Inhibitors targeting the allosteric site of dsba

Abstract

The present disclosure relates to inhibitors of A. baumannii, P. aeruginosa, and K. pneumoniae that target Disulfide oxidoreductase (DsbA).

Description

INHIBITORS TARGETING THE ALLOSTERIC SITE OF DSBACROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This International Patent Application claims priority to U.S. Provisional Patent Application No. 63 / 519,040, filed on 11 August 2023 and U.S. Provisional Patent Application No.63 / 667,673, filed 3 July 2024, the disclosures of each of which are incorporated herein in its entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to the use of DsbA13 [l-methyl-3- (methylcarbamoyloxymethyl)-5,6-dihydro-4 / / -pyrrolo[l,2-a][l]benzazepin-2-yl]methyl / V- methylcarbamate], for the treatment of multidrug-resistant (MDR) Acinetobacter baumannii infections.BACKGROUND

[0003] The worldwide emergence of multi drug-resistant (MDR) Acinetobacter baumannii strains has raised severe healthcare concerns for deployed military service members and the civilian population. MDR A. baumannii infections are of particular interest due to the limited therapeutic treatment options for wounded soldiers infected with these pathogens. To address this problem, novel antibacterials with new mechanisms of action are urgently required to treat infections caused by MDR 4. baumannii .

[0004] There exists a need in the art for new antibiotics.SUMMARY OF THE INVENTION

[0005] In an embodiment, DsbA13 is l-methyl-3-(methylcarbamoyloxymethyl)-5,6-dihydro-477- pyrrolo[ 1 ,2-a]

[0001] benzazepin-2-yl]methyl / V-methylcarbamate (F ormula I).

[0006] In an embodiment, PTHD7 is [l-(4-bromophenyl)-2,5-dimethyl-4- (methylcarbamoyloxymethyl)pyrrol-3-yl]methyl / V-methylcarbamate (Formula II).

[0007] In an embodiment, PTHD8 is [l-(3,4-dichlorophenyl)-2,5-dimethyl-4- (methylcarbamoyloxymethyl)pyrrol-3-yl]methyl A'-m ethylcarbamate (Formula III).

[0008] In an embodiment, DsbA13 has the structure of Formula I:Formula I.

[0009] An compound (DsbA13) of Formula I:Formula I.

[0010] In an embodiment, the compound is l-methyl-3-(methylcarbamoyloxymethyl)-5,6- dihydro-4 / / -py rrol o[ 1 ,2-a]

[0001] benzazepi n-2-yl ] methyLV-m ethyl carbamate.

[0011] In an embodiment, a composition can comprise an effective amount of the compound (DsbA13) of Formula I:Formula I, a compound (PTHD7) of Formula II:Formula II,a compound (PTHD8) of Formula III:Formula III, or a mixture thereof.

[0012] In an embodiment, the compound of Formula IV.

[0013] In an embodiment, the compound of Formula V.

[0014] In an embodiment, a composition can comprise an effective amount of l-methyl-3-(methylcarbamoyloxymethyl)-5,6-dihydro-4 / f-pyrrolo[l,2-a][l]benzazepin-2-yl]methylV- methylcarbamate. In an embodiment, a composition can comprise an effective amount of the compound Formula I, II, III, or a mixture thereof. In an embodiment, a composition can comprise an effective amount of the compound Formula I. In an embodiment, a composition can comprise an effective amount of the compound Formula II. In an embodiment, a composition can comprise an effective amount of the compound Formula III. The composition can be a pharmaceutical composition. The composition can be formulated as a liquid, semi-solid, matrix, gel, or paste. The composition can be formulated to be administered by subcutaneous, intramuscular, intravenous, intraperitoneal, vaginal, rectal, intrapleural, intravesicular, intrathecal, topical, nasal, inhalation, oral administration, or a combination of routes.

[0015] In an embodiment, the composition can be a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, carrier, diluent, vehicle, adjuvant, or a combination thereof.

[0016] In an embodiment, the effective amount of the compound is between about 1 pg and 1,000 pg. The effective amount of the compound can be between about 1 pg and 100 pg, 50 pgand 250 ng, 200 ng and 500 ng, 300 ng and 700 ng, 400 ng and 800 ng, 500 ng and 1,000 pg. The effective amount of the compound can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1,000 ng.

[0017] In an embodiment, the effective amount of the compound can be between about 1 mg and 1,000 mg. The effective amount of the compound can be between about 1 mg and 100 mg, 50 mg and 250 mg, 200 mg and 500 mg, 300 mg and 700 mg, 400 mg and 800 mg, 500 mg and 1,000 mg. The effective amount of the compound can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,970, 980, 990, or 1,000 mg.

[0018] In an embodiment, the effective amount of the compound can be about between about 1 g and 1,000 g. The effective amount of the compound can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,970, 980, 990, or 1,000 g.

[0019] In an embodiment, the composition can be formulated to comprise between about 0.01 mg / mL and 1.0 mg / mL of the compound. The composition can be formulated to comprise between about 0.02 mg / mL and 1.0 mg / mL, 0.05 mg / mL and 0.8 mg / mL, 0.2 mg / mL and 0.5 mg / mL, 0.1 mg / mL and 1.0 mg / mL, 0.5 mg / mL and 0.7 mg / mL, 0.03 mg / mL and 0.3 mg / mL, 0.07 mg / mL and 0.9 mg / mL, or 0.09 mg / mL and 0.4 mg / mL of the compound. The compositioncan be formulated to comprise about 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, and 1.0 mg / mL of the compound.

[0020] In an embodiment a method for treating or preventing an Acinetobacter baumannii infection can comprise administering to a mammal in need thereof an effective amount of the compound of Formula I:

[0021] In an embodiment a method for treating or preventing an Acinetobacter baumannii infection can comprise administering to a mammal in need thereof an effective amount of 1- methyl-3-(methylcarbamoyloxymethyl)-5,6-dihydro-477-pyrrolo[l,2-a][l]benzazepin-2- yl]methyl / V-methylcarbamate.

[0022] In an embodiment, a method for treating or preventing a Gram-negative bacterial infection comprises administering to a mammal in need thereof a composition comprising the compound(DsbA13) of Formula I:Formula I, a compound (PTHD7) of Formula II:a compound (PTHD8) of Formula III:Formula III, or a mixture thereof.

[0023] In an embodiment, a method for treating or preventing a Gram-negative bacterial infection, optionally an Acinetobacter baumannii infection, can comprise administering to a mammal in need thereof a composition comprising l-methyl-3-(methylcarbamoyloxymethyl)- 5,6-dihydro-4 / / -pyrrolo[ l ,2-a][ l]bcnzazepin-2-yl]mcthylA-methylcarbamatc.

[0024] In an embodiment, a method for treating or preventing a Gram-negative bacterial infection, optionally an Acinetobacter baumannii infection, can comprise administering to a mammal in need thereof a composition comprising [l-(3,4-dichlorophenyl)-2,5-dimethyl-4- (methylcarbamoyloxymethyl)pyrrol-3-yl]methyl A'-m ethyl carbamate.

[0025] In an embodiment, a method for treating or preventing a Gram-negative bacterial infection, optionally an Acinetobacter baumannii infection, can comprise administering to a mammal in need thereof a composition comprising [l-(4-bromophenyl)-2,5-dimethyl-4- (methylcarbamoyloxymethyl)pyrrol-3-yl]methyl V-methylcarbamate.

[0026] In an embodiment, a method for treating or preventing a Gram-negative bacterial infection, optionally an Acinetobacter baumannii infection, can comprise administering to a mammal in need thereof a composition comprising a compound Formula IV, V, or a combination thereof.

[0027] In an embodiment, the Gram-negative bacterial infection is an Acinetobacter baumannii Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or acombination thereof. The Gram-negative bacterial infection can be an Acinetobacter baumannii multi-drug resistant strain. The Gram-negative bacterial infection can be a Pseudomonas aeruginosa multi-drug resistant strain. The Gram-negative bacterial infection can be a Klebsiella pneumoniae multi-drug resistant strain.

[0028] In an embodiment, the composition can be administered by subcutaneous, intramuscular, intravenous, intraperitoneal, vaginal, rectal, intrapleural, intravesicular, intrathecal, topical, nasal, inhalation, oral administration, or a combination of routes.

[0029] In an embodiment, the composition can be administered subcutaneously, optionally injected subcutaneously.

[0030] In an embodiment, the composition can be administered within 0-10 days of the onset of the Acinetobacter baumannii infection.

[0031] In an embodiment, the composition can be administered within 0-24 hours of the onset of the Acinetobacter baumannii infection.

[0032] In an embodiment, the composition can be administered at least twice within 0-24 hours of the onset of the Acinetobacter baumannii infection.

[0033] In an embodiment, the composition can be administered within 0-3 hours of the Acinetobacter baumannii infection and again 24 hours after the onset of the Acinetobacter baumannii infection. The onset can comprise infection, the manifestation of symptoms, sign, a positive diagnostic test, or a combination thereof.

[0034] In an embodiment, the composition can be administered within 0-10 days of the onset of the Gram-negative bacterial infection, optionally Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or a combination thereof.

[0035] In an embodiment, the composition can be administered within 0-24 hours of the onset of the Gram-negative bacterial infection, optionally Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or a combination thereof.

[0036] In an embodiment, the composition can be administered at least twice within 0-24 hours of the onset of the Gram-negative bacterial infection, optionally Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or a combination thereof.

[0037] In an embodiment, the composition can be administered within 0-3 hours of the Gram-negative bacterial infection, optionally Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or a combination thereof, and again 24hours after the onset of the Gram-negative bacterial infection, optionally Acinetobacter haumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or a combination thereof. The onset can comprise infection, the manifestation of symptoms, sign, a positive diagnostic test, or a combination thereof.

[0038] In an embodiment, a composition for the treatment of Acinetobacter baumannii infection can comprise an effective amount of compound (DsbA13) of Formula I:Formula I.

[0039] In an embodiment, a composition for the treatment of Gram -bacterial infection can comprise an effective amount of compound (DsbA13) of Formula I:Formula I, a compound (PTHD7) of Formula II:a compound (PTHD8) of Formula III:Formula III, or a mixture thereof.

[0040] In an embodiment, a composition for the treatment of Gram-bacterial infection can comprise an effective amount of compound of Formula IV, V, or a combination thereof.

[0041] In an embodiment, the Gram-negative bacterial infection can be an Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or a combination thereof.

[0042] In an embodiment, an isolated crystal of Acinetobacter baumannii bacterial thiol disulfide oxidoreductase DsbA substantially free of E. coli elongation factor (EcEF-Tu). The isolated crystal can be free of EcEF-TU.

[0043] In an embodiment, a composition can comprise the crystal described herein. The composition can be a pharmaceutical composition.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1A depicts the peptide binding pocket (shaded) of the X-ray crystal structure of AbDsbA for in-silico screening of the National Cancer Institute (NCI) database.

[0045] FIG. IB depicts a ball-and-stick representation of compound DsbA13 (shaded) docked into the peptide binding pocket of the X-ray crystal structure of AbDsbA.

[0046] FIG. 2 depicts the inhibition of biofilm formation of A. baumannii strains AB5075 and AB5711 at various concentrations of compound DsbA13. AB5075 is a more virulent MDR- strain. AB5711 is an MDR-strain that forms substantial biofilms.

[0047] FIG. 3 depicts the inhibition of biofilm formation of A. baumannii strains AB5075 and AB5711 at various concentrations of compound DsbA13. AB5075 is a more virulent MDR- strain. AB5711 is an MDR-strain that forms substantial biofilms.

[0048] FIG. 4 depicts a SDS-PAGE of AbDshA free from bound EcEF-TU.

[0049] FIG. 5 depicts plate-shaped AbDsbA crystal (free of EcEf-TU).

[0050] FIG. 6 depicts the structure of the inhibitor described herein “DsbA13” (the compound of Formula I).

[0051] FIG. 7 depicts a schematic representation of the disulfide-bond-forming system.

[0052] FIG. 8A depicts the X-Ray crystal structure of A. baumannii Dithiol Oxidase DsbA bound to elongation factor EF-Tu which reveals a novel protein interaction site.

[0053] FIG. 8B depicts the SDS-PAGE of AbDsbA bound with Ec.Ef.-TU.

[0054] FIG. 9 depicts two potential allosteric pockets in AbDsbA for in silica screening of compounds.

[0055] FIG. 10 depicts the Peptide binding pocket in^^DbsA for in silico screening. DsbA is represented by cyan molecular surface and the peptide is shown by bonds and labeled. Six subpockets are labeled from SI to S6.

[0056] FIG. 11 depicts the homology model of PaDsbA (P. aeruginosa)' .

[0057] FIG. 12 depicts the homology model of KpDsbA (K. pneumoniae).

[0058] FIG. 13 depicts the structures of 5 analogs of KpDsbA 13 based on AbDesbA docking with inhibitory activity against a Gram-negative bacterial infection, optionally an Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or a combination thereof.

[0059] FIG. 14A-B depicts the inhibition of biofilm formation of A. baumannii strains AB5075 and AB5711 at various concentrations of compound of Formula I (KpDsbA13). AB5075 is a more virulent MDR-strain. AB5711 is an MDR-strain that forms substantial biofilms.

[0060] FIG. 15A-B depicts the inhibition of biofilm formation ofd. baumannii strains AB5075 and AB5711 at various concentrations of compound of Formula III (PTHD8). AB5075 is a more virulent MDR-strain. AB5711 is an MDR-strain that forms substantial biofilms

[0061] FIG. 16A-B depicts the effect of KpDsbA13 (compound of Formula I) on bacterial motility. Plates of motility of A. baumannii AB5075 strain in absence (A) or presence (B) of 32pg / mL KpDsbA13 (compound of Formula I) in semi-solid 0.4% TSA after 24 h growth at 37°C. The diameter of the motility zone decreased when medium is supplemented with 75 pM KpDsbA13.

[0062] FIG. 17A-B depicts effect of PTHD8 (compound of Formula III) on bacterial motility. Plates of motility of 4. baumannii AB5075 strain in absence (A) or presence (B) of 75 uM PTHD8 in semi-solid 0.4% TSA after 24 h growth at 37°C. The diameter of the motility zone decreased when medium is supplemented with 75 pM PTHD8.

[0063] FIG. 18A-B depict the calorimetric titration of peptide versus AbDsbA (A) and calorimetric titration of KpDsbl3 versus AbDsbA (B).Definitions

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0065] ‘Non-human mammals,” as used herein, refers broadly to non-human mammalian animals, including but not limited to dogs, cats, mice, rats, guinea pigs, rabbits, ferrets, cows, horses, sheep, goats, and pigs. Non-human mammals can be mammalian pets or companion animals, including but not limited to dogs and cats and also mice, rats, guinea pigs, ferrets, and rabbits. The non-human mammal can be a dog or a cat.

[0066] “Treatment” refers broadly to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. As used herein, the term “treating,” refers broadly to treating a disease, arresting, or reducing the development of the disease or its clinical symptoms, and / or relieving the disease, causing regression of the disease or its clinical symptoms. Therapy encompasses prophylaxis, treatment, remedy, reduction, alleviation, and / or providing relief from a disease, signs, and / or symptoms of a disease. Therapy encompasses an alleviation of signs and / or symptoms in patients with ongoing disease signs and / or symptoms.Therapy also encompasses “prophylaxis”. The term “reduced”, for purpose of therapy, refers broadly to the clinical significant reduction in signs and / or symptoms. Therapy includes treating relapses or recurrent signs and / or symptoms. Therapy encompasses but is not limited to precluding the appearance of signs and / or symptoms anytime as well as reducing existing signs and / or symptoms and eliminating existing signs and / or symptoms. Therapy includes treating chronic disease (“maintenance”) and acute disease. For example, treatment includes treating or preventing relapses or the recurrence of signs and / or symptoms.

[0067] ‘Effective amount,” as used herein, refers broadly to the amount of an agent, e.g., a compound, antibody, vector or cells that, when administered to a patient for treating a disease, is sufficient to affect such treatment for the disease. The effective amount can be an amount effective for prophylaxis, and / or an amount effective for prevention. The effective amount can be an amount effective to reduce, an amount effective to prevent the incidence of signs / symptoms, to reduce the severity of the incidence of signs / symptoms, to eliminate the incidence of signs / symptoms, to slow the development of the incidence of signs / symptoms, to prevent the development of the incidence of signs / symptoms, and / or effect prophylaxis of the incidence of signs / symptoms. The “effective amount” can vary depending on the disease and its severity and the age, weight, medical history, susceptibility, and pre-existing conditions, of the patient to be treated. The term “effective amount” is synonymous with “therapeutically effective amount” for purposes described herein.

[0068] ‘Mammal,” as used herein, refers broadly to any and all warm-blooded vertebrate animals of the class Mammalia, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. Mammals include, but are not limited to, humans, domestic and farm animals, and zoo, sports, or pet animals. Similarly, the term “subject” or “patient” includes both human and veterinary subjects and / or patients.ANTIBIOTICS TARGETING DsbA

[0069] Multi drug-resistant Acinetobacter baumannii is a significant cause of nosocomial infection. Decreased susceptibility of A. baumannii strains to currently available antibiotics has necessitated the development of novel antibacterials against new drug targets. A promising drug target in A. baumannii for inhibitor development is the disulfide bond (DSB) forming system, in particular DsbA, which is a key bacterial oxidative folding catalyst. Disulfide oxidoreductase (DsbA) plays a crucial role in promoting the correct assembly of a wide range of virulencefactors required during pathogenesis. Both disulfide bond forming, and chaperone-usher pathways are required for biofilm formation, a major virulence trait of A. baumannii. As a master virulence regulator of bacterial pathogens, DsbA (a bacterial thiol disulfide oxidoreductase) is a target for anti-virulence drug development. It is also bacteria specific. The X-ray crystal structure of A. baumannii DsbA (AbDsbA) in complex with the highly conserved prokaryotic E. coli elongation factor (EcEF-Tu), revealed a novel protein interaction site (PDB ID:4P3Y). The structure revealed that the EcEF-Tu switch I region binds to the non-catalytic surface of AbDsbA. In addition, an 11-amino acid peptide (46-AFDQIDNAPEE-56) (SEQ ID NO: 1) derived from the EcEF-Tu switch I region is shown to bind to AbDsbA. The inhibitors described herein (compounds of Formula I, II, and III) have activity against Gram -negative bacteria have anti-biofilm and anti -virulence activity.

[0070] The inventors identified inhibitors of A. baumannii, P. aeruginosa and K. pneumoniae by targeting DsbA, a key enzyme of the disulfide bond-forming (Dsb) system in Gram-negative bacteria. The inventors discovered that the AbDsbA, PaDsbA and KpDsbA inhibitors will have anti-biofilm activity and potential as anti-virulence drugs.

[0071] The worldwide emergence of multi drug-resistant (MDR) Acinetobacter baumannii strains has raised severe healthcare concerns for deployed military service members and the civilian population. MDR A. baumannii infections are of particular interest due to the limited therapeutic treatment options for wounded soldiers infected with these pathogens. To address this problem, novel antibacterials with new mechanisms of actions are urgently required to treat infections caused by MDR A. baumannii. Enzymes in the bacterial disulfide bond (Dsb) forming machinery provide excellent targets for new inhibitor development. One of the enzymes, DsbA, found in the periplasmic space, catalyzes the disulfide bond formation for proper folding of proteins in bacteria and represents a promising drug target. Research studies also indicate that DsbA plays crucial role in establishing attachment and biofilm formation. The objective of this study is to develop a novel inhibitor of A. baumannii by targeting DsbA using structure-based drug design. The inhibitor described herein (compound of FORMULA I) has anti-biofilm and potential anti-virulence activity:Formula I

[0072] The shallow active site of DsbA consists of two cysteine (34C and 37C) and limits in- silico screening against the active site.

[0073] Based on the AbDsbA X-ray crystal structure (PDB ID: 4P3Y), two allosteric pockets are present. (1) At the interface between the heterodimer (DsbA and DsbB) closer to the active site. (2) At the opposite side of the active site, formed by the binding of a hexapeptide (50- IDNAPE-55) of EcEF-Tu.

[0074] In-silico screening against the second allosteric site of AbDsbA yielded 9 compounds.

[0075] Anti -biofilm activities of the compounds were evaluated against virulent MDR. .4. baumannii strain AB5075 and another MDR A. baumannii strain that forms substantial biofilms, AB5711. None of the compounds were active against the strains by themselves.

[0076] To facilitate the entry of compounds into the cell, they were tested in media containing sub-MIC of polymyxin B (0.25 pg / mL). Compound DsbA13 completely inhibited AB5075 and AB5711 biofilm formation at 32 & 64 pg / mL respectively (Figures 2 & 3). Successfully purified AbDsbA protein free from EcEF-Tu using affinity and ion-exchange chromatography (Protein concentration: 21.07 mg / mL & Yield: 8.10 mg / L).

[0077] AbDsbA crystallization hit condition (0.2M Sodium thiocyanate and 20% (w / v) PEG 3350). AbDsbA crystals diffracted x-ray at a resolution of 1.4 A.

[0078] The inventors successfully identified a potential anti-virulence / anti-biofilm compound (DsbA13) by in-silico screening of the NCI Database. The inventors were also the first to crystallize AbDsbA free from the constitutively produced EcEF-Tu protein.

[0079] Further, the inventors optimized derivatives of DsbA13 to produce PTHD7 andPTHD8:Formula II,a compound (PTHD8) Formula III:Formula III

[0080] The inventors surprisingly discovered that PTHD7 and PTHD8 do not need polymyxin for activity.Compositions

[0081] The compositions comprising the compounds of Formula I, II, III, or mixtures thereof, described herein can be formulated into a pharmaceutical composition. The composition, including pharmaceutical compositions, can comprise an adjuvant, carrier, buffers, antioxidants, wetting agents, lubricating agents, gelling agents, thickening agents, binding agents, disintegrating agents, humectants, preservatives, diluent, stabilizer, filler, excipient, or a combination thereof.

[0082] The composition, optionally pharmaceutical compositions, can be in the form of a liquid, foam, or gel. The composition, optionally pharmaceutical compositions, can be in the form of a liquid.

[0083] The compositions described herein may be formulated as a pharmaceutical composition comprising compounds of Formula I, II, III, or mixtures thereof, described herein and pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, excipient, lubricant, emulsifier, stabilizer, solvent, diluent, buffer, vehicle, or a combination thereof.

[0084] Pharmaceutically acceptable carriers are well known in the art and include, forexample, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.Pharmaceutically acceptable carriers may be a liquid, including but not limited to water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. The pharmaceutical carriers may be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, or urea. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. For example, sugars and / or amino acids may be admixed into the pharmaceutical composition. The pharmaceutical composition can comprise water, glycerin, phospholipids, or a mixture thereof.

[0085] The pharmaceutical carrier can be either solid or liquid. Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. A solid carrier can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.

[0086] The pharmaceutical compositions can include the formulation of compounds of Formula I, II, III, or mixtures thereof, described herein with encapsulating material as a carrier providing a capsule in which the inhibitor (with or without other carriers) is surrounded by the carrier, such that the carrier is thus in association with the compounds. In a similar manner, cachets can also be included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.

[0087] Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, suspensions, and emulsions suitable for oral administration. Sterile water solutions of the active component, e.g., compounds of Formula I, II, III, or mixtures thereof, described herein, or sterile solutions of the active component in solvents comprising water, buffered water, saline, PBS, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration. The composition can comprise a compound of Formula IV, V, or a mixture thereof. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.

[0088] Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician.

[0089] The compositions comprising compounds of Formula I, IT, TIT, or mixtures thereof, described herein can be administered by a route selected from the group consisting of subcutaneous, intramuscular, intravenous, intraperitoneal, vaginal, rectal, intrapleural, intravesicular, intrathecal, topical, nasal, inhalation, oral administration, or a combination of routes.

[0090] The compositions may further comprise a permeability agent, for example a polymyxin, optionally Polymyxins B and E. The Gram-negative cell wall comprises an outer membrane. The inner leaflet consists of phospholipids, whereas the outer leaflet shows lipopolysaccharides, lipoproteins and porins. Lipopolysaccharide (LPS) is the target of polymyxins. Cationic polymyxins bind to the negatively charged components of lipopolysaccharides and disrupt outer membrane. Such property of polymyxins can be exploited to allow drugs, which are impermeable to the outer membrane.

[0091] Further details described herein can be found in the following example, which further defines the scope described herein. All references cited throughout the specification, and the references cited therein, are hereby expressly incorporated by reference in their entirety.EXAMPLESEXAMPLE 1IDENTIFICATION OF ANTIBIOTIC

[0092] Methods: The X-ray crystal structure of baumannii DsbA (AbDsbA, PDB:4P3Y) in complex with E. coli Elongation Factor Tu (EcEF-Tu) led to the identification of an allosteric site. Peptides derived from EcEF-Tu bind tightly to the allosteric site. Thus, the inventors performed in-silico screening of the NCI database for potential allosteric inhibitors using the X- ray crystal structure of AbDsbA. Compounds were tested for inhibition of A. baumannii biofilm formation at various inhibitor concentrations in CAMHB media by the Minimum Biofilm Eradication Concentration (MBEC) assay. Anti-biofilm activities of compounds were evaluated against a virulent MDR A. baumannii strain, AB5075, and the substantial biofilm-forming strain, AB5711. To mitigate permeability issues and facilitate compound entry into the cell, compounds were also tested in growth media containing sub-MIC of polymyxin B (0.25 pg / mL), a concentration at which there is no effect on bacterial viability or biofilm formation. Moreover, the inventors purified AbDsbA protein separate from EcEF-Tu using affinity and ion-exchangechromatography. Affinity purification was performed in a high salt (IM NaCl) containing buffer in order to separate EcEF-Tu, which is constitutively formed during expression of AbDsbA protein.

[0093] In-silico screening and molecular docking of chemical libraries: From the interaction of the 11 -amino acid peptide (46-AFDQIDNAPEE-56) with AbDsbA, we developed a 5-point 3D stereo-electronic pharmacophore. Queries from this pharmacophore were used to perform in- silico screening of the NCI database for potential inhibitors. Molecular docking studies of the in- silico identified compounds were performed in the peptide binding pocket of AbDsbA as shown in Figure 1 and rank-ordered based on scoring functions.

[0094] Biofilm Inhibition Assay: Efficacy of compounds against A. baumannii strains AB5075 and AB5711 biofilm formation was determined by MBEC assay. Compounds were tested for biofilm inhibition at drug concentrations ranging from 8-64 pg / mL in CAMHB media. Bacterial culture was incubated at 37°C with shaking at 60 rpm for 24 hrs. Due to permeability issues of the compounds, the assay was carried out in growth media containing sub-MIC of polymyxin B (0.25 pg / mL), a concentration at which there is no effect on bacterial viability. Inhibition of AB5075 and AB5711 biofilm formation on the MBEC plate pegs was quantified at OD = 595 nm using a crystal violet assay in 96-well plates.

[0095] Protein purification: The A. baumannii DsbA gene was synthesized and cloned into the pMCSG7 vector by Genscript. AbDsbA protein was first purified by affinity chromatography in 50 mM Tris-HcL buffer, pH 8.0 containing IM NaCl to separate EcEF-Tu (constitutively produced during protein expression) and AbDsbA. This was then followed by dialysis to remove NaCl. The protein sample was further purified by ion-exchange chromatography, and purity was checked by SDS-PAGE. AbDsbA crystallization conditions were determined using commercially available crystallization screening kits and standard vapor diffusion methods.

[0096] Results: Compound DsbA13 completely inhibited AB5075 biofilm formation at 32 pg / mL and 45% biofilm inhibition at 16 pg / mL in growth media containing polymyxin as stated above. The compound completely inhibited AB5711 biofilm formation at 64 pg / mL and 68% inhibition at 32 pg / mL in polymyxin containing media. In addition, the inventors successfully purified AbDsbA protein separate from EcEF-Tu, with a concentration of 21.1 mg / mL and yield of 8.10 mg / L.

[0097] Conclusion: The in-silico compound screening strategy described herein led to thediscovery ofDsbA13, a potential anti-virulence / anti-biofilm drug. The 2D Chemical structure of DsbA13 compound with IUPAC chemical name:[ 1 -methyl -3-(methylcarbamoyloxymethyl)-5,6-dihydro-4 / / -pyrrolo[ 1 ,2-a]

[0001] benzazepin-2- yl]methyW-methylcarbarnate]EXAMPLE 2 ANTIMICROBIAL SUSCEPTIBILITY TESTING OF IN SILICO DERIVED COMPOUNDS IN GROWTH MEDIA CONTAINING SUB-MIC OF POLYMYXIN

[0098] Compounds were tested against A baumannii strains (AB5075 & AB5711), K. pneumonaie (KP4640) and P. aeruginosa (PA01) to determine MIC. None of the above compounds was active against the respective pathogens (MIC >256 pg / mL). Drug Concentration range tested: 0.5-256 pg / mL. Permeability issue of the compounds.

[0099] TABLE 1 : In silico screening of chemical library

[0100] Due to permeability issues of the compounds, the AST was carried out in growthmedia containing sub-MIC of polymyxin B (0.25 pg / mL), a concentration at which there is no effect on bacterial viability. Out of the 38 in silico screened compounds only one was active against A baumannii, K. pneumoniae and P. aeruginosa strains.

[0101] TABLE 2

[0102] The inventors further modified compounds to arrive at a new set of compounds. 14 PTHD series of compounds and 13 THDsbA series of compounds were obtained and screened and then modified. Most of these compounds have a basic group to optimize permeability.

[0103] TABLE 3Table 4

[0104] The inventors also purified and crystallized AbDsbA, PaDsbA, and KpDsbA proteins. Plate-shaped AbDsbA crystal (free of EcEF-TU). Crystallization condition: 0.2M Sodium thiocyanate and 20% (w / v) PEG 3350. AbDsbA crystals diffracted to E4 A. Needle-shaped PaDsbA crystals. Hit condition: 2.0 M AmS04, lOOmM citric acid and 200mM Potassium Sodium tetra tryhydrate. KpDsbA crystallization condition: 0.1 M Tris-HCl pH8.6, 2.2 M Ammonium sulfate

Claims

What is claimed:

1. A method of treating or preventing a Gram-negative bacterial infection comprising administering to a mammal in need thereof an effective amount of a compound ofFormula I:Formula I, a compound of Formula II:a compound of Formula TIT:Formula ITT, or a mixture thereof.

2. A method of treating or preventing a Gram-negative bacterial infection comprising administering to a mammal in need thereof a composition comprising a compound of Formula I:Formula I,a compound of Formula II:Formula II, a compound of Formula III:Formula III, or a mixture thereof.

3. The method of claim 1 or 2, wherein the Gram-negative bacterial infection is an Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or a combination thereof.

4. The method of any one of claims 1-3, wherein the Acineiobacter baumannii is a multidrug resistant strain.

5. The method of any one of claims 1-3, wherein the Pseudomonas aeruginosa is a multidrug resistant strain.

6. The method of any one of claims 1-3, wherein the Klebsiella pneumoniae is a multi-drug resistant strain.

7. The method of any one of claims 1-6, wherein the composition is formulated as a liquid, semi-solid, gel, matrix, or paste.

8. The method of any one of claims 1-7, wherein the composition is formulated as a liquid.

9. The method of any one of claims 1-8, wherein the composition is administered by subcutaneous, intramuscular, intravenous, intraperitoneal, vaginal, rectal, intrapleural, intravesicular, intrathecal, topical, nasal, inhalation, oral administration, or a combination of routes.

10. The method of any one of claims 1-9, wherein the composition is administered subcutaneously, optionally injected subcutaneously.

11. The method of any one of claims 1-10, wherein the composition is administered within 0- 10 days of the onset of the Gram-negative bacterial infection.

12. The method of any one of claims 1-11, wherein the composition is administered within 0- 24 hours of the onset of the Gram-negative bacterial infection.

13. The method of any one of claims 1-12, wherein the composition is administered at least twice within 0-24 hours of the onset of the Gram-negative bacterial infection.

14. The method of any one of claims 1-13, wherein the composition is administered within 0- 3 hours of the Gram-negative bacterial infection and again 24 hours after the onset of the Gram-negative bacterial infection.

15. The method of any one of claims 1-14, wherein the onset comprises infection, the manifestation of symptoms, sign, a positive diagnostic test, or a combination thereof.

16. The method of any one of claims 1-15, wherein the method further comprises administering a polymyxin, optionally polymyxin B, polymyxin E, or a combination thereof.

17. A composition for treatment of a Gram-negative bacterial infection comprising an effective amount of a compound of Formula I:Formula I.

18. A composition for treatment of a Gram-negative bacterial infection comprising an effective amount of 1 -methyl -3-(methylcarbamoyloxymethyl)-5,6-dihydro-4 / Z- pyrrolo[ 1 ,2-a] [ 1 ]benzazepin-2-yl]methyLV-methylcarbamate.

9. A composition for treatment of a Gram-negative bacterial infection comprising an effective amount of a compound of Formula IFormula I, a compound of Formula II:a compound of Formula III:Formula III, or a mixture thereof.

20. The composition of any one of claims 17-19, wherein the Gram-negative bacterial infection is an Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterial infection, or a combination thereof.

21. The composition of any one of claim 17-20, wherein the Acinetobacter baumannii is a multi-drug resistant strain.

22. The composition of any one of claim 17-21, wherein the Pseudomonas aeruginosa is a multi-drug resistant strain.

23. The composition of any one of claim 17-22, wherein the Klebsiella pneumoniae is a multi-drug resistant strain.

24. An isolated crystal of Acinetobacter baumannii bacterial thiol disulfide oxidoreductase DsbA substantially free of E. coli elongation factor (EcEF-Tu).

25. The isolated crystal of claim 24, wherein the crystal of AbDsbA is free of EcEF-TU.

26. A composition comprising the crystal of claim 24 or 25.

27. The composition of claim 26, wherein the composition is a pharmaceutical composition.

28. A compound of Formula I:Formula I.

29. A compound having the structure l-methyl-3-(methylcarbamoyloxymethyl)-5,6-dihydro- 4#-pyrrolo[l,2-a][l]benzazepin-2-yl]methyLV-methylcarbamate.

30. A compound of Formula II:Formula II.

31. A compound having the structure [l-(4-bromophenyl)-2,5-dimethyl-4- (methylcarbamoyloxymethyl)pyrrol-3-yl]methyl .V-methylcarbamate.

32. A compound of Formula III:Formula III.

33. A compound having the structure [l-(3,4-dichlorophenyl)-2,5-dimethyl-4- (methylcarbamoyloxymethyl)pyrrol-3-yl]methyl .V-methylcarbamate.

34. A composition comprising an effective amount of the compound of any one of claims 28- 33 or a mixture thereof.

35. The composition of claim 34, wherein the composition is a pharmaceutical composition.

36. The composition of claim 34 or 35, wherein the composition is formulated as a liquid, semi-solid, matrix, gel, or paste.

37. The composition of any one of claims 34-36, wherein the composition is formulated to be administered by subcutaneous, intramuscular, intravenous, intraperitoneal, vaginal, rectal, intrapleural, intravesicular, intrathecal, topical, nasal, inhalation, oral administration, or a combination of routes.

38. The composition of any one of claims 34-37, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, carrier, diluent, vehicle, adjuvant, or a combination thereof.

39. The composition of any one of claims 34-38, wherein the effective amount of the compound is between about 1 pg and 1,000 pg.

40. The composition of any one of claims 34-39, wherein the effective amount of the compound is between about 1 pg and 100 pg, 50 pg and 250 pg, 200 pg and 500 pg, 300 pg and 700 pg, 400 pg and 800 pg, 500 pg and 1,000 pg.

41. The composition of any one of claims 34-40, wherein the effective amount of the compound is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,960, 970, 980, 990, or 1,000 pg.

42. The composition of any one of claims 34-41, wherein the effective amount of the compound is between about 1 mg and 1,000 mg.

43. The composition of any one of claims 34-42, wherein the effective amount of the compound is between about 1 mg and 100 mg, 50 mg and 250 mg, 200 mg and 500 mg, 300 mg and 700 mg, 400 mg and 800 mg, 500 mg and 1,000 mg.

44. The composition of any one of claims 34-43, wherein the effective amount of the compound is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,960, 970, 980, 990, or 1,000 mg.

45. The composition of any one of claims 34-44, wherein the effective amount of the compound is between about 1 g and 1,000 g.

46. The composition of any one of claims 34-45, wherein the effective amount of the compound is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1,000 g.

47. The composition of any one of claims 34-46, wherein the composition is formulated to comprise between about 0.01 mg / mL and 1.0 mg / mL of the compound.

48. The composition of any one of claims 34-47, wherein the composition is formulated to comprise between about 0.02 mg / mL and 1.0 mg / mL, 0.05 mg / mL and 0.8 mg / mL, 0.2 mg / mL and 0.5 mg / mL, 0.1 mg / mL and 1.0 mg / mL, 0.5 mg / mL and 0.7 mg / mL, 0.03 mg / mL and 0.3 mg / mL, 0.07 mg / mL and 0.9 mg / mL, or 0.09 mg / mL and 0.4 mg / mL of the compound.

49. The composition of any one of claims 34-48, wherein the composition is formulated to comprise about 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, and 1.0 mg / mL of the compound.

50. The composition of any one of claims 34-49, wherein the composition further comprises an effective amount of polymyxin, optionally Polymyxin B and / or E.

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