# Patent

## Title
Antibiotic compositions and related screening methods

## Abstract

Moenomycin inhibits bacterial growth by clocking the transglycosylase activity of class A penicillin-binding proteins (PBPs), which are key enzymes in bacterial cell wall synthesis. The binding affinities of moenomycin A with various truncated PBPs were compared showing that the transmembrane domain is important for moenomycin binding. Full-length class-A PBPs from 16 bacterial species were produced, and their binding activities showed a correlation with the antimicrobial activity of moenomycin against Enterococcus faecalis and Staphylococcus aureus. Moreover, a fluorescence anisotropy-based high-throughput assay was developed and used successfully for identification of transglycosylase inhibitors.

## Background
RELATED APPLICATION
This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 61/021,279, filed Jan. 15, 2008, Taiwan Application Serial No. 097125469, filed Jul. 4, 2008, and Japan Application Serial No. 2008-178187, filed Jul. 7, 2008, and the contents of each are incorporated by reference herein in their entirety.


BACKGROUND
This disclosure relates to methods for expressing and purifying full-length class A penicillin-binding protein from bacteria. This disclosure also relates to high-throughput screening methods for an antibiotic.
SUMMARY
According to a feature of the present disclosure, a method is disclosed comprising obtaining a candidate for screening; carrying out an anisotropy measurement assay with a class A penicillin-binding protein comprising at least a transmembrane and a transglycosylase domains; and determining the effectiveness of the candidate as a transglycosylase inhibitor.
According to a feature of the present disclosure, a high throughput device is contemplated, having multiple versions of a class A penicillin-binding protein comprising at least transmembrane and a transglycosylase domain for determining the effectiveness of a candidate agent as a transglycosylation inhibitor. The assays may be carried out as either competition displacement assays or direct binding assays or both.
According to a feature of the present disclosure, a method is disclosed comprising amplifying DNA sequence of full-length penicillin-binding protein from bacterial genomic DNA; cloning the DNA sequence into a vector, expressing the DNA sequence in a host cell to obtain a full-length penicillin-binding protein, solubilizing the protein with a detergent, and purifying the protein.
According to a feature of the present disclosure, a composition is disclosed comprising an agent having the formula selected from the group consisting of:







and a pharmaceutically acceptable carrier.

According to a feature of the present disclosure, a method is disclosed comprising treating a subject having a bacterial infection or susceptible for a bacterial infection with a pharmaceutical composition comprising an agent having the formula selected from the group consisting of:







and a pharmaceutically acceptable carrier.

According to implementations of the present disclosure, the antibiotic is a transglycosylase inhibitor, and, preferably, it is (Z)-5-(4-bromophenyl)-3-((5-nitrofuran-2-yl)methylene)furan-2(3H)-one; (Z)-1,3-diphenyl-4-(2-(thiazol-2-yl)hydrazono-1H-pyrazol-5(4H)-one; (E)-4-(2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-enyl)benzoic acid.
According to implementations of the present disclosure, the protein comprises three domains which, from N terminus to C terminus, are transmembrane, transglycosylase and transpeptidase.
According to implementations of the present disclosure, the moenomycin used as fluorescent probe binds the penicillin-binding protein when fluorescence-anisotropy-based assay is carried out.
According to implementations of the present disclosure, the hits screened by the method of present disclosure, which are antibiotics, inhibit the growth bacterium which is Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus (MRSA Mu50), Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica or Neisseria gonorrhoeae. 
According to implementations of the present disclosure, the bacterium is E. faecalis or S. aureus. 
According to implementations of the present disclosure, a method is provided for expressing and purifying full-length class A penicillin-binding protein from a bacterium comprising: (a) amplifying DNA sequence of full-length penicillin-binding protein from bacterial genomic DNA; (b) cloning the DNA sequence into a vector; (c) expressing the DNA sequence in a host cell to obtain a full-length penicillin-binding protein; (d) solubilizing the protein with a detergent; and (e) purifying the protein; wherein the detergent is n-decyl-β-D-maltopyranoside, n-undecyl-β-D-maltopyranoside, n-dodecyl-β-D-maltopyranoside, n-octyl-β-D-glucopyranoside, n-nonyl-β-D-glucopyranoside, n-tetradecylphosphocholine, n-dodedyl-N,N-dimethylamine-N-oxide, 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate or α-[4-(1,1,3,3-Tetramethylbutyl)phenyl]-w-hydroxy-poly(oxy-1,2-ethanediyl).
According to implementations of the present disclosure, the bacterium is Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus (MRSA Mu50), Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica or Neisseria gonorrhoeae. 
According to implementations of the present disclosure, the detergent used for purifying full length class A penicillin-binding protein is n-dodecyl-β-D-maltopyranoside.
According to implementations of the present disclosure, the full length protein comprises three domains, which, from N terminus to C terminus, are transmembrane, transglycosylase and transpeptidase.



DRAWINGS
The above-mentioned features and objects of the present disclosure will become more apparent with reference to the following description taken in conjunction with the accompanying drawings wherein like reference numerals denote like elements and in which:
FIG. 1 shows inhibition of transglycosylase by moenomycin and binding affinities of truncated PBP variants, according to implementations of the present disclosure. FIG. 1(A) shows Moenomycin A (1) inhibiting the transglycosylation step in the bacterial cell wall synthesis. FIG. 1(B) is a schematic representation of the PBP variants used for moenomycin binding studies;
FIG. 2 shows sequence alignment of full-length bi-functional PBPs from 16 bacterial strains (Seq. ID Nos. 1-16), according to implementations of the present disclosure;
FIG. 3 shows the SDS-PAGE analysis of purified protein of class A PBPs from sixteen bacterial species, according to implementations of the present disclosure;
FIG. 4 shows the design of a novel fluorescence anisotropy assay for transglycosylase, according to implementations of the present disclosure. FIG. 4(A) shows the chemical structures of the modified moenomycin A (2), and FIG. 4(B) shows the chemical structures of the fluorescein-labeled moenomycin, F-Moe (3). FIG. 4(C) shows the SPR analysis of the binding activity of moenomycin A to E. coli PBP1b, and FIG. 4(D) shows the SPR analysis of the binding activity of F-Moe to E. coli PBP1b;
FIG. 5 shows the development of high-throughput FA assay for transglycosylase. FIG. 5(A) shows the concentration-dependent changes in fluorescence anisotropy observed when E. coli PBP1b bound to F-Moe. FIG. 5(B) shows the improved FA assay with the Helicobacter pylori PBP1a. FIG. 5(C) shows the displacement of the PBP1a bound F-Moe complex by unlabeled moenomycin at various concentrations;
FIG. 6 illustrates chemical structures of moenomycin derivatives (4 and 5), according to implementations of the present disclosure; and
FIG. 7 illustrates screening for small molecules as TG inhibitors using the class A PBPs, according to implementations of the present disclosure. FIG. 7(A) shows the scheme of HTS for TG inhibitors using FA assay. FIG. 7(B) shows chemical structures of the HTS hits (compounds 6-8).



DETAILED DESCRIPTION
All scientific terms are to be given their ordinary meanings as understood by those of skill in the art, unless an alternate meaning is set forth below. In case of conflict, the definitions set forth in this specification shall control.
As used in this application, the term “host cell” means a cell that may be used to express a particular DNA molecule, vector, or plasmid other than the naturally occurring segments of the host cell's chromosomal DNA.
As used in this application, the term “vector” means a DNA molecule that is at least originally separate from the chromosomal DNA of a host cell, and may include plasmids.
As used in this application, the term “detergent” means any agent that causes a substance to become soluble or more soluble in a solution.
Many common bacterial pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis have become multidrug-resistant and emerged as a public health concern, demanding an unmet medical need for novel antibiotics.
The bacterial cell wall or peptidoglycan synthetic pathway has been targeted for the development of antibacterial agents. The synthesis of peptidoglycan consists of several steps including the synthesis of lipid I and lipid II followed by the final transglycosylation and transpeptidation of lipid II to form peptidoglycan. Many of the current antibiotics are β-lactam derivatives that target the transpeptidation. No medicines are yet developed to inhibit the transglycosylation process. The only known potent inhibitors for transglycosylase (TG) are moenomycin complexes (flavomycin), including moenomycin A (FIG. 1A, Moe A, 1), A12, C1, C3 and C4. Among them, Moe A is the most abundant agent in its family. FIG. 1 shows inhibition of transglycosylase by moenomycin and binding affinities of truncated PBP variants. FIG. 1(A) shows Moenomycin A (1) inhibiting the transglycosylation step in the bacterial cell wall synthesis. The unique antibacterial properties have prompted chemists to synthesize moenomycin fragments and derivatives. The first total synthesis of moenomycin A was achieved recently by Kahne and co-workers. Its biosynthesis pathway has also been elucidated. However, due to poor bioavailability, flavomycin is only employed as an antibiotic growth promoter in animal feeds.
Transglycosylation is mainly catalyzed by bi-functional class A penicillin-binding proteins (PBPs). PBPs are the major enzymes responsible for the last-step cell-wall formation by polymerization of N-acetylglucosamine-N-acetylmuramyl-pentapeptide, and cross-linking between penta-peptide, and thus have been considered as one of the important targets for antibiotics discovery. These membrane-anchored enzymes consist of three distinct protein domains, i.e., transmembrane (TM), transglycosylase (TG) and transpeptidase (TP) domains from their amino to carboxyl termini. The recent X-ray crystal structures of the PBP2 extracellular domain from S. aureus and the transglycosylase domain of PBP1a from A. aeolicus have provided invaluable structural insight into the plausible mechanism of cell-wall peptidoglycan polymerization, albeit lacking the information regarding the role of the transmembrane domain of PBPs in catalysis. The transmembrane domain of PBPs has been speculated to interact with the lipid moiety of moenomycin or lipid II.
The characterization of class A PBPs and the identification of TG inhibitors require functional PBP and lipid II as the substrate for the polymerization. The limited availability of lipid II has hampered the enzymatic study as well as the development of inhibitors. To get around the lipid II deficiency, a majority of screening methods to search for TG inhibitors rely mainly on moenomycin. Based on moenomycin activity, low-throughput inhibitor screening methods using SPR assays or radioactive assays have been developed. A TG activity assay platform that is amenable to high-throughput screening is thus desirable for inhibitor identification.
According to implementations, a high throughput device is contemplated. According to implementations, the high throughput device comprises a platform having multiple versions of a class A penicillin-binding protein comprising at least transmembrane and a transglycosylase domain for determining the effectiveness of a candidate agent as a transglycosylation inhibitor. FA assays may then be carried out on the platform, for example either competition displacement assays or direct binding assays or both, and a determination made of the effectiveness of the candidate agent determined.
According to implementations, a method is disclosed comprising obtaining a candidate for screening; carrying out an anisotropy measurement assay, such as fluorescent anisotropy, with a class A penicillin-binding protein comprising at least a transmembrane and a transglycosylase domains; and determining the effectiveness of the candidate as a transglycosylase inhibitor.
According to implementations, a method is provided for amplifying DNA sequence of full-length penicillin-binding protein from bacterial genomic DNA; cloning the DNA sequence into a vector, expressing the DNA sequence in a host cell to obtain a full-length penicillin-binding protein, solubilizing the protein with a detergent, and purifying the protein.
According to similar implementations of the present disclosure, a high-throughput screening method is provided for identification of TG inhibitors and antibiotic development. The method may comprise: (a) providing a candidate for screening; (b) carrying out fluorescence anisotropy measurement with fluorescent moenomycin and class A penicillin-binding protein comprising transmembrane and transglycosylase domains; (c) calculating fluorescence anisotropy value and KD value; (d) calculating KI, and IC50 value of an transglycosylase inhibitor; and (e) determining minimal inhibitory concentration of the inhibitor; wherein KD is the dissociation constant between the moenomycin and the protein; KI, is the dissociation constant between the inhibitor and the protein; IC50 is inhibitory concentration needed to inhibit bacterial growth by half; minimal inhibitory concentration is the minimal concentration of the inhibitor that prevents bacterial growth.
A high-throughput screening method according to implementations of the present disclosure may be used to determine the protein binding affinity for a candidate inhibitor or compound. Where a protein may be known or suspected to play a role in the activity of a target cell, the binding affinity of the candidate may correspond to the effectiveness of the candidate as an inhibitor of the protein with regard to the known or suspected cell activity.
An FA-based PBP binding assay according to implementations of the present disclosure has been demonstrated as a powerful HTS assay for the discovery of novel antibacterial agents. An FA-based assay according to implementations of the present disclosure can be used as a robust primary screening to quickly identify the potential hits from large antibiotic compound libraries. Furthermore, an efficient and economical TG assay in an HTS format according to implementations of the present disclosure may facilitate the identification of new TG inhibitors. The selected hits may be further screened using antibacterial assay or lipid II polymerizing activity analysis to identify leads.
According to implementations of the present disclosure, a method is provided for expressing and purifying full-length class A penicillin-binding protein from a bacterium comprising: (a) amplifying DNA sequence of full-length penicillin-binding protein from bacterial genomic DNA; (b) cloning the DNA sequence into a vector; (c) expressing the DNA sequence in a host cell to obtain a full-length penicillin-binding protein; (d) solubilizing the protein with a detergent; and (e) purifying the protein.
A method for expressing and purifying proteins according to implementations of the present disclosure may be applied to obtain a variety of proteins that are capable of being expressed in a host cell. Such proteins may correspond to proteins naturally generated within living organisms and may include proteins known or suspected to play a role in the activity of a cell of the living organism.
Proteins obtained by implementations of the disclosed method may be used for binding experiments for screening purposes. For example, proteins obtained may be used to evaluate the binding activity of candidate compounds to determine the effectiveness of the compound as an inhibitor of the protein obtained.
PBPs may be obtained by implementations of the disclosed method, including PBP variants including various combinations of transmembrane (TM), transglycosylase (TG) and transpeptidase (TP) domains. For example, the following variants of domain combinations may be obtained: (i) full-length protein containing the TM, TG, and TP domains (TM+TG+TP), (ii) TG+TP, (iii) TM+TG, (iv) TG alone, and (v) TP alone (see FIG. 1B).
A method for expressing and purifying full-length class A penicillin-binding protein from a bacterium according to implementations of the present disclosure may be used to obtain the PBPs from different species with purity greater than 80%. Implementations of the method may include selection of a DNA sequence from bacterial genomic DNA that is known to be active in certain activities of the corresponding bacterium.
Pharmaceutical compositions are also contemplated. The methods of the present disclosure allow for the rapid determination of agents that are useful as antibiotics. For example, as disclosed in more detail in Example 9 and 10, compounds 2 through 10 were tested for antibiotic activity. Moenomycin analogs 2 and 4, as well as small molecules 6-8, were confirmed to have both antibacterial and TG binding inhibition activities (see Table 2).
As disclosed above, pharmaceutical preparations are herein disclosed having antibiotic or TG binding inhibition activities. According to implementations, one or more of the following agents for a pharmaceutical comprised of a pharmaceutical carrier and one of the following agents:







and administered to a subject in need of an antibiotic due to bacterial infection or as a preventative measure.

Pharmaceutical Compositions

The instant disclosure also provides pharmaceutical compositions. In some implementations, the pharmaceutical compositions comprise agents, namely moenomycin analogs and small molecules (TG Inhibitors) shown to have antibiotic activity via inhibition of TG binding. In such pharmaceutical compositions, the TG Inhibitors form the “active compound” or “agent.” According to implementations, the pharmaceutical compositions are administered to a subject to in need of anti-bacterial therapy, including gram-negative bacteria. According to other implementations, the pharmaceutical compositions are administered to a subject having a bacterial infection to inhibit the transgylcosylation process during the synthesis of bacterial cell wall.
In addition to active compound, the pharmaceutical compositions preferably comprise at least one pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Subject as used herein refers to humans and non-human primates (e.g., guerilla, macaque, marmoset), livestock animals (e.g., sheep, cow, horse, donkey, pig), companion animals (e.g., dog, cat), laboratory test animals (e.g., mouse, rabbit, rat, guinea pig, hamster), captive wild animals (e.g., fox, deer) and any other organisms who can benefit from the agents of the present disclosure. There is no limitation on the type of animal that could benefit from the presently described agents. Human subjects are expressly contemplated. A subject regardless of whether it is a human or non-human organism may be referred to as a patient, individual, animal, host, or recipient.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Other delivery methods and devices common in the art, including mechanically actuated atomizing-like devices are expressly contemplated.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For epidermal, dermal, or transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one implementation, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811, incorporated by reference herein.
It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in subjects. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in subjects. Levels in plasma can be measured, for example, by high performance liquid chromatography.
As defined herein, a therapeutically effective amount of an active compound of the disclosure may range, for examples, from about 0.001 to 30 mg/kg body weight, about 0.01 to 25 mg/kg body weight, about 0.1 to 20 mg/kg body weight, or about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Without limitation, the active compound can be administered between one time per week and three or more times per day, for between about 1 to 10 weeks, for example between 2 to 8 weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a pharmaceutical composition of the disclosure can include a single treatment or, preferably, can include a series of treatments.
For those agents determined to be transglycosylation inhibitors or antibiotics, further testing may then be performed for the candidate agent to determine agents that are both good transglycosylation inhibitors and also having reasonably bioavailability.
EXAMPLES
The present disclosure and its implementations may be further illustrated by the following examples.
Example 1
Domain Requirement of Moenomycin Binding to Class A Penicillin-Binding Proteins (Bi-Functional Transglycosylases)
Protein constructs containing different domains of PBP1b from E. coli were expressed and purified with suitable detergents for moenomycin binding activities using Surface Plasmon Resonance (SPR). Five E. coli PBP1b variants were expressed and purified: 1) full-length protein containing transmembrane, transglycosylase and transpeptidase domains (TM+TG+TP), 2) TG+TP, 3) TM+TG, 4) TG, and 5) TP (FIG. 1B).
FIG. 1 shows inhibition of transglycosylase by moenomycin and binding affinities of truncated PBP variants. FIG. 1(B) is a schematic representation of the PBP variants used for moenomycin binding studies. The full-length PBP (TM+TG+TP) contains amino acid residues 1-844 of the E. coli PBP1b P02919. TG+TP (residues 88-844), TM+TG (residues 1-409), TG (residues 195-409), and TP (residues 444-736) are truncated variants with the part of the full-length PBP1b. The moenomycin binding constant (KD) for these variants were determined using SPR.
After immobilization of the target proteins on the sensor chip, different concentrations of moenomycin were passed through the surface and the binding affinities were determined. As shown in FIG. 1B, the moenomycin binding activity of the PBP without the transmembrane domain (TG+TP) was about 5-fold lower in comparison to that of the full-length PBP1b. On the other hand, the binding affinity of TM+TG is similar to that of the full-length protein. The results suggested that the transmembrane domain may play an important role in moenomycin binding.
The moenomycin binding affinity of the full-length was determined as 4.4×10−7 M, similar to the inhibition potency for the TG reaction. Using a TP variant as a control protein, it was confirmed that the determined binding affinities of moenomycin to the PBP1b variants is significant and specific. Binding studies using truncated PBP1b variants further showed that the binding affinities of the TM+TG variant is similar to that of the full-length protein. The role of the TM domain in Moe A binding was shown in the observation that the binding capacity of the TG+TP variant decreased five-fold compared with that of full-length PBP1b (FIG. 1B).
Example 2
Correlation of PBP Binding Activities and Minimal Inhibitory Concentration (MIC) of Moenomycin
Since the transmembrane domain contributes significantly to moenomycin binding, full-length PBPs were prepared from 16 Gram-negative and -positive bacteria (Table 1) for moenomycin binding studies. The genes of Class A PBPs were identified using NCBI database and all of them were confirmed to have TG and TP motifs (FIG. 2).
FIG. 2 shows sequence alignment of full-length bi-functional PBPs from 16 bacterial strains (Seq. ID Nos. 1-16). Each section A-L of FIG. 2 contains 16 rows, with each row corresponding to, in order, Seq. ID Nos. 1-16, respectively. The order of the aligned sequences, in rows, is identical to the order given in Table 1 and FIG. 3.
Each row of FIG. 2 is labeled with the following: “Bpe” (row 1) indicates PBP1b from Bordetella pertussis (Seq. ID No. 1); “Cfr” (row 2) indicates PBP1b from Citrobacter freundii (Seq. ID No. 2); “Eco” (row 3) indicates PBP1b from Escherichia coli (Seq. ID No. 3); “Hin” (row 4) indicates PBP1b from Haemophilus influenzae (Seq. ID No. 4); “Hpy” (row 5) indicates PBP1a from Helicobacter pylori (Seq. ID No. 5); “Kpn” (row 6) indicates PBP1b from Klebsiella pneumoniae (Seq. ID No. 6); “Ngo” (row 7) indicates PBP1 from Neisseria gonorrhoeae (Seq. ID No. 7); “Pae” (row 8) indicates PBP1b from Pseudomonas aeruginosa (Seq. ID No. 8); “Sen” (row 9) indicates PBP1b from Salmonella enterica (Seq. ID No. 9); “Sfl” (row 10) indicates PBP2 from Shigella flexneri (Seq. ID No. 10); “Bsu” (row 11) indicates PBP1a/1 b from Bacillus subtilis (Seq. ID No. 11); “Cdi” (row 12) indicates PBP from Clostridium difficile (Seq. ID No. 12); “Efa” (row 13) indicates PBP2a from Enterococcus faecalis (Seq. ID No. 13); “Efc” (row 14) indicates PBP1 from Enterococcus faecium (Seq. ID No. 14); “Sau” (row 15) indicates PBP2 from Staphylococcus aureus (Seq. ID No. 15); “Spn” (row 16) indicates PBP1b from Streptococcus pneumoniae (Seq. ID No. 16).
Each of the 16 rows of FIG. 2 represents a sequence that continues through each of sections A-L of FIG. 2. Each sequence contains a Transmembrane (TM) domain, a Transglycosylase (TG) domain, and a Transpeptidase (TP) domain. Each domain is represented by gray background shading, with the type of domain (“Transmembrane (TM)”, “Transglycosylase (TG)”, or “Transpeptidase (TP)”) identified by a label at the beginning of each domain (section of gray shading). Conserved residues are represented by boxes of thin black lines that encompass one or more columns. Where a set of conserved residues (i.e., conserved residues in a given column) contains some similarities among the 16 sequences, the text is shown in black or dark gray. Where a set of conserved residues contains identical entries across the 16 sequences, the column is represented with a black background and light text. Note that such columns represented with black background and light text indicate sets of conserved residues.
The sequences of FIG. 2 have been distributed such that beginning and end of each domain and the sets of conserved residues across the 16 sequences are aligned along vertical columns. To accomplish this, “gaps” have been provided, each represented by a (“.”). Such “gaps” do not represent entries in the structural continuity of each sequence or characteristics thereof, but rather are provided only to accomplish the alignment described herein. Accordingly, the numbering of sequence entries (excluding gaps) is specific to each sequence. The number corresponding to the first entry in each row of FIGS. 2A, 2C, 2E, 2G, 2I, and 2K is given to the left of each said first entry. The dots above the top row of each section are provided to indicate the numbering of the first sequence (“Bpe”), occurring at intervals of 10 entries with respect to sequence “Bpe”.
Stars above the top rows of FIG. 2 indicate the motif for TG: EDxxFxxHxxG, GxSTxTQQ, RKxxE, KxxILxxYxN, and RRxxVL, and the motif for TP: SxxK, SxN, and KTG. Underlined stars indicate catalytic residues. The 16 full-length PBP sequences used in this study were aligned with program Jalview and the figure was prepared with ESPript.
The target genes were amplified from respective genomic DNA from each individual bacterial species, and cloned into expression vectors for recombinant protein production using E. coli host. The enzymatic activities of the purified proteins were confirmed by lipid II polymerization (data not shown) and moenomycin binding were measured with SPR. As shown in Table 1, varied steady-state affinity (KD) values were found among PBPs from different species. Nonetheless, all the measured KD values fell into the range of 10−7 M, close to the reported inhibition concentration for the transglycosylation process. Among the Gram-positive bacteria tested, the KD values of PBP from E. faecalis and S. aureus correlate with the MIC values, suggesting that the moenomycin binding site of PBP may be a good target for development of antibiotics against these species.







TABLE 1







Correlation of antimicrobial activity and PBP binding affinity of moenomycin for 16 bacterial strains










Reported values†














Species
MIC, μM*
MIC, μM
Citation
Gene sequence ID‡
SPR_KD, nM§
FA_KD, nM¶










Gram-negative













Bordetella pertussis
0.011-0.021
—

PBP1a (NP_882163)
1,270
 594 ± 2.30


Citrobactor freudii
—
352
28
PBP1b (CAA90232)
728
—


Escherichia coli
80
 55-110
28
PBP1b (NP_414691)
440
54 ± 17


Haemophilus influenzae
—
—

P8P1b (AAX88775)
966
174 ± 13 


Helicobacter pylori
—
1.3
29
PBP1a (NP_207392)
334
25 ± 14


Klebsiella pneumoniae
80
13.75-27.5 
28
P8P1b (NTUH-2044)
819
78 ± 24


Neisseria gonorrhoeae
—
1.69
28
PBP1 (YP_207272)
450
47 ± 26


Pseudomonas aeruginosa
40
 55-110
28
PBP1b (YP_793163)
866
42 ± 29


Salmonella enterica
160
13.75-110  
38
PBP1b (YP_149541)
561
138 ± 113


Shigella flexneri
40
27.5-110 
28
PBP2 (YP688236)
290
—







Gram-positive













Bacillus subtilis
0.3-0.6
0.07-0.43
30
PBP1a/1b (NP_390113)
1,690
197 ± 80 


Clostridium difficile
>160
220-440
30
PBP (CAJ67615)
582
37 ± 33


Enterococcous faecalis
0.075-0.3 
0.1
31
PBP2a (NP_814430)
619
276 ± 14 


Enterococcous faecium
—
>40.5
10
PBP1 (EAN08787)
94
56 ± 11


Staphytococcus aureus
0.075
0.02-0.12
10
PSP2 (NP_371974)
393
30 ± 29


Streptococcus pneumoniae
0.625
—

PBP1b (NP_359500)
900
38 ± 24





*MIC of moenomycin against different bacterial species.


†MIC values of moenomycin from the literature.


‡Sequence ID of class A PBP genes from individual bacterial strains. Homologs of class A PBPs were identified by using BLAST against the NCBI database.


§KD of PBP homologs and moenomycin using SPR. Average values are shown.


¶KD of PBP homologs and moenomycin using our FA-based assay.






Example 3
Expression and Purification of E. coli PBP1b Variants
Full-length PBP1b from E. coli (Swiss-Prot accession number P02919) was amplified from genomic DNA, cloned into the pET vectors (EMD Sciences, San Diego, Calif.), and expressed with an N-terminal (His)6 tag. BL21(DE3) E. coli host cells were grown at 37° C. until OD at 600 nm reached 0.6 and protein expression was induced with 1 mM IPTG for 3 hours. Cell pellets were resuspended in 20 mM Tris at pH 8.0, 300 mM NaCl and broken by Microfluidizer (Microfluidics, Newton, Mass., USA). The solubilization and purification of the recombinant full-length membrane proteins were tested with various detergents (Anatrace, Maumee, Ohio, USA), including n-decyl-β-D-maltopyranoside, n-undecyl-β-D-maltopyranoside, n-dodecyl-β-D-maltopyranoside, n-octyl-β-D-glucopyranoside, n-nonyl-β-D-glucopyranoside, n-tetradecylphosphocholine, n-dodedyl-N,N-dimethylamine-N-oxide, CHAPS (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate) and Triton X-100 (α-[4-(1,1,3,3-Tetramethylbutyl)phenyl]-w-hydroxy-poly(oxy-1,2-ethanediyl)). Among them, DDM (n-dodecyl-β-D-maltopyranoside) was found to solubilize the full-length PBP1b and retain its moenomycin binding activities. Therefore, full-length PBP1b was solubilized with 20 mM DDM and purified by Nickel chelation chromatography following manufacturer's instruction in the presence of 1 mM DDM.
The domains of PBP1b are defined as: transmembrane (TM, residues 64-87), transglycosylase (TG, residues 195-409) and transpeptidase (TP, residues 444-736). Nucleotide primers were designed to amplify respective variants containing TG+TP (residues 88-844), TM+TG (residues 1-409), TG (residues 195-409), and TP (residues 444-736). The amplified products were cloned into pET vectors for expression and purification. The procedure to obtain the variant TM+TG was as described for full-length protein preparations. For the variants TG+TP, TG and TP, 13 mM n-tetradecylphosphocholine (Anatrace) was used for protein extraction and 0.25 mM for Nickel chelation chromatography. The detergent was exchanged to 1 mM DDM and concentrated using Amicon Ultra filter units (Millipore, Billerica, Mass., USA).
Example 4
Expression and Purification of Recombinant Full-Length Bi-Functional PBPs from 16 Bacterial Strains
Homologs of full-length bi-functional PBPs were identified with BLAST against NCBI database using PBP1b protein sequence from E. coli (Swiss-Prot accession number P02919). To confirm the presence of the transglycosylase motifs and active sites, the sequences were aligned using program Jalview (4) with MUSCLE multiple alignment algorithm (5), and the alignment output (FIG. 2) were prepared with ESPript (6). The following residues are omitted in the alignment for clarity, Bpe (B. pertussis): 354-443, Ngo (N. gonorrhoeae): 331-416, Bsu (B. subtilis): 615-643, 735-787, 797-835 and 897-914, and Cdi (C. difficile): 267-322 and 804-842.
FIG. 3 shows the SDS-PAGE analysis of purified protein of class A PBPs from sixteen bacterial species. Each lane contains 5 μg of protein and M denotes molecular weight markers in kDa. 1: PBP1a from Bordetella pertussis (Seq. ID No. 1), 2: PBP1b from Citrobacter freundii (Seq. ID No. 2), 3: PBP1b from Escherichia coli (Seq. ID No. 3), 4: PBP1b from Haemophilus influenzae (Seq. ID No. 4), 5: PBP1a from Helicobacter pylori (Seq. ID No. 5), 6: PBP1b from Klebsiella pneumoniae (Seq. ID No. 6), 7: PBP1 from Neisseria gonorrhoeae (Seq. ID No. 7), 8: PBP1b from Pseudomonas aeruginosa (Seq. ID No. 8), 9: PBP1b from Salmonella enterica (Seq. ID No. 9), 10: PBP2 from Shigella flexneri (Seq. ID No. 10), 11: PBP1a/1b from Bacillus subtilis (Seq. ID No. 11), 12: PBP from Clostridium difficile (Seq. ID No. 12), 13: PBP2a from Enterococcus faecalis (Seq. ID No. 13), 14: PBP1 from Enterococcus faecium (Seq. ID No. 14), 15: PBP2 from Staphylococcus aureus (Seq. ID No. 15), 16: PBP1b from Streptococcus pneumoniae (Seq. ID No. 16).
For expression of individual PBPs, genomic DNAs were purchased from American Type Culture Collection (ATCC, Manassas, Va., USA). The recombinant proteins of the full-length genes from 16 bacteria, including Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus (MRSA Mu50), Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica and Neisseria gonorrhoeae, were prepared using the same protocols as described for the full-length E. coli PBP1b. Most of them can be obtained with purity greater than 80% (FIG. 3) and used for binding experiments.
Example 5
Surface Plasmon Resonance Detection
Purified PBP and its variants were immobilized onto CM3 chip (GE healthcare, Uppsala, Sweden) to about 1,500 ̃2,000 RU via amine-coupling method. The chips were then passed over with different concentrations of moenomycin A (0-2000 nM). Immobilization and data collection were performed with BIAcore T100 (GE Healthcare, Uppsala, Sweden) at 25° C.
Example 6
Fluorescence Anisotropy Measurements
Fluorescence anisotropy measurements were carried out in triplicates in the wells of 384-well plates using a laser fluorimetry equipped with a 488 nm laser (IsoCyte, Blueshift Biotech, Inc., Sunnyvale, Calif., USA). Various buffers, salts, pH values, and divalent cations (Ca++, Mg++, Co++) were optimized for fluorescence anisotropy measurements. KD and KI, determinations were carried out in 100 mM NaCl, 10 mM Tris, pH 8.0. The scanning focus was above the plate bottom to avoid detection interferences. Data analysis was performed with the proprietary software, BlueImage (Blueshift Biotech Inc). Fluorescence anisotropy values (A) were calculated using the equation: A=(/∥−G*/⊥)/(/∥+2G*/⊥), where /∥ is the fluorescence intensity of emitted light parallel to excitation, /⊥ is the fluorescence intensity of emitted light perpendicular to excitation, and G is the gating factor that corrects for instrument bias. The G factor is experimentally determined for each run using the probe-only well as the basal an isotropy.
FIG. 4 shows the design of a novel fluorescence anisotropy assay for transglycosylase, according to implementations of the present disclosure.
Based on the conclusion that the full-length proteins shall be used for moenomycin binding studies, a fluorescence anisotropy (FA)-based assay was thus designed to monitor the binding affinities of small molecules towards TG. Previous studies on the complex crystal structure and the structure-activity relationship suggested that the modification of compound 1 to 2 (FIG. 4A) will not dramatically reduce the binding and antibacterial activities. FIG. 4(A) shows the chemical structures of the modified moenomycin A (2). Furthermore, the amino moiety in 2 allows the conjugation with any fluorophore as a moenomycin-based probe for binding studies. Indeed, compound 2 was readily linked with a fluorescein (6-carboxyfluoresein N-hydroxysuccinimide ester) under basic conditions to prepare the fluorescent probe 3 (F-Moe, FIG. 4B). FIG. 4(B) shows the chemical structures of the fluorescein-labeled moenomycin, F-Moe (3). One major concern about the fluorescent probe used in the FA assay is the probe itself; either the fluorophore or the structure modification, may interfere with the binding between the targeted protein and the small molecule. Therefore, the PBP binding affinities of Moe A and the fluorescent probe were compared using SPR. The determined steady-state affinity (KD) values are similar for Moe A and F-Moe (4.4×10−7 vs. 5.2×10−7 M) (FIGS. 4C and 4D).
FIG. 4(C) shows the SPR analysis of the binding activity of moenomycin A to E. coli PBP1b, and FIG. 4(D) shows the SPR analysis of the binding activity of F-Moe to E. coli PBP1b. Responses for moenomycin binding to immobilized E. coli PBP1b were shown. The data were analyzed using steady-state affinity and fitted to a 1:1 interaction model, as shown in the inset graph. The KD values deduced from the intercepts of X-axis and the dotted lines, are 4.4×10−7 M and 5.161×10−7 M for Moe A and F-Moe, respectively.
The result confirms that F-Moe is a valid fluorescence probe useful for the FA-based PBP binding assay.
FIG. 5 shows the development of high-throughput FA assay for transglycosylase. FIG. 5(A) shows the concentration-dependent changes in fluorescence anisotropy observed when E. coli PBP1b bound to F-Moe. FIG. 5(B) shows the improved FA assay with the Helicobacter pylori PBP1a. The concentration-dependent fluorescence anisotropy changes were performed similarly using the H. pylori PBP1a. The maximum anisotropy value was 0.2. FIG. 5(C) shows the displacement of the PBP1a bound F-Moe complex by unlabeled moenomycin at various concentrations. The changes in fluorescence anisotropy is defined as [(Aobs−Amin)/(Amax−Amin)×100%]. The KD and IC50 value was calculated as described in example 2.
The anisotropy of F-Moe increased significantly by incubation with E. coli PBP1b, supposedly due to the formation of F-Moe-PBP1b complex (FIG. 5A). In contrast, the anisotropy of F-Moe was unchanged when incubated with bovine serum albumin, up to 100 μM (data not shown). The protein concentrations were further titrated down to establish the dose-dependency of the anisotropy changes. The deduced dissociation constant (KD) for F-Moe was in the sub-micromolar range (54±17 nM) for E. coli PBP1b (FIG. 5A). However, the signal-to-noise ratio of FA assay using the E. coli PBP1b is of limited satisfaction with the maximum anisotropy equal to 0.120. Different class A PBPs were screened to improve the assay. Of all homologs tested, the FA assay using Helicobacter pylori PBP1a produced the best signal-to-noise ratio with KD of 25±14 nM, and with the anisotropy increases from 0.018 to 0.2 upon binding to F-Moe (FIG. 5B). For the development of an assay for inhibitor screening, F-Moe was pre-incubated with H. pylori PBP1a and then competed with unlabeled Moe A at various concentrations. A decrease in anisotropy was observed as the concentration of Moe A increased, resulting in the KI, (inhibition constant) and IC50 values as 0.47±0.10 μM and 0.36 μM, respectively (FIG. 5C). The result validates the FA assay to screen for inhibitors that displace the probe competitively from the moenomycin binding pocket of PBP.
Example 7
Determination of KD Values from Direct Binding Assay
For direct binding assay, the initial condition contained 40 μl of 100 nM F-Moe in 10 mM Tris, pH 8.0, 100 mM NaCl. After a small volume of PBP stock was added, the anisotropy change was measured after a 5-minute equilibration. We assume that the direct titration follows a simple binding equilibrium between Ligand (L) and Receptor (R)




  
    (
    
      
        
          R
          +
          
            L
            S
          
        
        ↔
        
          
            
              RL
              
                K
                D
              
            
            S
          
          ⁢
          
            
          
          ⁢
          with
          ⁢
          
            
          
          ⁢
          
            K
            D
          
        
      
      =
      
        R
        ·
        
          
            L
            S
          
          /
          
            RL
            S
          
        
      
    
    )
  
  .




The data was fitted to the [Eq. 1] using Prism (GraphPad Software, San Diego, Calif.) for the determination of KD (3):

(Aobs−Amin)/(Amax−Amin)=(KD+LST+RT)−[(KD+LST+RT)2−4·LST·RT]1/2  [Eq. 1]

where Aobs is the observed anisotropy; Amin is the minimum anisotropy; Amax is the maximum anisotropy; LST is the total concentration of F-Moe, RT is the total concentration of PBP, and KD is the dissociation constant between the ligand (F-Moe) and the receptor (PBP). The reported value is the average of four different runs.

Example 8
Determination of KI, and IC50 Values from Competitive Displacement Assay
For displacement assay, the initial condition contained 40 μl of 100 nM F-Moe, 10 μg/ml H. pylori PBP1a in 10 mM Tris, pH 8.0, 100 mM NaCl. Aliquots of compound stock solution were added and the anisotropy was monitored after 5 minutes of equilibration. The data from the displacement assay was used to calculate the inhibition constant (KI) and IC50 value of an inhibitor using the complete competitive binding model




  
    
      
        
          (
          
            
              RL
              +
              
                L
                S
              
            
            ⁢
            
              ↔
              
                K
                1
              
            
            ⁢
            
              R
              +
              
                L
                S
              
              +
              L
            
            ⁢
            
              ↔
              
                K
                D
              
            
            ⁢
            
              
                RL
                S
              
              +
              L
            
          
          )
        
        .
      
    
    
      
        (
        3
        )
      
    
  




The KI value was determined by plotting the total inhibitor concentration (C) versus the molar ratio of bound/free F-Moe and fitting the resulting curve to [Eq. 2],

C=[(KI/KD*X)+1][RT−(LST*X)/(X+1)−KD*X  [Eq. 2]

X=Bound/Free=(Aobs−Amin)/(Amax−Aobs)

where Aobs is the observed anisotropy at a particular concentration of a compound (C), Amin is the minimum anisotropy, Amax is the maximum anisotropy, LST is the total concentration of F-Moe, RT is the total concentration of PBP1a, KD is the dissociation constant between the ligand (F-Moe) and the receptor (PBP) and KI is the dissociation constant between the inhibitor and PBP1a. The reported value is the average of four different runs.

For IC50 determination, the fraction of bound fluorescent probe is plotted versus the log value of the competitor concentration (C). Fitting the resulting sigmoidal curve using [Eq. 3] would result in an IC50 value,

(Aobs−Amin)/(Amax−Amin)=1/(1+10^((C−log(IC50))*Hill slope)  [Eq. 3]

where Aobs is the observed anisotropy at a particular concentration of a compound (C), Amin is the minimum anisotropy, Amax is the maximum anisotropy.

Example 9
High-Throughput Screening for Transglycosylase Inhibitors
The FA assay was used to screen against 50,000 purchased small molecules (ChemBridge Inc., San Diego, Calif., USA) and 7,000 from proprietary collections. The compounds were transferred to 96-well plates (Freedom Evo, Tecan Schweiz AG, Männedorf, Switzerland) and then to 384-well plates using a multi-dispenser (Labcyte, Sunnyvale, Calif., USA) to prepare the compound plates for screening. The H. pylori PBP1a (10 μg/ml) in 100 nM F-Moe, 10 mM Tris, 100 mM NaCl, pH 8.0 at a final volume of 40 μl was added to 384-well plates (Freedom Evo 150, Tecan). One μl of 2 mM compound stocks were added to wells using a multi-dispenser (Labcyte). The last two columns of every plate were controls with 10 μM moenomycin and 2.5% DMSO, respectively. After a 30-minute incubation, changes in fluorescence anisotropy were determined with Isocyte (Blueshift Biotech Inc). Hits that showed greater than 75% reduction compared to the control anisotropy values were selected for further confirmation.
Several moenomycin analogues (2, 4, and 5) were prepared to evaluate the reliability of the FA-based assay. As shown in Table 2, the differential binding affinities (the IC50 values) of these moenomycin analogues by the FA-based assay are comparable with the published reports. The modification of moenomycin A to 2 resulted in a little decrease in TG binding affinities. Dimerization of 2 via an eight-carbon spacer produced 4 with a two-fold increase over 2 in both PBP binding and the antimicrobial potencies, although 4 is still less potent than Moe A (1). More significantly, compound 5 without the C25 lipid moiety could hardly displace the F-Moe probe 3, indicating that the hydrophobic part of moenomycin is crucial to the binding to the full-length PBP1b. It is noteworthy that the order in inhibition potency is in agreement with that of MIC values (Table 2).







TABLE 2







Inhibition of TG activity and the antibacterial determinations of selected hits









MIC, μM‡














B. subtilis
E. faecalis
S. aureus
S. pneumoniae


Compound*
IC50, μM†
(ATCC23857)
(ATCC29212)
(ATCC29213)
(ATCC49619)















1
0.36
0.33
0.04
<0.01
0.33


2
2.10
2.50
10.0
1.25
20.0


4
0.92
1.25
5.0
0.625
20.0


5
125.00
—
—
—
—


6
34.00
0.25
0.25
1.0
4.0


7
3.70
0.25
1.0
4.0
—


8
9.30
4.0
>4.0
>4.0
>4.0





*Compounds 2, 4, and 5 are moenomycin derivatives. Compounds 6-8 were HTS hits.


†IC50 values were determined by using the fluorescence anisotropy assay shown in FIG. 5A.


‡The MICs of moenomycin against different bacterial species were determined as described in Materials and Methods. ATCC, American Type Culture Collection.






The FA assay was used to screen a collection of 57,000 small molecules along with Moe A derivatives (FIG. 6) at 50 μM. FIG. 6 illustrates chemical structures of moenomycin derivatives (4 and 5), according to implementations of the present disclosure.
High controls are assays with 1 μM of moenomycin and low controls are assays with 2.5% DMSO. Other proteins, such as bovine serum albumin, were also included as a control to confirm that the anisotropy increase is not from non-specific binding.
FIG. 7 illustrates screening for small molecules as TG inhibitors using the class A PBPs, according to implementations of the present disclosure. FIG. 7(A) shows the scheme of HTS for TG inhibitors using FA assay. Protein structure graphics were created from PDB ID 2OLV (12). 7(B) shows chemical structures of the HTS hits (compounds 6-8).
Z′ value, a statistical parameter ranging from 0 to 1 to evaluate the robustness of high-throughput screening, was determined as 0.895 from more than 100 independent experiments. Eleven possible hits that showed at least 75% inhibition in the screening were selected for additional studies involving antimicrobial assays and IC50 values determinations for PBP binding (FIG. 7A). Among which, two moenomycin analogues (2 and 4) and three small molecules (compounds 6, 7 and 8) were confirmed to have both antibacterial and the TG binding inhibition activities (Table 2). Respectively, compounds 6, 7 and 8 are (Z)-5-(4-bromophenyl)-3-((5-nitrofuran-2-yl)methylene)furan-2(3H)-one(Z)-1,3-diphenyl-4-(2-(thiazol-2-yl)hydrazono-1H-pyrazol-5(4H)-one; (E)-4-(2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-enyl)benzoic acid.
Using the FA-based assay, a hit rate of 0.02% was achieved with the Z′ value equal to 0.895.
Example 10
Determination of Minimal Inhibitory Concentration (MIC)
The minimal inhibitory concentration (MIC) of tested compounds was determined following the NCCLS standard. The experiments were conducted in 96-well microtiter plates using two-fold dilutions in Muller-Hilton broth with (Streptococcus pneumonia) or without blood (Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus). Exponentially growing cells at 5×105 cells/ml were incubated with test compounds at various concentrations. After an 18 h to 24 h incubation at 37° C., MIC was determined as the minimal concentration of the compound that prevents bacterial growth.
While the apparatus and method have been described in terms of what are presently considered to be the most practical and preferred implementations, it is to be understood that the disclosure need not be limited to the disclosed implementations. It is intended to cover various modifications and similar arrangements included within the spirit and scope of the claims, the scope of which should be accorded the broadest interpretation so as to encompass all such modifications and similar structures. The present disclosure includes any and all implementations of the following claims.

## Claims

1. A method for high throughput screening (HTS) of antibacterial agents, the method comprising:
providing a candidate agent for screening;
providing a class A penicillin-binding protein, or fragment thereof (PBP), from a bacterial species, comprising at least a transmembrane (TM) domain and a transglycosylase (TG) domain;
determining a first binding affinity of the candidate agent to the at least transmembrane (TM) domain and transglycosylase (TG) domain-containing fragment of the class A penicillin-binding protein;
comparing the first binding affinity to a second binding affinity of the candidate agent to a class A penicillin-binding protein fragment that lacks the TM domain;
identifying a candidate agent as a putative transglycosylase inhibitor of the bacterial species as the antibacterial agent, wherein the candidate agent exhibits a higher first binding affinity to the transmembrane (TM) domain-containing class A penicillin-binding protein, or fragment thereof, as compared to the second binding affinity to the PBP fragment that lacks the TM domain; and
confirming the transglycosylase inhibitor activity of the putative transglycolase inhibitor by testing for an ability to inhibit growth of the same bacterial species whose PBP transmembrane domain-containing fragment it binds with higher affinity,
wherein the binding affinity between the candidate agent and the transmembrane (TM) domain of the class A penicillin-binding protein is determined by anisotropy measurement assay.

2. The method of claim 1, wherein the anisotropy measurement assay is at least one of a competition displacement assay or a direct binding assay.

3. The method of claim 2, wherein the competition displacement assay competes a labeled moenomycin with the candidate.

4. The method of claim 1, wherein the class A penicillin-binding protein comprises three domains which, from N terminus to C terminus, are transmembrane, transglycosylase, and transpeptidase domains.

5. The method of claim 1, wherein the bacterial species is Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus (MRSA Mu50), Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica or Neisseria gonorrhoeae.

6. The method of claim 1, further comprising:
using a high throughput device comprising a platform with a plurality of versions of class A penicillin-binding protein, or fragment thereof, comprising at least transmembrane and a transglycosylase domain from a plurality of bacterial species;
determining the binding affinity of the candidate agent to the multiple versions of class A penicillin-binding protein, or fragment thereof; and
identifying a candidate agent as an effective transglycosylation agent for the bacterial species based on a binding affinity to the corresponding class A penicillin-binding protein, or fragment thereof.

7. The method of claim 6, wherein the plurality of class A penicillin-binding proteins, or fragments thereof are immobilized on a solid substrate or chip.

8. The method of claim 7, wherein the plurality of class A penicillin-binding proteins, or fragments thereof are immobilized on the solid substrate or chip by amine coupling.

9. The method of claim 1, wherein the anisotropy measurement of PBP binding, comprises a fluorescence anisotropy (FA) assay.

10. The method of claim 9, wherein a fluorescein-labeled moenomycin is used in the fluorescence anisotropy (FA) assay.

11. The method of claim 2, further comprising:
determining the inhibition constant (KI) and IC50 values from the competition displacement assay.

12. The method of claim 2, further comprising:
determining the dissociation constant (KD) value from the direct binding assay.

13. The method of claim 1, wherein the candidate agent is a small molecule.