL) Glu UUC, and the codon GAA, to code for the amino acid Glu.
  • ASL Glu UUC is AGGGC%U ⁇ CU6AUCCCU.
  • a representative mRNA sequence comprising the box sequence, a Shine Delgamo sequence, a codon for Met, and a codon for Glu, is 5’- GGGCGAUAACACUCAGAAAGGAGGAUAAUAAAUG GAA
  • ASL Lys UUU is ACGGACUSUU6ATCCGC
  • mRNA sequence is 5’- GGGCGAUAACACUCAGAAAGGAGGAUAAUAAAUG AAA
  • Streptomyces and Mycobacterium non-specifically use the ASL Gin UUG and the codon UUG, to code for the amino acid Gin.
  • the sequence for ASL Gin UUG is UCUGAJUNUG/PUCAGA, and the mRNA sequence is 5’- GGGCGAUAACACUCAGAAAGGAGGAUAAUAAAUG CAA
  • Mycobacterium selectively use the ASL Arg UCU, and the codon UCU, selectively code for Arg.
  • the ASL sequence for ASL Arg UCU is AGGGC%U ⁇ CU6AUCCCU, and the mRNA sequence is 5’-GGGCGAUAACACUCAGAAAGGAGGAUAAUAAAUG AGA ACAGCUGAUCAAUCGUGCAUCC-3 ’ .
  • M. tuberculosis specifically uses the ASL Leu UAG, and the codon UAG, to specifically code for Leu.
  • the sequence for ASL Leu UAG is CCGGUUU5AGKTGCCGG, and the mRNA is 5’-GGGCGAUAACACUCAGAAAGGAGGAUAAUAAAUG CUA
  • the sequence can be AGGGC%U ⁇ CU6AUCCCU-Label
  • the sequence can be CCGGUUU5AGKTGCCGG-Label. The same holds true for the other ASLs listed above.
  • the tRNA fragment above can be modified with one or more modified nucleosides, so long as it maintains its selectivity for the amino acids discussed herein, and the modified tRNA is still specific for Mycobacteria (and in some cases, Streptomyces), for example, M. tuberculosis) over other bacteria.
  • the tRNA fragment incorporates one, two, three, or more modified nucleosides into the nucleic acid sequence. In another aspect, the tRNA fragments incorporate three modified nucleosides into their nucleic acid sequence.
  • Modified nucleosides that can be incorporated into the tRNA fragments include any modified nucleotide, including, but not limited to unknown modified adenosine (?A), 1 -methyladenosine (mlA), 2-methyladenosine (m2A), N 6 - isopentenyladenosine (i6A), 2-methylthio-N 6 -isopentenyladenosine (ms2i6A), N 6 - methyladenosine (m6A), N 6 -threonylcarbamoyladenosine (t6A), N 6 -methyl- N 6 threonylcarbomoyladenosine (m6t6A), 2-methylthio-N 6 -threon
  • the tRNA fragment may or may not be capable of forming a secondary structure.
  • the tRNA fragment is not capable of forming a stem loop structure with itself.
  • the fragment is a linear fragment of a tRNA that is not capable of forming a stem loop structure with itself.
  • the specific tRNA referred to herein with respect to host tRNA is also preferably a unique or unusual tRNA: that is, one that contains one or more modified bases other than adenine, guanine, cytosine, or uracil in the anticodon binding region (including both the stem and loop thereof), as set forth above, and/or preferably one that is the only tRNA available in that host for binding to RNA for priming of translation of A aureus proteins.
  • a method of screening for compounds useful for inhibiting S. aureus propagation involves contacting one of the specific tRNAs discussed above, such as a specific tRNA Leu , to a ribosome that binds that tRNA in the presence of the test compound.
  • the contacting step is typically carried out in vitro in an aqueous solution, with the tRNA, the ribosome, an appropriate messenger RNA, and the test compound in the aqueous solution.
  • the contacting step may be carried out with a single test compound or with a library of probes or test compounds in any of a variety of combinatorial chemistry systems, as discussed in greater detail below.
  • the appropriate messenger RNA comprises the Shine-Dalgarno sequence appropriate for the ribosome.
  • the ribosome can be, for example, a Mycobacterium ribosome, such as a tuberculosis ribosome or E. coh ribosome, and each has its own anti-Shine- Dalgarno sequence that interacts with the Shine-Dalgarno sequence.
  • the messenger RNA also comprises a series of 5-8 base pairs to the right of the Shine-Dalgarno sequence, then the codon AUG, which codes for methionine, then the codon for Arg, Gin, Glu, Leu or Lys, as discussed herein.
  • the mRNA can optionally, but preferably, include from 1 to 100 base pairs to the left of the Shine-Dalgarno sequence, and from 1 to 100 base pairs to the right of the codon for Arg, Gin, Glu, Leu or Lys.
  • the next step involves determining whether the compound inhibits the binding of the specific tRNA to the ribosome (e.g., the binding of tRNA Leu for M. tuberculosis) at the appropriate position(s) on the ribosome for incorporation of the appropriate amino acid into a growing peptide or protein.
  • the determining step can be carried out by any suitable means, such as the filter binding assays disclosed below, or in any of the binding detection mechanisms commonly employed with combinatorial libraries of probes or test compounds as discussed below. Inhibition of ribosomal binding by the test compound indicates that the test compound is useful for inhibiting bacterial propagation.
  • Compounds identified by this technique are sometimes referred to as “active compounds” herein.
  • the method is particularly useful for identifying compounds that inhibit bacterial growth, preferably bacteria that contain a single tRNA for a particular amino acid, such as a single arginine tRNA that is specific for Mycobacteria (and in some cases, Streptomyces), such as M. tuberculosis, over other bacteria.
  • a method of screening for compounds useful for inhibiting Mycobacterial, such as tuberculosis, propagation in a host comprises contacting the specific host tRNA to the Mycobacterium RNA, such as tuberculosis RNA, in the presence of the test compound.
  • the contacting step is typically carried out in vitro in an aqueous solution, with the tRNA, the bacterial RNA, and the test compound in the aqueous solution.
  • Mycobacterium RNA is intended to encompass both a complete Mycobacterium genome and fragments thereof that contain the tRNA binding portions (such fragments will typically be at least 10 or 12 to 50 or more nucleotides in length).
  • the contacting step may again be carried out with a single test compound or with a library of probes or test compounds in any of a variety of combinatorial chemistry systems, as discussed in greater detail below.
  • the next step involves determining whether the compound inhibits the binding of the specific host tRNA to the Mycobacterium RNA in the presence of the test compound.
  • the determining step can be carried out by any suitable means, such as gel shift assays, chemical and enzymatic footprinting, circular dichroism and NMR spectroscopy, equilibrium dialysis, or in any of the binding detection mechanisms commonly employed with combinatorial libraries of probes or test compounds as discussed below.
  • the inhibition or antagonism of binding indicates that the test compound is useful for inhibiting propagation of the S. aureus in the host.
  • Such compounds are also sometimes referred to as “active compounds” herein.
  • the method may be carried out with Mycobacterium.
  • the specific host tRNA is mammalian, preferably primate or specifically human, such as tRNA arg suu
  • the determining step comprises determining whether the compound inhibits the binding of tRNA arg suu to the Mycobacterium RNA.
  • Non- oligomers include a wide variety of organic molecules, such as heterocyclics, aromatics, alicyclics, aliphatics and combinations thereof, comprising steroids, antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids, opioids, benzodiazepenes, terpenes, prophyrins, toxins, and combinations thereof.
  • the resulting mixture is incubated under conditions that allow binding of the tRNA fragment and the target nucleic acid in the absence of the test compound.
  • the method further involves detecting whether the test compound inhibits or antagonizes the binding of the tRNA fragment to the target nucleic acid, where the absence or antagonism of binding of the tRNA ASL fragment and the target nucleic acid molecule is indicative of the test compound being an inhibitor of Mycobacterium propagation.
  • RNA nucleotide base modifications are frequently found near catalytic sites of RNAs and many of the proteins that are responsible for the modification are encoded by essential genes (Zhang 2004).
  • the researchers in Agris and Malkiewicz labs have taken an approach to study model systems produced by chemical synthesis methods rather than using modified nucleotide bases obtained from biochemical methods (Agris et. al. 1995). This synthetic approach provides far more control to investigate, in detail, the significant contributions made by modified nucleotides.
  • the screening methods described herein can use translation as a biochemical target, where the ASL of tRNA containing modified nucleotides is essential to translation, the modified bases and RNA oligomers containing these modified bases can be synthesized using the techniques described herein, and these oligomers bind to programmed ribosomes.
  • a series of experiments can be conducted to demonstrate that a fluorescently-labeled synthetic oligomer containing 17 nucleotide bases, 2 or 3 of which are modified, will bind to programmed ribosomes isolated from Mycobacteria, such as M. tuberculosis, and that this binding can be detected by monitoring the change in fluorescence.
  • the compounds described herein can be employed as part of a pharmaceutical composition with other compounds intended to prevent or treat a particular microbial infection, i.e., combination therapy.
  • the pharmaceutical compositions can also include various other components as additives or adjuncts.
  • the combination therapy may be administered as (a) a single pharmaceutical composition which comprises an inhibitor as described herein, at least one additional pharmaceutical agent described herein, and a pharmaceutically acceptable excipient, diluent, depot, such as a polyethylene glycol depot, or carrier; or (b) two separate pharmaceutical compositions comprising (i) a first composition comprising an inhibitor as described herein and a pharmaceutically acceptable excipient, diluent, depot, or carrier, and (ii) a second composition comprising at least one additional pharmaceutical agent described herein and a pharmaceutically acceptable excipient, diluent, depot, or carrier.
  • the pharmaceutical compositions can be administered simultaneously or sequentially and in any order.
  • the inhibitors can be administered together with at least one other antimicrobial agent as part of a unitary pharmaceutical composition. Alternatively, it can be administered apart from the other antimicrobial agents. In this embodiment, the inhibitors and the at least one other antimicrobial agent are administered substantially simultaneously, i.e. the compounds are administered at the same time or one after the other, so long as the compounds reach therapeutic levels for a period of time in the blood.
  • Aztreonam is a representative monobactam.
  • antibacterial agents include, for example, Arsphenamine, Chloramphenicol, Clindamycin, Lincomycin, Ethambutol, Fosfomycin, Fusidic acid, Furazolidone, Isoniazid, Linezolid, Metronidazole, Mupirocin, Nitrofurantoin, Platensimycin, Pyrazinamide, Quinupristin/Dalfopristin, Rifampin or Rifampicin, and Tinidazole. Tuberculosis is commonly treated with combination therapy, so as to minimize the possibility that the bacteria will develop drug resistance.
  • Latent TB is commonly treated with either isoniazid or rifampin alone, or a combination of isoniazid with either rifampicin or rifapentine. The treatment takes three to nine months depending on the medications used.
  • the recommended treatment of new-onset pulmonary tuberculosis is six months of a combination of antibiotics containing rifampicin, isoniazid, pyrazinamide, and ethambutol for the first two months, and only rifampicin and isoniazid for the last four months. Where resistance to isoniazid is high, ethambutol may be added for the last four months as an alternative.
  • Treatment with anti-TB drugs for at least 6 months results in higher success rates when compared with treatment less than 6 months.
  • Shorter treatment regimens may be recommended for those with compliance issues, though there is also no evidence to support shorter antituberculosis treatment regimens when compared to a 6-month treatment regimen, other than a four- month daily treatment regimen containing high-dose, or "optimized,” rifapentine with moxifloxacin (2PHZM/2PHM).
  • MDR-TB multiple drug-resistant TB
  • Medication Resistance Primary resistance occurs when a person becomes infected with a resistant strain of TB. A person with fully susceptible MTB may develop secondary (acquired) resistance during therapy because of inadequate treatment, not taking the prescribed regimen appropriately (lack of compliance), or using low-quality medication.
  • MDR-TB is defined as resistance to the two most effective first-line TB drugs: rifampicin and isoniazid. Extensively drug-resistant TB is also resistant to three or more of the six classes of second-line drugs. Totally drug-resistant TB is resistant to all currently used drugs. There is some efficacy for linezolid to treat those with XDR- TB, but side effects and discontinuation of medications has been common. Bedaquiline is tentatively supported for use in multiple drug-resistant TB.
  • Compounds identified using the screening assays described herein can be used to treat drug-resistant TB.
  • tuberculosis treatment is that a hydrophobic rifamycin antibiotic is commonly used because it is hydrophobic, and is thus able to enter the macrophage, which is a reservoir for tuberculosis.
  • active compounds identified using the assays described herein are tested for their hydrophobicity (for example, using an octanol/water partition coefficient test), and hydrophobic compounds used to replace rifamycins.
  • Replacing rifamycins can be beneficial, particularly where a patient is also infected with HIV. That is, rifamycins, such as rifampicin, can affect the metabolism of anti-HIV agents.
  • Rifampicin is a well-characterized drug with established strong CYP3A4/2C19 inducing capabilities.
  • the compounds identified using the screening assays described herein can also be tested for their ability to induce CYP3 A4/2C19, or other genes controlling drug metabolism, to ensure that the drug used to replace rifampicin does not have a similar effect on HIV treatments.
  • Methods for determining the impact of a drug on CYP450 are well known to those of skill in the art. IV. Methods of Using the Compounds and/or Pharmaceutical Compositions
  • the compounds can be used to treat or prevent microbial infections caused by Mycobacterium, such as M. tuberculosis, and, in some cases, also Streptomyces.
  • the compounds can also be used as adjunct therapy in combination with existing therapies in the management of the aforementioned types of infections. In such situations, it is preferably to administer the active ingredients to a patient in a manner that optimizes effects upon the these bacteria, including drug resistant versions, while minimizing effects upon normal cell types. While this is primarily accomplished by virtue of the behavior of the compounds themselves, this can also be accomplished by targeted drug delivery and/or by adjusting the dosage such that a desired effect is obtained without meeting the threshold dosage required to achieve significant side effects.
  • Bacterial propagation can be inhibited by inhibiting ribosomal binding of a specific tRNA useful for incorporation of an amino acid, such as Arg, Gin, Glu, Leu, and Lys, as described elsewhere herein, into a growing peptide or protein in Mycobacterium, such as M. tuberculosis, by an amount sufficient to inhibit propagation.
  • Inhibition of ribosomal binding may be carried out by contacting an active compound to the ribosome in an amount effective to inhibit binding sufficiently to inhibit propagation.
  • the Mycobacterium such as M.
  • Mycobacterial propagation in a host can be inhibited by inhibiting the binding, or stabilizing the binding, of the specific host tRNA to the Mycobacterial RNA at one of the binding sites by an amount sufficient to inhibit propagation of the Mycobacteria in the host.
  • Formulations of active compounds can be prepared and administered in accordance with known techniques, as discussed below.
  • Subjects to be treated by the methods of the present invention are typically human subjects, although the methods may be carried out with animal subjects (dogs, cats, horses, cattle, etc.) for veterinary purposes.
  • the present invention provides pharmaceutical formulations comprising the active compounds, including pharmaceutically acceptable salts thereof, in pharmaceutically acceptable carriers for aerosol, oral, and parenteral administration as discussed in greater detail below.
  • the therapeutically effective dosage of any specific compound, the use of which is in the scope of present invention, will vary somewhat from compound to compound, patient to patient, and will depend upon the condition of the patient and the route of delivery.
  • an active compound or a pharmaceutically acceptable salt thereof may be administered orally or through inhalation as a solid, or may be administered intramuscularly or intravenously as a solution, suspension, or emulsion. Alternatively, the compound or salt may also be administered by inhalation, intravenously or intramuscularly as a liposomal suspension.
  • the active compound or salt should be in the form of a plurality of solid particles or droplets having a particle size from about 0.5 to about 5 microns, preferably from about 1 to about 2 microns.
  • LFA Lateral flow assays
  • gold-labeled antibodies or other color-labeled antibodies, that bind to antigens, and also to a test strip.
  • a biological sample from a patient such as a blood, plasma, sputum, saliva, or other suitable sample
  • the medium in which the incubation takes place can be placed in the well of a suitable LFA, which includes a test line that is “striped” with a compound that binds to the labeled antibodies to the ASLs, and, ideally, a control line.
  • a Mycobacterium such as M. tuberculosis, or, in some cases, a Streptomyces, can be identified using these screens.
  • a multiplex assay is used. For example, if there are two test lines (and, ideally, a control line), one which indicates the presence of a Mycobacterium, and the other which indicates the presence of M. tuberculosis (for example, by using antibodies to the ASL Leu UAG), one can identify patients who have a Mycobacterium infection, and also determine whether the Mycobacterium is M. tuberculosis (or a different Mycobacteria bacteria, such as M. avian).
  • test lines one of which is non-selective for Streptomyces and Mycobacteria, and another which is selective for Mycobacteria, can be used to determine whether a patient is infected with Streptomyces or Mycobacteria.
  • Assay tool ASL of AT. tuberculosis tRNA Leu with FL label
  • This assay was designed to identify inhibitors and agonists of M. tuberculosis specific Leu ASL (anticodon stem loop) RNA oligomer interaction with programmed ribosomes, where inhibitors inhibit the complex formed between the ribosomes and the RNA oligomer and agonists stabilize the complex formed between the ribosomes and the RNA oligomer.
  • Leu ASL arachidon stem loop
  • 25 g frozen MRE600 E. coli cells (Cell Culture Facility, UAB) can be re-suspended in 100 ml buffer A (20 mM Tris-HCl, pH7.5, 200 mM NH4Q, 20 mM MgCb, 3 mM b-mercaptoethanol), and passed through French press twice; cell debris was removed by 20 min centrifugation at 16,000 rpm in Beckman Ti 45 rotor.
  • the mRNA analog 5’-GGGCGAUAACACUCAGAAAGGAGGAUAAUAAAUG CUA ACAGCUGAUCAAUCGUGCAUCC-3’ can be synthesized in vitro. Before addition to reaction 10 pm of single stranded DNA template
  • GGATGCACGATTGATCAGCTGTTCTCATTTATTATCTCCTGAGTGTTATCGCCCT ATAGTGAGTCGTATTA can be annealed to 10 pm T7 DNA primer in 2 pl MEGAscript Kit (Ambion) reaction buffer for 5 min at 95° C. and 15 min at room temperature. Each standard 20 pl reaction can be supplemented with additional 200p of T7 polymerase and incubated overnight as recommended for short transcripts by the manufacturer. After DNase treatment, mRNA can be phenol extracted, ethanol precipitated twice and analyzed on 8% polyacryamide gel for purity and stored in water.
  • the ASL Leu UAG with the sequence CCGGUUU5AGKTGCCGG, can first be heated for 2 min at 90° C in water, after addition of 1/10 V of 10x buffer incubation continued on ice for at least 10 min.
  • 70S ribosomes and tRNA can be reactivated in buffer for 5 min at 45° C, followed by 10 min at 37° C; mRNA can be heated in buffer at 37° C for 5 min.

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Abstract

La présente invention concerne des procédés d'inhibition de la propagation de mycobactéries, y compris de M. tuberculosis, et de criblage de composés qui inhibent la propagation mycobactérienne. Un procédé d'inhibition de la propagation mycobactérienne consiste soit à inhiber soit à stabiliser la liaison ribosomique d'un ARNt spécifique dans les mycobactéries par une quantité suffisante pour inhiber l'expression de protéines. Un procédé de criblage de composés utiles pour inhiber la propagation mycobactérienne consiste à mettre en contact un ARNt de mycobactérie spécifique avec un ribosome qui se lie à cet ARNt en présence du composé d'essai et d'un ARNm qui code pour la méthionine, et code également pour un acide aminé à l'aide d'un codon spécifique pour des mycobactéries, puis à déterminer si le composé inhibe la liaison de cet ARNt. Des anticorps spécifiques à des ASL spécifiques aux mycobactéries peuvent être utilisés dans des LFA pour diagnostiquer une infection.
PCT/US2024/010438 2023-01-05 2024-01-05 Procédés de criblage pour identifier des inhibiteurs spécifiques de mycobactéries, y compris m. tuberculosis Ceased WO2024148237A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030129679A1 (en) * 2002-01-10 2003-07-10 Siddiqi Salman H. Methods and devices for collecting and preparing specimens for detection of mycobacteria and antigens
US20100167294A1 (en) * 2008-09-23 2010-07-01 Ying Huang Methods for detecting nucleic acids in a sample
US20130137598A1 (en) * 2010-07-15 2013-05-30 University Of Pretoria Method of detecting surrogate markers in a serum sample
US20190293645A1 (en) * 2016-09-22 2019-09-26 Pace Diagnostics, Inc. Mycobacterium tuberculosis proteins in diagnostic assays and devices for tuberculosis detection and diagnosis
US20210025887A1 (en) * 2018-03-29 2021-01-28 Foundation Of Innovative New Diagnostics Antibody or antibody combination and method using same for detection of an antigen related to mycobacterium in a urine sample of a subject
WO2022109435A1 (fr) * 2020-11-23 2022-05-27 Thomas Jefferson University Fragments d'arnt et leurs procédés d'utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030129679A1 (en) * 2002-01-10 2003-07-10 Siddiqi Salman H. Methods and devices for collecting and preparing specimens for detection of mycobacteria and antigens
US20100167294A1 (en) * 2008-09-23 2010-07-01 Ying Huang Methods for detecting nucleic acids in a sample
US20130137598A1 (en) * 2010-07-15 2013-05-30 University Of Pretoria Method of detecting surrogate markers in a serum sample
US20190293645A1 (en) * 2016-09-22 2019-09-26 Pace Diagnostics, Inc. Mycobacterium tuberculosis proteins in diagnostic assays and devices for tuberculosis detection and diagnosis
US20210025887A1 (en) * 2018-03-29 2021-01-28 Foundation Of Innovative New Diagnostics Antibody or antibody combination and method using same for detection of an antigen related to mycobacterium in a urine sample of a subject
WO2022109435A1 (fr) * 2020-11-23 2022-05-27 Thomas Jefferson University Fragments d'arnt et leurs procédés d'utilisation

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