WO2019174277A1

WO2019174277A1 - Phosphoethanolamine transferase inhibitors

Info

Publication number: WO2019174277A1
Application number: PCT/CN2018/114069
Authority: WO
Inventors: Sheng Chen; Kin-Fai Chan; Chen Xu
Original assignee: The Hong Kong Polytechnic University
Priority date: 2018-03-13
Filing date: 2018-11-06
Publication date: 2019-09-19

Abstract

Provided herein are methods and compounds useful as bactericides. The compounds and methods can be used for inhibiting the function of mobilized colistin resistance (MCR) enzymes and are useful in the treatment of bacterial infections caused by bacteria that express a mcr gene.

Description

Phosphoethanolamine Transferase Inhibitors

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of United States provisional application 62/642,080, filed on March 13, 2018, the contents of which being hereby incorporated by reference in there entirety for all purposes.

BACKGROUND

Clinical and healthcare problems due to multidrug-resistant bacterial infections have worsened in recent years following the emergence of bla _NDM-1 and bla _KPC-2, mobile elements that can mediate development of carbapenem resistance in the host strain, and possess the ability to disseminate rapidly among various species of bacterial pathogens. Current descriptions such as “superbug” and “post-antibiotic era” reflect the seriousness of the antimicrobial resistance issue.

Among the major multidrug-resistant organisms that emerged within the past two decades, carbapenem-resistant Enterobacteriaceae (CRE) , which commonly cause untreatable and hard-to-treat infections among hospitalized patients, is considered an urgent threat according to a report by the Center for Diseases Control and Prevention (CDC) in 2013 on antibiotic resistance threats in the United States, where more than 9,000 healthcare-associated infections are being caused by CRE each year, resulting in up to 50% mortality rate, especially among patients who suffer from CRE-related bloodstream infections ⁴.

Phosphate polymyxins (polymyxins) are considered the last resort antibiotics for treatment of CRE infections due to its high efficacy and low resistance rate in clinical CRE. Polymyxins are polycationic antimicrobial peptides which bind to negatively charged phospholipids in cell membranes of Gram-negative bacteria, leading to structural damage and functional alteration of the cell membrane, and hence bacterial death. This antibiotic class includes polymyxin A through E, with polymyxin B being the major type in the market. Despite being discovered more than 50 years ago, they are rarely used clinically and have been discontinued briefly due to their nephrotoxic and neurotoxic side effects. As a result of the increasing prevalence of carbapenem-resistant Enterobacteriaceae (CRE) , pan-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, however, polymyxins were re-evaluated recently and are currently considered one of the few remaining choices for treatment of multidrug-resistant Gram-negative bacterial infections. In fact, polymyxins have become the most reliable choice to treat critically ill patients infected with strains of the CRE in recent years.

Bacterial resistance to polymyxins was previously thought to be rare and mainly attributed to chromosomal mutations in the two component regulatory systems (e.g., pmrAB, phoPQ, and its negative regulator mgrB in the case of K. pneumoniae) , leading to modification of lipid A and total loss of the lipopolysaccharide. Recently, a new plasmid-mediated colistin resistance mechanism encoding the MCR-1 protein, a phosphoethanolamine transferase that structurally modifies lipid A, has been discovered. The mcr-1 gene has been reported in various species of bacteria including E. coli, Klebsiella pneumoniae, Salmonella and Pseudomonas aeruginosa collected from both clinical and environmental sources in various parts of the world. The gene has since been detectable in various conjugative plasmids transmissible among different bacterial species. These plasmids were found to be stably inherited in bacteria even without polymyxin selective pressure. Through development and utilization of a specific method for the isolation of mcr-1-bearing bacteria, we recently reported that mcr-1-bearing organisms were commonly detectable in the ecosystem, with as much as ～50% of animal and meat products being contaminated by such organisms. Furthermore, mcr-1-bearing bacteria were found to have colonized the GI tract of both hospital patients and healthy individuals, including children as young as two months old, suggesting that the mcr-1 gene could be readily transferred to other bacterial pathogens in the human GI tract or disseminated to different environmental niches and acquired by environmental organisms. Worse still, the mcr-1 gene was also reported to be co-harbored by CRE carrying bla _KPC, bla _NDM and bla _VIM rendering such organisms a veritable "superbug” . These findings suggest that development of effective inhibitors for MCR-1 may be the only effective strategies to prolong the use of colistin as a last-line antibiotic to treat life-threatening bacterial infections. Thus, there exists a need to identify new inhibitors of MCR-1.

SUMARY OF THE INVENTION

Provided herein are compounds useful as bactericides. The compounds described herein can at least partially inhibit the function of MCR enzymes and thus can be used in reversing polymyxin resistance in bacteria expressing mcr genes.

In a first aspect, provided herein is a method of treating a bacterial infection in a patient comprising the step of co-administering a pharmaceutically acceptable amount of a polymoxin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable amount of a compound of formula I to a patient in need thereof, wherein the compound of formula I has the structure:

or a pharmaceutically acceptable salt thereof, wherein

Ar is aryl or heteroaryl;

X is -O-, -NR ¹-, or a bond;

A is - (CR ² ₂) _m-;

Y is -O-, -NR ¹-, - (NR ¹ ₂) -, or a bond;

Z is - (CR ² ₂) _n-;

R is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl;

R ¹ for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; or two instances of R ¹ taken together with the nitrogen to which they are attached form a 3-6 membered heterocyclic ring;

R ² for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; or two instances of R ² taken together with the carbons to which they are attached form a 3-6 membered heterocyclic ring; m for each instance is independently a whole number selected from 2-14; and n for each instance is independently a whole number selected from 2-14.

In a first embodiment of the first aspect, provided herein is the method of the first aspect, wherein Ar is selected from the group consisting of aryl, pyrimidine, and imidazole.

In a second embodiment of the first aspect, provided herein is the method of the first aspect, wherein X is -O-; Y is - (NR ¹ ₂) ; m is a whole number selected from 2-6; and n is a whole number selected from 6-14.

In a third embodiment of the first aspect, provided herein is the method of the second embodiment of the first aspect, wherein Ar is aryl and R ₁ for each instance is independently alkyl.

In a fourth embodiment of the first aspect, provided herein is the method of the first aspect, wherein X and Y are a bond; n and m are whole numbers independently selected from 3-8 and Ar is represented by the structure:

In a fifth embodiment of the first aspect, provided herein is the method of the first aspect, wherein X is -O-; Y is - (NR ¹) -or -O-; m is a whole number selected from 2-4; and n is a whole number selected from 6-14.

In a sixth embodiment of the first aspect, provided herein is the method of the fifth embodiment of the first aspect, wherein R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl.

In a seventh embodiment of the first aspect, provided herein is the method of the first aspect, wherein X is - (NR ¹) -; Y is -O-; m is a whole number selected from 2-4; and n is a whole number selected from 6-14.

In an eighth embodiment of the first aspect, provided herein is the method of the seventh embodiment of the first aspect, wherein R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl.

In a ninth embodiment of the first aspect, provided herein is the method of the first aspect, wherein the polymyxin is colistin or a pharmaceutically acceptable salt thereof.

In a tenth embodiment of the first aspect, provided herein is the method of the first aspect, wherein the bacterial infection is caused by a bacteria that expresses a mobilized colistin resistance (mcr) gene.

In an eleventh embodiment of the first aspect, provided herein is the method of the ninth embodiment of the first aspect, wherein the method comprises co-administering a pharmaceutically effective amount of a polymyxin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable amount of a compound of formula I to a patient in need thereof, wherein the bacterial infection is caused by a bacteria that expresses mcr-1 and the compound of formula I has the structure:

or a pharmaceutically acceptable salt thereof, wherein

Ar is aryl; X is -O-; Y is - (NR ¹ ₂) ; R ₁ for each instance is independently alkyl; m is a whole number selected from 2-6; and n is a whole number selected from 6-14;

X is -O-; Y is - (NR ¹) -or -O-; R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl; m is a whole number selected from 2-4; and n is a whole number selected from 6-14; or

X is - (NR ¹) -; Y is -O-; R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl; m is a whole number selected from 2-4; and n is a whole number selected from 6-14.

In a twelfth embodiment of the first aspect, provided herein is the method of the eleventh embodiment of the first aspect, wherein the bacteria is an E. coli strain selected from the group consisting of 7, 119, WZ3951, WZ3903, 101, CX48, WZ3955, XH69, WZ2431, WZ3920, J53, and BL21.

In a thirteenth embodiment of the first aspect, provided herein is the method of the eleventh embodiment of the first aspect, wherein the compound of formula I is a salt of cetylpyridinium or domiphen.

In a fourteenth embodiment of the first aspect, provided herein is the method of the thirteenth embodiment of the first aspect, wherein the polymyxin is colistin or a pharmaceutically acceptable salt thereof.

In a second aspect, provided herein is a method of inhibiting the function of a MCR enzyme comprising the step of contacting a compound of formula I with the MCR enzyme, wherein the compound of formula I has the structure:

or a pharmaceutically acceptable salt thereof, wherein

Ar is aryl or heteroaryl;

X is -O-, -NR ¹-, or a bond;

A is - (CR ² ₂) _m-;

Y is -O-, -NR ¹-, - (NR ¹ ₂) -, or a bond;

Z is - (CR ² ₂) _n-;

In a first embodiment of the second aspect, provided herein is the method of the second aspect, wherein Ar is aryl; X is -O-; Y is - (NR ¹ ₂) ; R ₁ for each instance is independently alkyl; m is a whole number selected from 2-6; and n is a whole number selected from 6-14;

In a second embodiment of the second aspect, provided herein is the method of the second aspect, wherein the compound of formula I is a salt of cetylpyridinium or domiphen.

In a third embodiment of the second aspect, provided herein is the method of the second aspect, wherein the step of contacting the compound of formula I and the MCR enzyme occurs in a bacterial cell.

In a fourth embodiment of the second aspect, provided herein is the method of the third embodiment of the second aspect, wherein the step of contacting the compound of formula I and the MCR enzyme occurs in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which:

Figure 1 depicts Killing curves of E. coli J53 expressing MCR-1 upon treatment with colistin alone (A) , 20μM of CET in combination with a range of concentrations of colistin (B) , 40μM of DOM in combination with a range of concentrations of colistin (C) .

Figure 2 depicts microscopic image of mcr-1-bearing E. coli J53 strain in the presence or absence of MCR-1 inhibitor and colistin.

Figure 3 depicts the enzymatic activity assay of MCR-1-ED. Thin layer chromatography assay results showing fluorescent lipid substrate NBD-PEA, Acyl 12: 0 NBD-glycerol-3-phosphoethanolamine (A) , which was used as substrate and incubated with the soluble domain of MCR-1, MCR-1-ED in the absence of inhibitor (B) and in the presence of compounds, CET (C) and DOM (D) with an Enzyme: CET/DOM molar ratio of 1: 100.

Figure 4 depicts the observation of Lipid A and its modified products in MCR-1 activity assay by MALDI-TOF mass spectrometry. (A) Lipid A extracted from E. coli was observed on a Bruker UltrafleXtreme MALDI-TOF-TOF Mass Spectrometer at m/z 1796 using 2, 5-Dihydroxybenzoic acid as matrix (negative-ion mass spectra) confirming that the lipid A has the correct molecular weight; (B) Same lipid A was observed on a Bruker UltrafleXtreme MALDI-TOF-TOF Mass Spectrometer at m/z 1686 using α-Cyano-4-hydroxycinnamic acid as matrix (positive-ion mass spectra) , that was used for activity assay due to the high sensitivity of detection probably due to that the modified lipid A becomes more hydrophilic and is more compatible with this matrix. It is noted that the signal of lipid A in α-Cyano-4-hydroxycinnamic acid is much stronger than in 2, 5-Dihydroxybenzoic acid, which is probably due to the improved solubility of lipid A in the former matrix. (C) A portion of the positive-ion MALDI-TOF mass spectrum of Lipid A (1mg/ml) treated with MCR-1-ED (48μg/ml) and substrate PE (1mg/ml) for 3h. (D) A portion of the positive-ion MALDI-TOF mass spectrum of Lipid A (1mg/ml) treated with MCR-1 (48μg/ml) and substrate PE (1mg/ml) for 3h. (E) A portion of the positive-ion MALDI-TOF mass spectrum of Lipid A (1mg/ml) treated with MCR-1-ED (48μg/ml) and substrate PE (1mg/ml) for 3h. (F) MALDI-TOF mass spectrum of Lipid A (1mg/ml) in the presence of CET or DOM (D) . (G) A portion of the positive-ion MALDI-TOF mass spectrum of Lipid A (1mg/ml) treated with MCR-1 (L ¹⁰⁹R) (48μg/ml) and substrate PE (1mg/ml) for 3h. (H) A portion of the positive-ion MALDI-TOF mass spectrum of Lipid A (1mg/ml) treated with MCR-1 (L ¹⁰⁶R, A1 ⁰⁹R) (48μg/ml) and substrate PE (1mg/ml) for 3h.

Figure 5 depicts the structural basis of inhibition of MCR-1 by CET and DOM. (A) Alignment of nmEptA and modeled MCR-1. (B) Binding of detergent dodecyl-beta-d-maltoside to the hydrophobic pocket channel of nmEptA. (C) Binding of CET or DOM (D) to the hydrophobic pocket channel of MCR-1 based on molecular docking. MCR-1 was at close mode. (E) Insertion of the lipid chain of lipid A to the hydrophobic pocket channel of MCR-1 to stabilize lipid A for further modification. MCR-1 was at open mode. (F) The A ¹⁰⁹R and L ¹⁰⁶R substitutions in MCR-1 block the hydrophobic pocket channel of MCR-1, thereby inhibiting MCR-1 activity.

Figure 6 depicts the mechanism of action of MCR-1. (A) As a membrane protein, the transmembrane domain of MCR-1 is anchored onto the inner membrane of Gram-negative bacteria such as E. coli. (1) Upon stimulation by membrane lipid, the active domain and the transmembrane domain open up along the linker between these two domains to expose the active site of MCR-1. (2) Substrate PE binds to the active site domain of MCR-1 and transfers PEA from PE to T ²⁸⁵. (3) Another substrate, lipid A, binds to various residues in the active site domain and the lipid chain of lipid A inserts into the hydrophobic channel pocket of TDM to allow lipid A to stably anchor into MCR-1, thereby facilitating modification of lipid A. (4) Upon transferal of PEA from T ²⁸⁵ to lipid A, the substrate leaves MCR-1 and starts another cycle of substrate recognition and cleavage.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are compounds that act synergistically with colistin to significantly enhance its potency and re-sensitize mcr-1-bearing bacterial pathogens through specific binding and inhibition of MCR-1 activity. Furthermore, utilization of the compounds described herein allowed us to depict, for the first time, the mechanisms of action of MCR-1 and other phosphoethanolamine transferases.

Definitions

The definitions of terms used herein are meant to incorporate the present state-of-the-art definitions recognized for each term in the chemical and pharmaceutical fields. Where appropriate, exemplification is provided. The definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.

Where stereochemistry is not specifically indicated, all stereoisomers of the inventive compounds are included within the scope of the invention, as pure compounds as well as mixtures thereof. Unless otherwise indicated, individual enantiomers, diastereomers, geometrical isomers, and combinations and mixtures thereof are all encompassed by the present invention. Polymorphic crystalline forms and solvates are also encompassed within the scope of this invention.

As used herein, the term "amino acid" refers to molecules containing both a carboxylic acid moiety and an amino moiety. The carboxylic acid and amino moieties are as defined below. Both naturally occurring and synthetically derived amino acids are encompassed in the scope of this invention.

The term "heteroatom" is art-recognized and refers to an atom of any element other than carbon or hydrogen. Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term "alkyl" is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C ₁-C ₃₀ for straight chain, C ₃-C ₃₀ for branched chain) , and alternatively, about 20 or fewer. Likewise, cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, "lower alkyl" refers to an alkyl group, as defined above, but having from one to about ten carbons, alternatively from one to about six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

The term "aralkyl" is art-recognized and refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group) .

The terms "alkenyl" and "alkynyl" are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The term "aryl" is art-recognized and refers to 5-, 6-and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, naphthalene, anthracene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics. " The aromatic ring may be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF ₃, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings" ) wherein at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and para are art-recognized and refer to 1, 2-, 1, 3-and 1, 4-disubstituted benzenes, respectively. For example, the names 1, 2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms "heterocyclyl", "heteroaryl" , or "heterocyclic group" are art-recognized and refer to 3-to about 10-membered ring structures, alternatively 3-to about 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles may also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring may be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF ₃, -CN, or the like.

The term "optionally substituted" refers to a chemical group, such as alkyl, cycloalkyl aryl, and the like, wherein one or more hydrogen may be replaced with a with a substituent as described herein, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF ₃, -CN, or the like

The term "nitro" is art-recognized and refers to NO ₂; the term "halogen" is art-recognized and refers to -F, -Cl, -Br or -I; the term "sulfhydryl" is art-recognized and refers to -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" is art-recognized and refers to -SO ₂-. "Halide" designates the corresponding anion of the halogens, and "pseudohalide" has the definition set forth on 560 of "Advanced Inorganic Chemistry" by Cotton and Wilkinson.

The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formulas:

wherein R ₅₀, R ₅₁ and R ₅₂ each independently represent a hydrogen, an alkyl, an alkenyl, - (CH ₂) _mR ₆₁, or R ₅₀ and R ₅₁, taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R ₆₁ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In other embodiments, R ₅₀ and R ₅₁ (and optionally R ₅₂) each independently represent a hydrogen, an alkyl, an alkenyl, or - (CH ₂) _mR ₆₁. Thus, the term "alkylamine" includes an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R ₅₀ and R ₅₁ is an alkyl group.

The terms "alkoxyl" or "alkoxy" are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O- (CH ₂) _mR ₆₁, where m and R ₆₁ are described above.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations.

Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. In addition, polymers of the present invention may also be optically active. The present invention contemplates all such compounds, including cis-and trans-isomers, R-and S-enantiomers, diastereomers, (D) -isomers, (L) -isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.

The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The term "pharmaceutically acceptable salt" or "salt" refers to a salt of one or more compounds. Suitable pharmaceutically acceptable salts of compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, benzoic acid, acetic acid, citric acid, tartaric acid, phosphoric acid, carbonic acid, or the like. Where the compounds carry one or more acidic moieties, pharmaceutically acceptable salts may be formed by treatment of a solution of the compound with a solution of a pharmaceutically acceptable base, such as lithium hydroxide, sodium hydroxide, potassium hydroxide, tetraalkylammonium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, ammonia, alkylamines, or the like.

The term "subject" as used herein, refers to an animal, typically a mammal or a human, that will be or has been the object of treatment, observation, and/or experiment. When the term is used in conjunction with administration of a compound or drug, then the subject has been the object of treatment, observation, and/or administration of the compound or drug.

The terms "co-administration" and "co-administering" refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents) , as long as the therapeutic agents are present in the patient to some extent at the same time.

The term "therapeutically effective amount" as used herein, means that amount of active compound or pharmaceutical agent that elicits a biological or medicinal response in a cell culture, tissue system, animal, or human that is being sought by a researcher, veterinarian, clinician, or physician, which includes alleviation of the symptoms of the disease, condition, or disorder being treated.

The term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.

The term "pharmaceutically acceptable carrier" refers to a medium that is used to prepare a desired dosage form of a compound. A pharmaceutically acceptable carrier can include one or more solvents, diluents, or other liquid vehicles; dispersion or suspension aids; surface active agents; isotonic agents; thickening or emulsifying agents; preservatives; solid binders; lubricants; and the like. Remington's Pharmaceutical Sciences, Fifteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1975) and Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe ed. (American Pharmaceutical Assoc. 2000) , disclose various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

Eight different mobile colistin resistance genes (mcr) and their variants have been identified, namely mcr-1 (11 variants) , mcr-2 (three variants) , mcr-3 (ten variants) , mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8. The mcr genes encode MCR enzymes, classified as phosphatidylethanolamine transferases, which act to transfer the phosphatidylethanolamine residue to lipid A of the bacterial cell membrane

Lipid A of the bacterial outer membrane is the target binding site for colistin, which upon binding to it, disrupts it by displacing magnesium and calcium, thus causing cell death. The presence of the phosphatidylethanolamine residue lowers the affinity of the lipid A to colistin and related polymyxins thereby rendering the antimicrobial inactive or less effective and making the bacteria resistant.

Provided herein are compounds and methods useful in the inhibition of MCR enzymes and re-sensitizing resistant bacteria (e.g., bacteria that express a mcr gene) to polymyxins and related compounds.

The compounds described herein are capable of binding to and at least partially inhibiting the function of MCR enzymes, thus restoring the sensitivity of bacterial cells expressing a mcr gene to treatment with a polymyxin or salt thereof. The compounds described herein can generally be represented by the compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein Ar is aryl or heteroaryl; X is -O-, -NR ¹-, or a bond; A is - (CR ² ₂) _m-; Y is -O-, -NR ¹-, - (NR ¹ ₂) -, or a bond; Z is - (CR ² ₂) _n-; R is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; R ¹ for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; or two instances of R ¹ taken together with the nitrogen to which they are attached form a 3-6 membered heterocyclic ring; R ² for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; or two instances of R ² taken together with the carbons to which they are attached form a 3-6 membered heterocyclic ring; m for each instance is independently a whole number selected from 2-14; and n for each instance is independently a whole number selected from 2-14.

Ar can represent an aryl or a heteroaryl. In instances in which Ar represents an aryl, the aryl can be any carbocyclic aromatic group. In certain embodiments, the carbocylic aromatic group contains 6-14 carbons in the aromatic ring structure. In certain embodiments, the aryl is selected from the group consisting of phenyl, biphenyl, naphthalene, anthracene, phenanthrene, and the like. The aryl can be optionally substituted at one or more ring positions with such substituents as described above.

In instances in which Ar represents heteroaryl, the heteroaryl can be any heteroaromatic group. In certain embodiments, the heteroaromatic group contains between 4-14 carbons in the ring structure and whose ring structure includes one to four heteroatoms selected from the group consisting of oxygen, sulfur, nitrogen, and phosphorus. In certain embodiments, the heteroaryl is selected from the group consisting of pyrrole, furan, thiophene, imidazole, thiazole, thioimidazole, triazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, benzothiophene, benzofuran, and the like. The heteroaryl can be optionally substituted at one or more ring positions with such substituents as described above.

In certain embodiments, Ar is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, wherein R ³ is amino, carboxylic acid, or hydroxyl.

In certain embodiments, Ar is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, wherein R ³ is NH ₂, OH, or CO ₂H.

In instances in which X is a bond, Ar is directly covalently bonded to group A, as shown below:

Likewise, in instances in which Y is a bond, the groups A and Z are directly covalently bonded as shown below:

In instances where both X and Y are each a bond, Ar is directly covalently bonded to group A and the groups A and Z are directly covalently bonded, as shown below:

In certain embodiments, m is a whole number selected from 2-12; 2-10; 2-8; 2-6; or 2-4. In such embodiments, A can be represented by - (CR ² ₂) _2-12-; - (CR ² ₂) _2-10-; - (CR ² ₂) _2-8-; - (CR ² ₂) _2-6-; or - (CR ² ₂) _2-4-.

In certain embodiments, n is a whole number selected from 2-12; 2-10; 2-8; 2-6; or 2-4. In such embodiments, Z can be represented by - (CR ² ₂) _2-12-; - (CR ² ₂) _2-10-; - (CR ² ₂) _2-8-; - (CR ² ₂) _2-6-; or - (CR ² ₂) _2-4-.

In instances in which Y is - (NR ¹ ₂) -, Y can be represented by the structure:

When Y is - (NR ¹ ₂) -and in the absence of moieties that have a negative charge, the compound of formula I has a net positive charge and would thus exist in the presence of an anion as a salt.

In certain embodiments, R is selected from the group consisting of hydrogen, C _1-C ₆ alkyl, C _2-C ₆ alkenyl, C _2-C ₆ alkynyl, C _3-C ₆ cycloalkyl, aryl containing 6-10 carbons in the ring structure, and heteroaryl containing 3-9 carbons and 1 or 2 heteroatoms in the ring structure, wherein the heteroatoms are selected from the nitrogen, oxygen, sulfur, and phosphorus. In certain embodiments, R is selected from the group consisting of hydrogen, C _1-C ₆ alkyl, and C _3-C ₆ cycloalkyl. In certain embodiments, R is selected from the group consisting of hydrogen and C _1-C ₆ alkyl.

In certain embodiments, R ¹ for each instance is independently selected from the group consisting of hydrogen, C _1-C ₆ alkyl, C _2-C ₆ alkenyl, C _2-C ₆ alkynyl, C _3-C ₆ cycloalkyl, aryl containing 6-10 carbons in the ring structure, and heteroaryl containing 3-9 carbons and 1 or 2 heteroatoms in the ring structure, wherein the heteroatoms are selected from the nitrogen, oxygen, sulfur, and phosphorus. In certain embodiments, R ¹ is selected from the group consisting of hydrogen, C _1-C ₆ alkyl, and C _3-C ₆ cycloalkyl. In certain embodiments, R ¹ is selected from the group consisting of hydrogen and C _1-C ₆ alkyl.

In certain embodiments, R ² for each instance is independently selected from the group consisting of hydrogen, C _1-C ₆ alkyl, C _2-C ₆ alkenyl, C _2-C ₆ alkynyl, C _3-C ₆ cycloalkyl, aryl containing 6-10 carbons in the ring structure, and heteroaryl containing 3-9 carbons and 1 or 2 heteroatoms in the ring structure, wherein the heteroatoms are selected from the nitrogen, oxygen, sulfur, and phosphorus. In certain embodiments, R ² is selected from the group consisting of hydrogen, C _1-C ₆ alkyl, and C _3-C ₆ cycloalkyl. In certain embodiments, R ² is selected from the group consisting of hydrogen and C _1-C ₆ alkyl.

In certain embodiments,

represents a moiety selected from the group consisting of:

wherein n is a whole number selected from 6-13.

In certain embodiments, Ar is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof,

wherein R ³ is amino, carboxylic acid, or hydroxyl; X is -O-, -NR ¹-, or a bond; A is A is - (CR ² ₂) _m-; Y is -O-, -NR ¹-, - (NR ¹ ₂) -, or a bond; Z is - (CR ² ₂) _n-; R is selected from the group consisting of hydrogen or alkyl; R ¹ for each instance is independently selected from the group consisting of hydrogen and alkyl; andR ² for each instance is independently selected from the group consisting of hydrogen and alkyl.

In certain embodiments, Ar is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, wherein R ³ is amino, carboxylic acid, or hydroxyl; and

represents a moiety selected from the group consisting of:

wherein n is a whole number selected from 6-13.

In certain embodiments, the compound of formula I is represented by the following formula:

wherein n is a whole number selected from 12-20; and Q is a pharmaceutically acceptable anion. In certain embodiments, n is a whole number selected from 12-18; 14-18; 14-20; or 16-20. In certain embodiments, Q is selected from the group consisting of chloride, bromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, benzoate, and the like.

wherein n is a whole number selected from 8-16; R ¹ for each instance is independently alkyl; and Q is a pharmaceutically acceptable anion. In certain embodiments, n is a whole number selected from 10-16 or 10-14. In certain embodiments, R ¹ for each instance is independently lower alkyl. In certain embodiments, Q is selected from the group consisting of chloride, bromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, benzoate, and the like.

In certain embodiments, the compound of formula I comprises cetylpyridinium and domiphen and one or more pharmaceutically acceptable anions. In certain embodiments, the one or more pharmaceutically acceptable anions is selected from the group consisting of chloride, bromide, sulfate, phosphate, acid phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, and combinations thereof.

In certain embodiments, the compound of formula I is cetylpyridinium chloride or domiphen bromide.

Methods of Preparation

The compounds provided herein can be prepared using any number of well-known methods, such as by nucleophilic substitution, reductive alkylation, electrophilic aromatic substitution, nucleophilic aromatic substitution, metal catalyzed carbon-carbon or carbon-heteroatom bond formation, condensation reactions, and the like.

Cetylpyridinium chloride is available commercially or can alternatively be prepared by, e.g., alkylation of pyridine with 1-chlorohexadecane.

Likewise, domiphen bromide is commercially available. In the alternative, domiphen bromide can be prepared by reaction of 2-phenoxyethyl bromide with dimethyl amine to form dimethyl- (2-phen-oxyethyl) amine. Dimethyl- (2-phen-oxyethyl) amine can then be alkylated with 1-bromododecane.

The as prepared salts can optionally be subjected to anion exchange to substitute the anion with a different anion using methods well known to those skilled in the art.

Pharmaceutical Compositions

The present disclosure also provides a pharmaceutical composition comprising any one of the aforementioned compounds and at least one pharmaceutically acceptable excipient.

The compounds described herein and their pharmaceutically acceptable salts can be administered to a mammalian subject either alone or in combination with pharmaceutically acceptable carriers or diluents in a pharmaceutical composition according to standard pharmaceutical practice. The compounds can be administered orally or parenterally. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous and topical, the preferred method being intravenous administration.

Accordingly, the present disclosure provides pharmaceutically acceptable compositions, which comprise a therapeutically-effective amount of one or more of the compounds described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. The pharmaceutical compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; and (2) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions) , tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue. The preferred method of administration of compounds of the present invention is parental administration (intravenous) .

As set out herein, certain embodiments of the compounds described herein may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term "pharmaceutically-acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the bromide, chloride, sulfate, bisulfate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.

The pharmaceutically acceptable salts of the compounds of the present disclosure include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from nontoxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

In other cases, the compounds described herein may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, solubilizing agents, buffers and antioxidants can also be present in the compositions.

Methods of preparing these formulations or compositions include the step of bringing into association a compound described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers (liquid formulation) , liquid carriers followed by lyophylization (powder formulation for reconstitution with sterile water or the like) , or finely divided solid carriers, or both, and then, if necessary, shaping or packaging the product.

Pharmaceutical compositions of the present disclosure suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, chelating agents, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. In the examples, the active ingredients are brought together with the pharmaceutically acceptable carriers in solution and then lyophilized to yield a dry powder. The dry powder is packaged in unit dosage form and then reconstituted for parental administration by adding a sterile solution, such as water or normal saline, to the powder.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) , and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the compounds of the present invention may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration, " "administered systemically, " "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

Methods of Use

The present disclosure provides methods of treating bacterial infections, wherein the compounds and compositions described herein are used in combination with at least one polymyxin or an analog thereof. The methods described herein are useful for killing bacteria that express at a mcr gene. Also provided are methods for inhibiting the action of an MCR enzyme comprising the step of contacting the MCR enzyme with a compound as described herein. In certain embodiments, the step of contacting the MCR enzyme with a compound as described herein occurs in a bacterial cell. In certain embodiments, the step of contacting the MCR enzyme with a compound as described herein occurs in a patient. Also provided is a method of inhibiting the function of a phosphoethanolamine transferase comprising the step of contacting the phosphoethanolamine transferase with a compound as described herein.

The compounds and methods described herein are useful for treating bacterial infections caused by bacteria that express one or more mcr genes. The mcr gene can be mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, mcr-8 and other variants that may evolved in the future.

The results provided below demonstrate the efficacy of the compounds described herein in the inhibition of mcr and the resulting resensitization of colistin resistant E. coli to colistin. Since the mcr gene is carried on a variety of plasmids, which allow the gene to be readily transferred between different strains of bacteria by horizontal gene transfer, other bacteria have been shown to express mcr besides E. coli.

Thus, the compounds and methods described herein can be used to treat bacterial infection caused by Gram-negative bacteria other than E. coli, such as for example, Klebsiella, such as Klebsiella pneumoniae, Proteus, such as Proteus mirabilis or Proteus vulgaris, Salmonella, such as Salmonella typhosa, Shigella, such as Shigella sonnef, Enterobacter, such as Enterobacter aerogenes or Enterobacter cloacae, Serratia, such as Serratia marcescens, Pseudomonas, such as Pseudomonas aeruginosa, Acinetobacter, such as Acinetobacter baumanii, Nocardia, such as Nocardia autotrophica, or Mycobacterium, such as Mycobacterium fortuitum, and combinations thereof.

Polymyxins useful in the methods described herein include, but are not limited to, polymyxin A, polymyxin B, polymyxin C, polymyxin D, polymyxin E, and polymyxin A. The polymyxin can also be a polymyxin analog. In such instances, the polymyxin analog can be, for example, the polymyxin analogs described in publications WO 2015/149131, WO 2015/135976, US 2015/0031602, WO 2014/188178, WO 2014/108469, US 2014/0162937, WO 2013/072695, WO 2012/168820, WO 2012051663, US 2012/0283176, US 2010/0160215, US 2009/0215677, WO 2008/017734, U.S. Pat. No. 6,380,356, and U.S. Pat. No. 3,450,687, the contents of which are hereby incorporated by reference.

In certain embodiments, the polymyxin is colistin A (polymyxin E1) or colistin B (polymyxin E2) . In certain embodiments, the colistin A is colistin A sulfate or colistimethate A sodium.

The compounds described herein can be administered according to therapeutic protocols well known in the art. It will be apparent to those skilled in the art that the administration of the compounds described herein and the polymyxin can be varied depending on the disease being treated and the known effects of the polymyxin on that disease. Also, in accordance with the knowledge of the skilled clinician, the therapeutic protocols (e.g., dosage amounts and times of administration) can be varied in view of the observed effects of the administered therapeutic agents (i.e., polymyxin) on the patient, and in view of the observed responses of the disease to the administered therapeutic agents.

Also, in general, compounds described herein and the polymyxin do not have to be administered in the same pharmaceutical composition, and may, because of different physical and chemical characteristics, have to be administered by different routes. For example, the compounds described herein may be administered orally to generate and maintain good blood levels, while the polymyxin may be administered intravenously. The determination of the mode of administration and the advisability of administration, where possible, in the same pharmaceutical composition, is well within the knowledge of the skilled clinician. The initial administration can be made according to established protocols known in the art, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the skilled clinician.

The particular choice of the polymyxin will depend upon the diagnosis of the attending physicians and their judgment of the condition of the patient and the appropriate treatment protocol.

A compound described and the polymyxin may be administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the bacterial infection, the condition of the patient, and the actual choice of the polymyxin to be administered in conjunction (i.e., within a single treatment protocol) with a compound described herein.

If a compound described herein and the polymyxin are not administered simultaneously or essentially simultaneously, then the optimum order of administration of the compound described herein and the beta-lactam antibiotic, may be different for different bacterial infections. Thus, in certain situations the compound described herein may be administered first followed by the administration of the polymyxin; and in other situations the polymyxin may be administered first followed by the administration of a compound described herein. This alternate administration may be repeated during a single treatment protocol. The determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is well within the knowledge of the skilled physician after evaluation of the disease being treated and the condition of the patient. For example, the polymyxin may be administered first and then the treatment continued with the administration of a compound described herein followed, where determined advantageous, by the administration of the polymyxin, and so on until the treatment protocol is complete.

Thus, in accordance with experience and knowledge, the practicing physician can modify each protocol for the administration of a component (compound described herein and the polymyxin) of the treatment according to the individual patient's needs, as the treatment proceeds.

Assays for Identifying MCR-1 inhibitors

To obtain compounds that can reduce the MIC of colistin in clinical E. coli strains, an assay was developed using the E. coli J53 strain carrying a natural mcr-1-carrying IncI2 plasmid (33kb, KX711706.1) obtained from a mcr-1-bearing clinical isolate of E. coli. The IncI2 plasmid only carried one antimicrobial resistance gene, i.e., mcr-1. In this screening assay, the mcr-1-bearing J53 strain was incubated at 37℃ in Mueller-Hinton Broth (MHB) medium supplemented with serial concentrations (0.5-2 μg/ml) of colistin alone, the test compound alone (10, 20, or 40 μM) and different combinations of colistin and the test compounds. The J53 cultures were incubated at 37 ℃ overnight and the minimum inhibitory concentration (MIC) of colistin in the presence of different concentrations of the compounds was determined.

Determination of bactericidal effects of MCR-1 inhibitor /colistin combinations

The bactericidal effects of the selected MCR-1 inhibitors alone or in combination with colistin on an E. coli J53 strain, which harbored a mcr-1-bearing plasmid were determined. The overnight E. coli cultures was diluted 100-fold in 3ml fresh Lysogeny broth (LB) and incubated for 2 hours until the OD reached 0.6. The cultures were then incubated with a series of concentrations of colistin, MCR-1 inhibitors, and different combinations of colistin and MCR-1 inhibitors, at 37 ℃. Viable counts were recorded at 0, 1, 2, 3, 5 and 8 hrs as previously described with duplication. Killing curves were drawn by plotting log ₁₀ CFU/ml against time (h) using the software GraphPad Prism 5.0 (San Diego, CA) .

Examination of effect of colistin and MCR-1 inhibitor on cellular morphology of E.coli

The overnight cultures of E. coli J53 carrying the mcr-1 gene was diluted 100-fold in 3ml of fresh LB and then incubated in 37 ℃ for 2 hours until it reached the exponential phase. The cultures were treated with colistin, MCR-1 inhibitors or different combinations of colistin and MCR-1 inhibitors by incubation at 37 ℃ for 3 hours. The culture was then subjected to centrifugation and washed twice with PBS, followed by resuspension of the pellet in 100μL of PBS. One μM of FM4-64 dye ( (N- (3-triethylammoniumpropyl) -4- (6- (4- (diethylamino) phenyl) hexatrienyl) pyridinium dibromide) ) was then added to the cell suspension and incubated at room temperature for 5 minutes. FM4-64 was reported to be the best dye for imaging the dynamic changes in size and morphology because of its staining specificity, higher photo-stability and low cytotoxicity. FM4-64 has been reported to selectively and permanently stain yeast or bacterial membranes with red fluorescence, and is almost non-fluorescent in water. Upon staining, the cells were washed with PBS twice to remove extra dyes and then resuspended in 20 μL of PBS. Two μL of the cell suspension were used for imaging with a Leica TCS SP8 MP Multiphoton Microscope (excitation/emission maxima～515/640nm) .

Expression and purification of Enzymatic Domain of MCR-1 (MCR-1-ED) and full length of MCR-1

Expression and purification of MCR-1-ED (200-541) was performed as previously described. For full length MCR-1 expression, the expression vector pGEX-6p-mcr-1 was transformed into the E. coli BL21 (DE3) strain. Transformed cells were cultured in LB at 37 ℃. Protein expression was induced by adding 0.75 mM IPTG until OD600 reached 0.6-0.8 at 16 ℃ overnight. Cell culture was centrifuged at 5000 rpm at 4 ℃ for 30 mins. Cell pellet was collected, re-suspended in Lysis buffer (50 mM Tris-HCl pH = 7.4, 150 mM NaCl, 2mM Protein Inhibitor Cocktail, 10 μg/ml DNase) , and lysed by sonication. Cell debris was removed by low-speed (8000 rpm) centrifugation for 30 min. The supernatant was collected and ultra-centrifuged at 38,000 rpm (HITACHI, P40ST) for 1 hour. Membrane fraction was collected and incubated with 2%(w/v) dodecyl-β-D-maltopyranoside (DDM, Sigma) for 4h at 4℃. After another ultracentrifugation step at 38,000 rpm for 1 hour, the supernatant was collected and incubated with 1 ml of 50 % suspension of glutathione Sepharose 4B (GS4B) beads (GE Healthcare) on a rotating wheel at 4℃ for 30 min, followed by washing with wash buffer (50 mM Tris-HCl pH =7.4, 150 mM NaCl, 0.023 %DDM) . The GS4B beads were pre-equilibrated with wash buffer. The GST tag was removed by PreScission Protease (GE Healthcare) . Amino acid substitutions in MCR-1 were introduced with primers shown in Table S1, using the QuickChange (Stratagene) commercial kit, and confirmed by sequencing.

Table S1 (ST1) . Primers used to clone MCR-1 and site-directed mutagenesis

Determination of inactivation constants of MCR-1 inhibitors

Isothermal titration calorimetry (ITC) experiments were performed to investigate the binding affinity between MCR-1 and the test compounds using MicroCal PEAQ-ITC Automated (Malvern Instruments Ltd. ) . The degree of interaction between 50μM of MCR-1 and 500 μM of the test compound (e.g., CET /DOM) in a buffer of 50 mM Tris-HCl containing 150 mM NaCl and 0.023 %DDM at pH 7.4 was investigated.

MCR-1 activity assay

A lipid substrate with a fluorescence label (1-acyl-2- {12- [ (7-nitro-2-1, 3-benzoxadiazol-4-yl) amino] dodecanoyl} -sn-glycero-3-phosphoethanolamine, Avanti Lipids) (NBD-PEA) was used to test the activity of MCR-1-ED, where the PEA group could be cleaved from PE. One hundred μg/ml of NBD-PEA was added to 800μg/ml of purified MCR-1-ED in buffers containing 50mM Hepes PH 7.5, 100mM NaCl and 0.023% DDM, and equilibrated at room temperature for 5 hours. The reaction was stopped by freezing the reaction at -20℃. To identify the inhibitory effect of MCR-1 inhibitors, 100-fold of inhibitor was added to the reaction (MCR-1-ED: inhibitor of 1: 100 molar ratio) . The reactions were resolved on thin-layer chromatography (TLC) plate and developed by use of ethyl acetate: methanol: water (7: 2: 1) . The fluorescence signal on the plate was visualized in the fluorescence emitter. For confirmation of formation of cleaved product of NBD-PEA (1-acyl-2- {12- [ (7-nitro-2-1, 3-benzoxadiazol-4-yl) amino] dodecanoyl} -sn-glycerol) , the appropriate material on the TLC plate was scraped off and washed with methanol. The material was centrifuged, and the supernatant was analyzed using an Agilent 6460 Liquid Chromatography-Electrospray Ionization Quadrupole-TOf Mass Spectrometer.

To test the activity of MCR-1, another assay containing the MCR-1 enzyme, lipid A extracted from E. coli and phosphatidylethanolamines (PE) from E. coli (Sigma-Aldrich, St. Louis, MO) were used. E. coli lipid A was extracted as previously described. Purified Lipid A was detected using a Bruker UltrafleXtreme MALDI-TOF-TOF Mass Spectrometer at m/z 1796 using 2, 5-dihydroxybenzoic acid as matrix and at m/z 1686 using α-cyano-4-hydroxycinnamic acid as matrix that was used for activity assay due to the high sensitivity of detection. In the activity assay, 1 mg/ml of Lipid A and the substrate PE (1mg/ml) were suspended with 48μg/ml of MCR-1 in 50mM Hepes buffer which contained 100mM NaCl and 0.023 % of DDM at pH 7. The reaction was incubated at room temperature with gently shaking for 3h. The reaction was stopped by freezing after incubation. The relative abundance of lipid A and modified lipid A was analyzed by a Bruker UltrafleXtreme MALDI-TOF-TOF Mass Spectrometer. In brief, 10mg of matrix (α-cyano-4-hydroxycinnamic acid) were dissolved in 1ml of buffer (70% ACN, 29.9% water and 0.1% TFA v/v) , 10μl of which was mixed with 10μl of reaction mixture. The lipid in the mixture was desalted and purified using ZipTip with C ₁₈ resin and 2μl of the purified lipid sample were loaded for analysis. To identify the inhibition effect of inhibitors on MCR-1-mediated lipid A modification, the compound was added to the assay reaction to produce a 1: 100 molar ratio of MCR-1: inhibitors (80μM) prior to incubation.

MCR-1 structure modeling and molecular docking

The crystal structure of the integral membrane protein lipooligosaccharide phosphoethanolamine transferase A (nmEptA) of Neisseria meningitidis, which formed a complex with dodecyl-β-d-maltoside, was downloaded from the PDB database (PDB entry: 5FGN; resolution:

) . The homologue model of E. coli MCR-1 was constructed by using SWISS MODEL. Molecular modeling was performed using CLC Drug Discovery Workbench (Version 2.5) . The modeled MCR-1 structure was prepared for docking using an automated procedure after removing water molecules and co-crystal ligands. The structure of DOM and CET were sketched in 2D and converted into 3D by ChemDraw. Automated docking studies were then carried out. The top-scoring fits were visually inspected and selected.

Cetylpyridinium chloride and domiphen bromide reduced MIC of colistin in E. coli carrying the mcr-1 gene

Cetylpyridinium chloride (CET) and domiphen bromide (DOM) were shown to reduce colistin MIC of E. coli J53 carrying the mcr-1 gene by over 64-fold (Table 1) . When measured in combination with 10 μM of CET or DOM, colistin MIC of the test strain was found to be reduced from 8 μg/ml to 0.5 μg/ml and 0.25 μg/ml respectively. Increasing the concentration of CET and DOM to 20 μM and 40 μM resulted in further reduction in the MIC of colistin (Table 1) . Each of CET and DOM exhibited no antibacterial effect at 10 or 20 μM respectively, but slight antibacterial effect on E. coli J53 strain was observable at concentrations of 40 μM and 80 μM respectively. This data clearly demonstrated that these two compounds can re-sensitize mcr-1-bearing E. coli to colistin. Consistently, synergistic antimicrobial effect was also observed when E. coli strain BL21 carrying the plasmid pET15-mcr-1 was treated with a combination of colistin and either of these two compounds (Table 1) . Interestingly, these two compounds also exhibited synergistic antimicrobial effect on E. coli J53 and E. coli BL21 strains that lacked the mcr-1 gene by dramatically reducing the MICs of colistin. At a concentration of 10μM or higher, both compounds were able to cause reduction of colistin MIC from 1 or 2μg/ml to <0.125 μg/ml in strain E. coli J53 and E. coli BL21 (Table 1) . To further confirm the effect of the two compounds on MCR-1-producing clinical bacterial strains, various clinical E. coli isolates that expressed MCR-1 were tested for colistin MIC in the presence of these two compounds, with results showing that both compounds restored the antimicrobial activity of colistin on these drug resistant clinical isolates by causing a 16-128-fold reduction in the MIC of colistin (Supplementary Table S2) . It should also be noted that colistin sensitive strains (mcr-1 negative) of these species also exhibited much lower MIC in the presence of CET or DOM.

Table 1. MICs of colistin against E. coli expressing MCR-1 and its parent strain.

^a CT, colistin; ^b CET, Cetylpyridinium chloride; ^c DOM, Domiphen bromide; ^dRF: Relative fold calculated by using the equation (MIC of CT alone) / (MIC of CT in the presence of inhibitor) .

Supplementary Table 2 (ST2) . Change of MICs of colistin in the presence of MCR-1 inhibitor, cetylpyridinium chloride and domiphen bromide, on clinical E. coli isolates with and without producing MCR-1.

+, MCR-1 positive; -, MCR-1 negative; CT, colistin; CET, Cetylpyridinium chloride; DOM, Domiphen bromide.

Synergistic antimicrobial effect of colistin and cetylpyridinium chloride /domiphen bromide combination

To further characterize the synergistic antimicrobial effect of colistin and CET and DOM, time killing assays were performed. Only at a concentration of 32μg/ml or higher was colistin able to inhibit the growth of E. coli J53 strain carrying the mcr-1 gene and completely eradicate the organism after 8-hour incubation. In the presence of 20μM of CET, however, the effective bactericidal concentration of colistin was surprisingly reduced to 2 μg/ml and killing was achieved within 3-hours of incubation. In the presence of 40 μM of DOM, the killing effect of colistin could surprisingly be observed at a concentration of as low as 1 μg/ml. At 4 μg/ml, colistin could kill all mcr-1 –bearing E. coli J53 strains in 3 hours. An antibacterial effect was not observable with CET at 20 μM or DOM at 40 μM alone, suggesting that the antimicrobial effect of CET or DOM must be exerted in the presence of colistin (Figure 1) .

To investigate the underlying bactericidal mechanism of the colistin and CET /DOM combinations, microscopy studies were performed to observe the morphological and physical change of the test strains upon treatment with various agents. Bacterial membrane staining using the FM4-64 dye allowed us to observe that the morphology of E. coli J53 carrying the mcr-1 gene remained unchanged when incubated with 8μg/ml of colistin or 30μM of CET alone. However, upon incubation with 20μM CET and 8μg/ml colistin, the test organisms started to exhibit gradual aggregation of cell cytosol at both sides of the cells. Increasing the concentration of CET to 30 μM led to membrane disruption and eventual cell death in the same manner as that observed with a high concentration of colistin (32μg/ml) alone. These findings confirmed that CET could significantly enhance the bactericidal effects of colistin (Figure 2) . Similar effect was also observed for DOM (data not shown) .

Cetylpyridinium chloride and domiphen bromide bind directly to MCR-1, but not MCR-1-ED

To confirm whether the two compounds act by binding directly to MCR-1, the recombinant forms of MCR-1-ED and full length of MCR-1 were produced in E. coli. ITC assay was then performed using both MCR-1-ED and MCR-1, with results showing that both CET and DOM were not able to bind to MCR-1-ED, but exhibited binding affinity to MCR-1, with a K _d of 102mM and 334μM respectively. The data suggested that CET and DOM could specifically bind to the full length MCR-1 protein but not MCR-1-ED. It should be noted that MCR-1 could only be made soluble in the presence of the detergent used in ITC assay, which generated small bubbles that prevented accurate measurement of binding kinetic constants. However, the ITC assays were still able to differentiate between compounds with or without exhibiting binding affinity on MCR-1.

Cetylpyridinium chloride and domiphen bromide inhibited the enzymatic activity of MCR-1 but not MCR-1-ED

To further confirm the ability of these two compounds to inhibit MCR-1 activity, in vitro activity assay was developed and performed as described previously, using NBD-PEA as substrate. Our data showed that MCR-1-ED exhibited the ability to cleave NBD-PEA to NBD-glycerol, which exhibited different polarity from NBD-PEA and could be separated by TLC plate, suggesting that MCR-1-ED alone was able to mediate enzymatic cleavage of the substrate NMD-PEA (Figure 3) . However, both CET and DOM were not able to inhibit the activity of MCR-1- ED.Upon incubation with MCR-1-ED and NBD-PEA, CET /DOM at a concentration 100-fold that of MCR-1-ED was not able to inhibit cleavage of NDB-PEA by MCR-1-ED, thus confirming that CET /DOM could not bind to the MCR-1-ED (Figure 3) . These data were consistent with the ITC binding data in that both compounds did not exhibit binding affinity to MCR-1-ED.

An in vitro activity assay using the full length of MCR-1 was also developed by using commercially available PE and lipid A extracted from E. coli as substrate. In this assay, upon incubation of a mixture containing 1 mg/ml of lipid A from E. coli, 1mg/ml of PE and 48μg/ml of MCR-1, a peak at m/z 1685.759 representing lipid A and a peak at m/z 1810.629 representing PPEA-4’ -lipid A that contained a molecule of lipid A and a single PEA group (123Da) , were observed, suggesting that MCR-1 was able to mediate the transfer of the PPEA group from PE to lipid A in the in vitro assay (Figure 4C) . Using this assay, we tested whether MCR-1-ED alone was able to transfer PEA from PE to lipid A, and confirmed that MCR-1-ED failed to catalyze such transfer; such finding was therefore consistent with that of two earlier reports (Figure 4D) . In the presence of 80μM of DOM or CET with the molecular ratio of MCR-1: DOM being 1: 100, the amount of PPEA-4’ -lipid A decreased dramatically, whereas the amount of lipid A increased, suggesting that DOM and CET could inhibit the activity of MCR-1 (Figures 4E, F) . This data is consistent with the idea that DOM and CET inhibited MCR-1 activity through binding to the full length of MCR-1, but not the enzymatic domain.

Cetylpyridinium chloride and domiphen bromide bind to a hydrophobic pocket located in the transmembrane domain (TMD) of MCR-1, which might be essential for lipid A binding

To further investigate the mechanism of inhibition of MCR-1 by DOM and CET, we modeled the structure of MCR-1 using the published structure of EptA of Neisseria meningitidis, namely nmEptA (PDB ID code 5FGN) , as template (Figure 5A) . The crystal structure of MCR-1-ED superimposed very well to the MCR-1-ED of the modeled structure of MCR-1 suggesting that confidence level of the modeled MCR-1 structure was very high. The modeled structure of MCR-1 was then used to conduct molecular docking for the two inhibitors. The highest docking score positioned both ligands, DOM and CET, in a pocket formed by three helixes (H1-H3) , as shown in Figures 5C and 5D. Both ligands were predicted to bind deeply inside the pocket, with the pyridinium ring of CET and the phenyl ring of DOM facing towards the Zn ion. The linear hydrocarbon chain of both ligands was predicted to be inserted into a hydrophobic pocket formed by the three helixes. Consistently, in the previously published structure of nmEptA, the detergent molecule dodecyl-beta-d-maltoside was found to have inserted into this hydrophobic pocket (Figure 5B) . Considering the structural similarity of the tail of lipid A and the inhibitors and without wishing to be bound by theory, it is hypotheized that lipid A is anchored into MCR-1 through inserting the lipid tail into this hydrophobic pocket (Figure 5E) . To test this hypothesis, we investigated the effect of interference of the L ¹⁰⁶R and A ¹⁰⁹R substitutions on MCR-1 activity. Both L ¹⁰⁶ and A ¹⁰⁹ were located at the edge of the pocket. We therefore expected that substitutions by residues with a larger side chain such as Arg would block the pocket, preventing insertion of the lipid A tail into the pocket (Figure 5F) . Our data showed that MCR-1 (L ¹⁰⁶R) and MCR-1 (A ¹⁰⁹R) indeed exhibited dramatic reduction in the ability to catalyze modification of Lipid A (Figure 4G) . Double mutations that lead to formation of the MCR-1 (L ¹⁰⁶R/A ¹⁰⁹R) mutant protein were also found to completely abolish the activity of MCR-1, thereby further suggesting that this hydrophobic channel is critical for the docking of lipid A for further modification (Figure 4H) . Furthermore, ITC assay using recombinant MCR-1 (L ¹⁰⁶R/A ¹⁰⁹R) and CET /DOM showed that these two inhibitors were not able to bind to MCR-1 double mutant (Data not shown) further confirming that CET/DOM indeed bound to the hydrophobic pocket in the TMD of MCR-1.

Based on findings described above and without wishing to be bound by theory, we propose the mechanism of substrate recognition and modification for MCR-1 and other structurally related phosphoethanolamine transferases (Figure 6) . As a membrane protein, the transmembrane domain of MCR-1 was anchored onto the inner membrane of Gram-negative bacteria such as E. coli. Upon stimulation by the membrane lipid, the linker between the enzymatic domain (ED) and the transmembrane domain (TMD) opens up to expose the active site of the active site of MCR-1. The divalent zinc ion and phosphate group acceptor Thr ²⁸⁵ are essential for MCR-1 activity as reflected by the observation that replacement of the zinc coordination ligand residues E ²⁴⁶, H ⁴⁶⁶ or D ⁴⁶⁵ and the acceptor T ²⁸⁵ by alanine led to reduction of the colistin MIC to a susceptible level. We therefore speculate that the zinc ion in the active site acts as an essential cofactor to catalyze the addition of a phosphoethanolamine group at T ²⁸⁵ by nucleophilic attack of hydroxyl group of T ²⁸⁵ on the phosphate group of phosphatidylethanolamine, forming an intermediate Figure 5B (2) . Another bulky MCR-1 substrate, lipid A, was coordinated to the active site with the help of various lipid A coordinating ligands, such as P ⁴⁸¹ and Y ²⁸⁷ which acted by holding both D-glucosamines molecules, H ⁴⁷⁸ and H ³⁹⁵ whose function was to hold the phosphate group, and the hydrophobic channel located in the TMD of MCR-1, into which the lipid chain was anchored. The nucleophilic attack of phosphate group of lipid A on the phosphoethanolamine group of T ²⁸⁵ resulted in addition of a phosphoethanolamine group to the bis-phosphorylated hexa-acylated lipid A.

It should be noted that, apart from the synergistic antimicrobial effect of colistin and cetylpyridinium chloride /domiphen bromide on MCR-1-producing bacteria, these drug combinations also exhibited much stronger antimicrobial effect on mcr-1-negative, colistin susceptible bacterial strains than colistin alone. Enhancement of the potency of colistin by these compounds might be due to their ability to inhibit other phosphoethanolamine transferases, such as EptA, that are also involved in lipid A biosynthesis, thereby elevating the sensitivity of the organisms to colistin. This data suggests that a significantly lower dosage of colistin could eradicate colistin-susceptible bacteria when used in combination with cetylpyridinium chloride /domiphen bromide, thus drastically reducing the side effect of colistin.

Thus, also included within the scope of the present disclosure is a method for treating bacterial infection in a patient caused by a bacteria that does not express an mcr gene comprising the step of co-administering a pharmaceutically effective amount of a compound described herein and a pharmaceutically acceptable amount of a polymyxin to a patient in need thereof. Also provided, are methods for killing bacteria and/or inhibiting the growth of bacteria comprising the step of contacting the bacteria with a compound as described herein and a polymyxin, wherein the bacteria does not express a mcr gene.

Molecular docking experiments further confirmed the prediction that the compounds described herein bind to a hydrophobic channel in MCR-1 by showing that they were well-docked in the hydrophobic channel, preventing insertion of the lipid A tail and hence recognition and modification of lipid A by MCR-1. Another elegant part of these kinds of inhibitor was that MCR-1 is very difficult to develop resistance to these inhibitors, because any mutation in MCR-1 that blocks the binding of these inhibitors to MCR-1 could simultaneously block the binding of lipid A substrate to MCR-1 preventing its modification of the substrate.

Mechanisms of action of lipid-to-lipid transferase are less understood. The structure of a lipid-to-lipid glycosyltransferase, 4-amino-4-deoxy-L-arabinose transferase (ArnT) that transfers the carbohydrate, 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the 1 and 4′phosphate groups of lipid A, the same site of modification upon interaction with MCR-1 and EptA, reveals the existence of three cavities that are supposed to house the lipid substrates lipid A and undecaprenyl phosphate-α-L-Ara4N. Based on this structure and various biochemical data, it suggests that ArnT needs to bind to both substrates in order to enable direct transfer of L-Ara4N from undecaprenyl phosphate to lipid A. Unlike ArnT, our model showed that the TMD and enzymatic domain of MCR-1 were separated to enable the binding of both substrates to this cavity of MCR-1, with PE binding to the active site residues and the Zn ion, lipid A interacting with several important residues in the active site, and stabilization effect being achieved through inserting the lipid chains into the hydrophobic pocket in the TMD. MCR-1 may still adopt the previously proposed “ping-pong” substrate recognition model of nmEptA, which involves formation of a T ²⁸⁰-PEA enzyme intermediate prior to transferring the PEA moiety to lipid A. T data suggested that transferring PEA to T ²⁸⁵ of MCR-1 to form T ²⁸⁵-PEA could be completed by the enzymatic domain of MCR-1, which could not transfer the incorporated PEA to lipid A without the help of the TMD, suggesting that anchoring of lipid A into the correct position in MCR-1 is essential for initiation of the modification process. Although several residues in MCR-1-ED might be involved in the coordination of lipid A, it is speculated that these interactions are not strong enough to stabilize lipid A into the active site of MCR-1 due to the large size of lipid A, and that recognition of lipid chain of lipid A by the hydrophobic pocket in the TMD facilitates anchoring of lipid A into the correct position for modification by MCR-1. Confirmation of this mechanism still requires evidence to show that MCR-1-ED could accept PEA by T ²⁸⁵ in the presence of PE. This possibility is currently being tested in our laboratory.

To conclude, this study has identified two FDA-approved drugs as well as additional compounds that act as inhibitors of MCR enzymes and other phosphoethanolamine transferases. The mode of inhibitor binding was elucidated to provide important insight into the mechanism of action of this important class of enzymes, which are involved in lipid A modification and colistin resistance. Importantly, the structural model and results of mutagenesis studies showed that resistance to these inhibitors could not be developed without a loss in enzymatic activity. These findings suggest that phosphoethanolamine transferase inhibitors can be utilized as an adjuvant to significantly enhance the antimicrobial potency of colistin in treatment of infections caused by multidrug resistant Gram-negative bacterial pathogens.

Claims

A method of treating a bacterial infection in a patient comprising the step of co-administering a pharmaceutically acceptable amount of a polymoxin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable amount of a compound of formula I to a patient in need thereof, wherein the compound of formula I has the structure:

or a pharmaceutically acceptable salt thereof, wherein

Ar is aryl or heteroaryl;

X is -O-, -NR ¹-, or a bond;

A is - (CR ² ₂) _m-;

Y is -O-, -NR ¹-, - (NR ¹ ₂) -, or a bond;

Z is - (CR ² ₂) _n-;

R is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl;

R ¹ for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; or two instances of R ¹ taken together with the nitrogen to which they are attached form a 3-6 membered heterocyclic ring;

R ² for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; or two instances of R ² taken together with the carbons to which they are attached form a 3-6 membered heterocyclic ring;

m for each instance is independently a whole number selected from 2-14; and

n for each instance is independently a whole number selected from 2-14.
The method of claim 1, wherein Ar is selected from the group consisting of aryl, pyrimidine, and imidazole.
The method of claim 1, wherein X is -O-; Y is - (NR ¹ ₂) ; m is a whole number selected from 2-6; and n is a whole number selected from 6-14.
The method of claim 3, wherein Ar is aryl and R ₁ for each instance is independently alkyl.
The method of claim 1, wherein X and Y are a bond; n and m are whole numbers independently selected from 3-8 and Ar is represented by the structure:
The method of claim 1, wherein X is -O-; Y is - (NR ¹) -or -O-; m is a whole number selected from 2-4; and n is a whole number selected from 6-14.
The method of claim 6, wherein R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl.
The method of claim 1, wherein X is - (NR ¹) -; Y is -O-; m is a whole number selected from 2-4; and n is a whole number selected from 6-14.
The method of claim 8, wherein R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl.
The method of claim 1, wherein the polymyxin is colistin or a pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the bacterial infection is caused by a bacteria that expresses a mobilized colistin resistance (mcr) gene.
The method of claim 10, wherein the method comprises co-administering a pharmaceutically effective amount of a polymyxin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable amount of a compound of formula I to a patient in need thereof, wherein the bacterial infection is caused by a bacteria that expresses mcr-1 and the compound of formula I has the structure:

or a pharmaceutically acceptable salt thereof, wherein

Ar is aryl; X is -O-; Y is - (NR ¹ ₂) ; R ₁ for each instance is independently alkyl; m is a whole number selected from 2-6; and n is a whole number selected from 6-14;

X is -O-; Y is - (NR ¹) -or -O-; R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl; m is a whole number selected from 2-4; and n is a whole number selected from 6-14; or

X is - (NR ¹) -; Y is -O-; R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl; m is a whole number selected from 2-4; and n is a whole number selected from 6-14.
The method of claim 12, wherein the bacteria is an E. coli strain selected from the group consisting of 7, 119, WZ3951, WZ3903, 101, CX48, WZ3955, XH69, WZ2431, WZ3920, J53, and BL21.
The method of claim 12, wherein the compound of formula I is a salt of cetylpyridinium or domiphen.
The method of claim 14, wherein the polymyxin is colistin or a pharmaceutically acceptable salt thereof.
A method of inhibiting the function of a MCR enzyme comprising the step of contacting a compound of formula I with the MCR enzyme, wherein the compound of formula I has the structure:

or a pharmaceutically acceptable salt thereof, wherein

Ar is aryl or heteroaryl;

X is -O-, -NR ¹-, or a bond;

A is - (CR ² ₂) _m-;

Y is -O-, -NR ¹-, - (NR ¹ ₂) -, or a bond;

Z is - (CR ² ₂) _n-;

R is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl;

R ¹ for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; or two instances of R ¹ taken together with the nitrogen to which they are attached form a 3-6 membered heterocyclic ring;

R ² for each instance is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroaryl; or two instances of R ² taken together with the carbons to which they are attached form a 3-6 membered heterocyclic ring; m for each instance is independently a whole number selected from 2-14; and

n for each instance is independently a whole number selected from 2-14.
The method of claim 16, wherein Ar is aryl; X is -O-; Y is - (NR ¹ ₂) ; R ₁ for each instance is independently alkyl; m is a whole number selected from 2-6; and n is a whole number selected from 6-14;

X is -O-; Y is - (NR ¹) -or -O-; R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl; m is a whole number selected from 2-4; and n is a whole number selected from 6-14; or

X is - (NR ¹) -; Y is -O-; R ¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, and cycloalkyl; m is a whole number selected from 2-4; and n is a whole number selected from 6-14.
The method of claim 16, wherein the compound of formula I is a salt of cetylpyridinium or domiphen.
The method of claim 16, wherein the step of contacting the compound of formula I and the MCR enzyme occurs in a bacterial cell.
The method of claim 19, wherein the step of contacting the compound of formula I and the MCR enzyme occurs in a subject.