WO2018204322A1

WO2018204322A1 - Multimeric inhibitors of bacterial type iii secretion system

Info

Publication number: WO2018204322A1
Application number: PCT/US2018/030389
Authority: WO
Inventors: Zachary D. Aron; Matthew C. TORHAN; Nicholas O. BOWLIN; John D. Williams; Terry L. Bowlin; Donald T. Moir
Original assignee: Microbiotix, Inc.
Priority date: 2017-05-02
Filing date: 2018-05-01
Publication date: 2018-11-08

Abstract

Organic compounds showing the ability to inhibit effector toxin secretion or translocation mediated by bacterial type III secretion systems (T3SS) are disclosed. The disclosed type III secretion system inhibitor compounds are useful for combating infections by Gram-negative bacteria such as Pseudomonas aeruginosa and other bacteria having such type III secretion systems.

Description

MULTIMERIC INHIBITORS OF BACTERIAL TYPE III SECRETION SYSTEM

Statement Regarding Federally Sponsored Research

This invention was made with government support under R43 AI068185 and R01 AI099269 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

This invention is in the field of therapeutic drugs to treat bacterial infection and disease. In particular, the invention provides organic compounds that inhibit the type III secretion system of one or more bacterial species.

Background of the Invention

The bacterial type III secretion system (T3SS) is a complex multi-protein apparatus that facilitates the secretion and translocation of effector proteins from the bacterial cytoplasm directly into the mammalian cytosol. This complex protein delivery device is shared by over 15 species of Gram-negative human pathogens, including Salmonella spp., Shigella flexneri, Pseudomonas aeruginosa, Yersinia spp., enteropathogenic and

enteroinvasive Escherichia coli, and Chlamydia spp. (Hueck, 1998, Type III protein secretion systems in bacterial pathogens of animals and plants, Microbiol. Mol. Biol. Rev. , 62:379-433; Keyser, et al., 2008, Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria, /. Intern. Med. , 264: 17-29.)

In the opportunistic pathogen P. aeruginosa, the T3SS is the major virulence factor contributing to the establishment and dissemination of acute infections (Hauser, 2009, The type III secretion system of Pseudomonas aeruginosa: infection by injection, Nat. Rev. Microbiol. , 7:654-65). Four T3SS effectors have been identified in P. aeruginosa strains - ExoS, ExoT, ExoY, and ExoU. ExoS and ExoT are bifunctional proteins consisting of an N- terminal small G-protein activating protein (GAP) domain and a C-terminal ADP ribosylation domain; ExoY is an adenylate cyclase; and ExoU is a phospholipase (see review in Engel and Balachandran, 2009, Role of Pseudomonas aeruginosa type III effectors in disease, Curr. Opin. Microbiol , 12:61-6). In studies with strains producing each effector separately, ExoU and ExoS contributed significantly to persistence, dissemination, and mortality while ExoT produced minor effects on virulence in a mouse lung infection model, and ExoY did not appear to play a major role in the pathogenesis of P. aeruginosa (Shaver and Hauser, 2004, Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung, Infect. Immun. , 72:6969-77). While not a prototypical effector toxin, flagellin (FliC) may also be injected into the cytoplasm of host cells from P. aeruginosa via the T3SS machinery, where it triggers activation of the innate immune system through the nod-like receptor NLRC4 inflammasome. (Franchi, et al., 2009, The inflammasome: a caspase-1 -activation platform that regulates immune responses and disease pathogenesis, Nat. Immunol. , 10:241-7; Miao, et al., 2008, Pseudomonas aeruginosa activates caspase 1 through Ipaf, Proc. Natl. Acad. Set USA, 105:2562-7.)

The presence of a functional T3SS is significantly associated with poor clinical outcomes and death in patients with lower respiratory and systemic infections caused by P. aeruginosa (Roy-Burman, et al., 2001, Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections, /. Infect. Dis. , 183: 1767- 74). In addition, T3SS reduces survival in P. aeruginosa animal infection models (Schulert, et al., 2003, Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia, /. Infect. Dis. , 188: 1695-706), and is required for the systemic dissemination of P. aeruginosa in a murine acute pneumonia infection model (Vance, et al., 2005, Role of the type III secreted exoenzymes S, T, and Y in systemic spread of Pseudomonas aeruginosa PAOl in vivo, Infect. Immun., 73: 1706-13). T3SS appears to contribute to the development of severe pneumonia by inhibiting the ability of the host to contain and clear bacterial infection of the lung. Secretion of T3SS toxins, particularly ExoU, blocks phagocyte-mediated clearance at the site of infection and facilitates establishment of an infection (Diaz, et al., 2008,

Pseudomonas aeruginosa induces localized immuno-suppression during pneumonia, Infect. Immun. , 76:4414-21). The result is a local disruption of an essential component of the innate immune response, which creates an environment of immunosuppression in the lung. This not only allows P. aeruginosa to persist in the lung, but it also facilitates superinfection with other species of bacteria. While several antibacterial agents are effective against P. aeruginosa, the high rates of mortality and relapse associated with serious P. aeruginosa infections, even in patients with hospital-acquired pneumonia (HAP) receiving antibiotics active against the causative strain, reflect the increasing incidence of drug-resistant strains and highlights the need for new therapeutic agents. (See, e.g., El Solh, et al., 2007, Clinical and hemostatic responses to treatment in ventilator-associated pneumonia: role of bacterial pathogens, Crit. Care Med. , 35:490-6; Rello, et al., 1998, Recurrent Pseudomonas aeruginosa pneumonia in ventilated patients: relapse or reinfection?, Am. J. Respir. Crit. Care Med. , 157:912-6; and Silver, et al., 1992, Recurrent Pseudomonas aeruginosa pneumonia in an intensive care unit., Chest, 101: 194-8.) Conventional bacteriostatic and bactericidal antibiotics appear insufficient to adequately combat these infections, and new treatment approaches such as inhibitors of P. aeruginosa virulence determinants may prove useful as adjunctive therapies (Veesenmeyer, et al., 2009, Pseudomonas aeruginosa virulence and therapy: evolving translational strategies, Crit. Care Med. , 37: 1777-86).

The potential for the type III secretion system as a therapeutic target has prompted several groups to screen for inhibitors of T3SS in various bacterial species, including Salmonella typhimurium, Yersinia pestis, Y. pseudotuberculosis, and E. coli. (Reviewed in Dickey SW, et al., 2017, Different drugs for bad bugs: antivirulence strategies in the age of antibiotic resistance. Nat Rev Drug Discov doi: 10.1038/nrd.2017.23. [advance online publication], Keyser, et al., 2008, Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria, /. Intern. Med. , 264: 17-29; and

Clatworthy, et al., 2007, Targeting virulence: a new paradigm for antimicrobial therapy, Nat. Chem. Biol. , 3:541-8). High levels of sequence conservation among various proteins comprising the T3SS apparatus suggest that inhibitors of T3SS in one species may also be active in related species. Broad spectrum activity of T3SS inhibitors identified in a screen against Yersinia has been demonstrated in Salmonella, Shigella, and Chlamydia (Hudson, et al., 2007, Inhibition of type III secretion in Salmonella enterica serovar Typhimurium by small-molecule inhibitors, Antimicrob. Agents Chemother., 51:2631-5; Veenendaal, et al., 2009, Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton, /. Bacteriol., 191:563-70; Wolf, et al., 2006, Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle, Mol. Microbiol , 61 : 1543-55). Screening for P. aeruginosa T3SS inhibitors has been reported, leading to several selective inhibitors of P. aeruginosa T3SS-mediated secretion, one of which is comprised of a phenoxyacetamide scaffold and reproducibly inhibits both T3SS-mediated secretion and translocation (Aiello, et al., 2010, Discovery and Characterization of Inhibitors of

Pseudomonas aeruginosa Type III Secretion, Antimicrob. Agents Chemother. , 54: 1988- 1999). SAR-driven experimentation of the phenoxyacetamide scaffold has led to new families of aryl and heteroaryl phenoxyacetamide derivatives showing T3SS inhibitory activity against multiple Gram-negative bacterial targets including Pseudomonas, Yersinia or Chlamydia. See, e.g., International patent publications WO 2010/118046, WO 2013/010082, and WO 2013/010089.

Further work to understand possible mechanisms of action for various T3SS inhibitors has inspired the experimental work leading to the development of a family of multimeric T3SS inhibitors having improved potency, as disclosed herein.

Summary of the Invention

The present invention provides novel antibacterial/antivirulence agents with surprisingly high potency against the P. aeruginosa T3SS, including the T3SS in drug- resistant strains. The novel compounds of the present invention show a level of potency, in comparison to previously reported T3SS inhibitor compounds, that make them particularly advantageous as antibacterial/antivirulence agents.

The present invention provides new bacterial type III secretion system (T3SS) inhibitor compounds. Preparation of multimeric (dimeric and trimeric) forms of

aryl/heteroaryl phenoxyacetamide analogues has led to the discovery of novel compounds showing a surprising increase in potency as inhibitors of, for example, the Pseudomonas aeruginosa type III secretion system in comparison to monomeric inhibitors of similar structure. The invention provides additional compounds with T3SS inhibition potencies surprisingly improved by about 100-fold over previous phenoxyacetamides or other small molecule T3SS inhibitors, and these novel compounds exhibit comparable or decreased cytotoxicity over previous compounds.

The surprisingly increased potencies of the compounds disclosed herein may be related to the polymeric nature of the molecular target of the phenoxyacetamides, namely, the T3SS needle subunit protein PscF. Previously, only genetic evidence suggested that polymeric PscF is the phenoxyacetamide target, but these new potency improvement results provide biochemical evidence that the target is a polymer such as the T3SS needle with multiple binding sites. Furthermore, the multimeric analogs of the present invention provide significant inhibition potency against P. aeruginosa strains carrying pscF alleles such as pscF(R75H) that confer resistance to phenoxyacetamide monomers. See, Figure 1 of Bowlin, N.O., et al., "Mutations in the Pseudomonas aeruginosa Needle Protein Gene pscF Confer Resistance to Phenoxyacetamide Inhibitors of the Type III Secretion System," Antimicrob. Agents Chemother. , 58:2211-2220 (2014). Approximately 100 PscF molecules polymerize to form the P. aeruginosa T3SS needle, resulting in multiple apparent phenoxyacetamide binding sites in close proximity.

Accordingly, the T3SS inhibitor compounds described herein inhibit T3SS-mediated secretion of a bacterial exotoxin (effector) from a bacterial cell. More preferably, a T3SS inhibitor compound described herein inhibits T3SS-mediated secretion of an effector from a bacterial cell and also inhibits T3SS-mediated translocation of the effector from the bacterial cell to a host cell (e.g., human or other animal cell).

In a preferred embodiment, a T3SS inhibitor compound described herein inhibits the T3SS in a bacterium of the genus Pseudomonas. Due to the similarities among Gram- negative bacterial secretion systems, it is contemplated that the presently disclosed inhibitors will also exhibit inhibitory activity against other pathogenic species, such as Salmonella spp., Shigella flexneri, Yersinia spp., enteropathogenic and enteroinvasive Escherichia coli, and Chlamydia spp.

The present invention provides a family of bacterial type III secretion system (T3SS) inhibitor compounds of Formula I:

wherein the multimer of Formula I has a linear or branched structure of n + 1 monomeric moieties, V;

n is an integer of at least 1 and may be up to 20 or more;

each V is, independently, a moiety having the structure of Formula Ila or lib:

wherein,

A is independently CH or N;

X is independently selected from hydrogen or halogen;

Z is O, S, NH; or NR³, where R³ is alkyl;

R¹, R^1', and R^1" are selected independently from: hydrogen, halogen, alkyl, hydroxy, alkoxy, alkylthio, or cyano;

R² is hydrogen or alkyl;

Y is a divalent straight-chain, branched, or cyclic alkyl, alkenyl or alkynyl radical of from 1 to 6 carbon atoms, which Y radical may be unsubstituted or substituted with up to four substituents selected from halo, cyano, hydroxyl, amino, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy;

and

W is an aryl or heteroaryl radical forming a five-membered or six-membered ring which may be additionally fused with from 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, which W radical may be unsubstituted or substituted with up to four substituents selected from halo, hydroxyl, amino, carboxamido, carboxyl, cyano, sulfonamide, sulfonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, and wherein any two substituents together may form an aromatic or non-aromatic ring structure fused with said aryl or heteroaryl radical W, or alternatively wherein substituents on W may be optionally bonded covalently to either Y or R², or both Y and R², to form a heterocyclic or carbocyclic, aromatic or non-aromatic ring systems;

and wherein,

each L is, independently, a linker comprising one or more divalent, optionally substituted radicals selected from C1-C6 alkylene, C1-C6 alkenylene, cumulenylene (such as a divalent radical derived from butatriene, -HC=C=C=CH-), arylene (such as phenylene or naphthylene), heteroarylene, ether (-R-0-R-, where R and R' are, independently, C1-C6 alkylene, C1-C6 alkenylene, arylene such as phenylene, or heteroarylene such as 2,5-pyridinylene), thioether (-R-S-R-, where R and R' are, independently, C1-C6 alkylene, C1-C6 alkenylene, arylene such as phenylene, or heteroarylene such as 2,5-pyridinylene), or -C:0-NH-.

In another embodiment, the present invention provides a family of bacterial type III secretion system (T3SS) inhibitor compounds wherein V may be the same or different moiety having the structure of Formula III:

Formula (III) wherein

A is CH or N;

X is independently selected from hydrogen or halogen;

R is hydrogen or methyl;

Y is a divalent straight-chain, branched, or cyclic alkyl, alkenyl or alkynyl radical of from 1 to 6 carbon atoms;

Z is O, S, or NH or NR³; and

W is an aryl or heteroaryl radical forming a five-membered or six-membered ring which may be additionally fused with from 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, which W radical may be unsubstituted or substituted with up to four substituents selected from halo, hydroxyl, amino, carboxamido, carboxyl, cyano, sulfonamido, sulfonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, and wherein any two substituents together may form an aromatic or non- aromatic ring structure fused with said aryl or heteroaryl radical W, or alternatively wherein substituents on W may be optionally bonded covalently to either Y or R, or both Y and R, to form a heterocyclic or carbocyclic, aromatic or non-aromatic ring systems.

In another embodiment, the present invention provides a family of bacterial type III secretion system (T3SS) inhibitor compounds wherein V may be the same or different moiety having the structure of Formula IV:

Formula (IV) wherein

A is CH or N;

X is independently selected from hydrogen or halogen;

Y is -CH₂- -CH(CH₃)-, or -C(CH₃)₂-; and

W is an aryl or heteroaryl radical forming a five-membered or six-membered ring which may be additionally fused with from 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, which W radical may be unsubstituted or substituted with up to four substituents selected from halo, hydroxyl, amino, carboxamido, carboxyl, cyano, sulfonamido, sulfonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, and wherein any two substituents together may form an aromatic or non- aromatic ring structure fused with said aryl or heteroaryl radical W, or alternatively substituents on W may be optionally bonded covalently to either Y or N, or both Y and N, to form a heterocyclic or carbocyclic, aromatic or non-aromatic ring systems.

Multimers linking together monomeric components of the foregoing Formulae Ila, lib, III and IV may have any of a number of isomeric configurations, considering the asymmetric carbon (a carbon) of the monomeric components (V in Formula I). Since each monomer may conform as an R-isomer or an S-isomer, the multimers may take multiple configurations, and compositions of compounds having a given structural formula may comprise a mixture of multiple isomeric forms, particularly where the polymeric forms are heteropolymers (as opposed to homopolymers of V). R-isomers are usually the active conformation of the monomer; the pure S-isomer is usually inactive. Thus, preferred compounds according to the invention will be isolated R-isomers or mixtures where the R- following formulas:

MBX-4103

MBX-4129

11

MBX-4915

In another embodiment, the present invention is directed to a composition for inhibiting the bacterial T3SS secretion system, the composition comprising a novel multimeric phenoxyacetamide inhibitor described herein. The compositions described herein are suitable for the inhibitors of, e.g., the Pseudomonas aeruginosa type III secretion system in mammals, and in particular, humans.

In another embodiment, the present invention is directed to a method for treating or preventing bacterial infections in a mammal by administration of the novel multimeric phenoxyacetamide bacterial T3SS system inhibitors of the present invention. In a preferred embodiment, the mammal is a human.

Also disclosed are pharmaceutical compositions comprising a therapeutically effective amount of a novel multimeric phenoxyacetamide bacterial T3SS inhibitor compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical compositions are suitable for use in the disclosed methods for treating or preventing bacterial T3SS infections in a mammal. The pharmaceutical compositions may be formulated for both parenteral and/or nonparenteral administration to a subject or patient in need thereof.

More particularly, the invention is related to the identification of novel multimeric phenoxyactamide inhibitors for treating and/or preventing bacterial T3SS infections caused by the genus Pseudomonas. Due to the similarities among Gram-negative bacterial secretion systems, it is contemplated that the presently disclosed inhibitors will also exhibit inhibitory activity against other pathogenic species, such as Salmonella spp., Shigella flexneri, Yersinia spp., enteropathogenic and enteroinvasive Escherichia coli, and Chlamydia spp.

In another embodiment, the T3SS inhibitors of the present invention may be administered to a subject in need thereof optionally in combination with one or more known antibacterial agents.

In another embodiment, the multimeric phenoxyacetamide bacterial T3SS inhibitors of the present invention are formulated into a pharmaceutically-acceptable carrier and are applied/administered to a subject in need thereof by an injection, including, without limitation, intradermal, transdermal, intramuscular, intraperitoneal and intravenous.

According to another embodiment of the invention, the administration is oral and the compound may be presented, for example, in the form of a tablet or encased in a gelatin capsule or a microcapsule, which simplifies oral application. The production of these forms of administration is within the general knowledge of a skilled practitioner in the field.

Multiple routes of administration are envisioned for these drug-like molecules, and highly cost-effective production strategies can be easily achieved.

In practice, the compounds of the invention, or pharmaceutically acceptable salts thereof, of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g. , oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the invention, and/or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and/or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention can include a

pharmaceutically acceptable carrier and a compound or a pharmaceutically acceptable salt of the compounds of the invention. The compounds of the invention, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds.

In a preferred embodiment, the multimeric phenoxyacetamide bacterial T3SS inhibitors of the present invention will specifically target the T3SS needle subunit protein PscF and thus prevent secretion of effector elements into a host cell.

In preferred embodiments, the T3SS inhibitor compounds of the present invention exhibit inhibit T3SS effector transcription by at least 50% at a concentration of 1 μΜ and/or exhibit at least 50% inhibition of effector secretion at a concentration of 1 μΜ or less (IC50≤1 μΜ).

Compounds according to the foregoing formulae were tested using assays showing specific inhibition of the T3SS of P. aeruginosa.

Desirable T3SS inhibitor multimeric compounds described herein inhibit T3SS effector transcription by at least 50% at a concentration of 1 μΜ using a transcriptional reporter assay or exhibit at least 50% inhibition of effector secretion at a concentration of 1 μΜ or less (IC50≤1 μΜ) in an effector secretion assay. The multimeric compounds described herein show T3SS-specific inhibition in Pseudomonas aeruginosa of greater than 50% at 0.1 μΜ using an exoT-lux transcriptional reporter construct transferred into Pseudomonas aeruginosa PAOl (reporter strain MDM852, described herein) and/or show an IC50 of 0.1 μΜ or less for T3SS as measured in an assay of T3SS-mediated secretion of an ExoS effector toxin- β -lactamase reporter fusion protein assay described herein using P. aeruginosa strain MDM1746 (PAOl exoSv.bloM) consisting of strain PAOl in which an exoS effector gene fused in-frame to a fully functional TEM-1 β-lactamase gene was added via miniCTX (Bowlin, N.O., et al., supra; Hoang, T.T., et al., 2000, "Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains," Plasmid, 43:59-72). See Example 1 and Table 3, infra. Compounds inhibiting effector transcription by less than 50% at 1 μΜ or inhibiting effector secretion with an IC50 greater than 1 μΜ are not generally useful as T3SS inhibitors in the compositions and methods described herein.

In a particularly preferred embodiment, a T3SS inhibitor compound useful in the compositions and methods described herein has an IC50 of less than 1 μΜ as measured in a T3SS-mediated effector toxin- β-lactamase reporter fusion protein secretion assay described herein (or comparable assay) and also has a relatively low cytotoxicity toward human cells, such as a CC50 value >50-fold higher than the IC50 for T3SS secretion inhibition, providing a selectivity index (CC50/IC50)≥50, as measured in a standard cytotoxicity assay as described herein or as employed in the pharmaceutical field for antibiotics. Such standard cytotoxicity assays may employ any human cell typically employed in cytotoxicity assays for antibiotics, including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, Hep-2 cells, human embryonic kidney (HEK) 293 cells, 293T cells, and the like.

Even more preferably, a multimeric T3SS inhibitor compound described herein has an IC50 value <0.1 μΜ as measured in a T3SS-mediated effector toxin- β-lactamase reporter fusion protein secretion assay as described herein or in a comparable assay.

In yet another embodiment, a multimeric T3SS inhibitor compound described herein has a sufficiently high minimal inhibitory concentration (MIC) to indicate that it inhibits T3SS specifically.

In a particularly preferred embodiment of the invention, a T3SS inhibitor compound blocks T3SS-mediated secretion and translocation of one or more toxin effectors from cells of P. aeruginosa.

The T3SS compounds described herein are useful as anti-virulence agents and may be used to treat bacterial infections. Accordingly, an individual infected with or exposed to bacterial infection, especially Pseudomonas, Yersinia, or Chlamydia infection, may be treated by administering to the individual in need an effective amount of a multimeric compound according to the present invention. Use of one or more or a combination of the multimeric compounds disclosed herein to treat infection by bacteria having a type III secretion system is contemplated herein.

Especially, use of one or more or a combination of the above compounds to treat

Pseudomonas, Yersinia, or Chlamydia infection is contemplated herein. In particular, use of one or more or a combination of the above compounds for the treatment of Pseudomonas aeruginosa, Yersinia pestis, or Chlamydia trachomatis infections is advantageously carried out by following the teachings herein.

The present invention also provides pharmaceutical compositions containing one or more of the multimeric T3SS inhibitor compounds disclosed herein and a pharmaceutically acceptable carrier or excipient. The use of one or more of the multimeric T3SS inhibitor compounds in the preparation of a medicament for combating bacterial infection is disclosed.

A multimeric T3SS inhibitor compound or combination of said T3SS inhibitor compounds described herein may be used as a supporting or adjunctive therapy for the treatment of bacterial infection in an individual (human or other animal). In the case of an individual with a healthy immune system, administration of a multimeric T3SS inhibitor compound described herein to inhibit the T3SS of bacterial cells in or on an individual may be sufficient to permit the individual's own immune system to effectively clear or kill infecting or contaminating bacteria from the tissue of the individual. Alternatively, a multimeric T3SS inhibitor compound described herein may be administered to an individual in conjunction (i.e., in a mixture, sequentially, or simultaneously) with an antibacterial agent, such as an antibiotic, an antibody, or immunostimulatory agent, to provide both inhibition of T3SS and inhibition of growth of invading bacterial cells.

In yet another embodiment, a composition comprising a multimeric T3SS inhibitor compound or a combination of said T3SS inhibitors described herein may also comprise a second agent (second active ingredient, second active agent) that possesses a desired therapeutic or prophylactic activity other than that of T3SS inhibition. Such a second active agent includes, but is not limited to, an antibiotic, an antibody, an antiviral agent, an anticancer agent, an analgesic (e.g., a non-steroidal anti-inflammatory drug (NSAID), acetaminophen, an opioid, a COX-2 inhibitor), an immunostimulatory agent (e.g., a cytokine), a hormone (natural or synthetic), a central nervous system (CNS) stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an antipsychotic agent, and combinations thereof.

Compositions comprising a T3SS inhibitor described herein may be formulated for administration to an individual (human or other animal) by any of a variety of routes including, but not limited to, intravenous, intramuscular, subcutaneous, intra-arterial, parenteral, intraperitoneal, sublingual (under the tongue), buccal (cheek), oral (for swallowing), topical (epidermis), transdermal (absorption through skin and lower dermal layers to underlying vasculature), nasal (nasal mucosa), intrapulmonary (lungs), intrauterine, vaginal, intracervical, rectal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrarenal, nasojejunal, and intraduodenal.

Brief Description of the Drawing

Figure 1 is a graph showing the effects of phenoxyacetamide monomer MBX-2359 and phenoxyacetamide dimer MBX-4129 on ExoS-βΕΑ secretion from P. aeruginosa strains MDM1746 (pscF WT) and MDM2446 [(pscF(R75H)] (Table 1). Concentration-dependence for MBX-2359 and MBX-4103 were determined by the rate of nitrocefin cleavage by secreted ExoS-βΕΑ and calculated as the fraction of cleavage in the absence of inhibitor.

Inhibition of wild-type (WT) secretion by the dimer MBX-4103 (■, dashed line) and monomer MBX-2359 (·, solid line), are shown. Also shown are inhibition of

phenoxyacetamide-resistant secretion [pscF(R75H)] by the dimer MBX-4103 (□, dashed line) and monomer MBX-2359 (O, solid line).

Detailed Description of the Invention

The invention described herein provides multimeric organic compounds that inhibit a bacterial type III secretion system ("T3SS") that secretes and translocates bacterially produced effectors (also referred to as effector toxins, exotoxins, cytotoxins, bacterial toxins) from the bacterial cell into animal host cells. Effectors translocated into host cells can effectively inactivate the host immune response, such as by killing phagocytes and thereby disabling the host innate immune response. The T3SS is thus a critical virulence factor in the establishment and dissemination of bacterial infections in an individual (human or other animal) and is particularly critical to Pseudomonas aeruginosa opportunistic infections of human patients with compromised immune systems or that otherwise have been made susceptible to infection by bacteria such as P. aeruginosa.

That the invention may be more clearly understood, the following abbreviations and terms are used as defined below.

Abbreviations for various substituents (side groups, radicals) of organic molecules are those commonly used in organic chemistry. Such abbreviations may include "shorthand" forms of such substituents. For example, "Ac" is an abbreviation for an acetyl group, "Ar" is an abbreviation for an "aryl" group, and "halo" or "halogen" indicates a halogen radical (e.g., F, CI, Br, I). "Me" and "Et" are abbreviations used to indicate methyl (CH3-) and ethyl (CH3CH2-) groups, respectively; and "OMe" (or "MeO") and "OEt" (or "EtO") indicate methoxy (CH3O-) and ethoxy (CH3CH2O-), respectively. Hydrogen atoms are not always shown in organic structural diagrams (e.g., at the end of a drawn line representing a CH3 group ) or may be only selectively shown in some structural diagrams, as the presence and location of hydrogen atoms in organic molecular structures are understood and known by persons skilled in the art. Likewise, carbon atoms are not always specifically abbreviated with "C", as the presence and location of carbon atoms in structural diagrams are known and understood by persons skilled in the art,. Minutes are commonly abbreviated as "min"; hours are commonly abbreviated as "hr" or "h".

A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as

"comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of" (or which "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of" (or which "consists of") the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step. It is also understood that an element or step "selected from the group consisting of" refers to one or more of the elements or steps in the list that follows, including combinations of any two or more of the listed elements or steps.

The terms "bacterial type III secretion system inhibitor", "bacterial T3SS inhibitor",

"bacterial T3SS inhibitor compound", "T3SS inhibitor compound", and as used herein are interchangeable and denote compounds exhibiting the ability to specifically inhibit a bacterial type III secretion system by at least 25% at a concentration of 50 μΜ, for example, as measured in a T3SS effector transcriptional reporter assay or the ability to inhibit a bacterial T3SS, for example, as measured in a T3SS-mediated effector toxin secretion assay.

In the context of therapeutic use of the multimeric T3SS inhibitor compounds described herein, the terms "treatment", "to treat", or "treating" will refer to any use of the T3SS inhibitor compounds calculated or intended to arrest or inhibit the virulence or the T3SS -mediated effector secretion or translocation of bacteria having type III secretion systems. Thus, treating an individual may be carried out after any diagnosis indicating possible bacterial infection, i.e., whether an infection by a particular bacterium has been confirmed or whether the possibility of infection is only suspected, for example, after exposure to the bacterium or to another individual infected by the bacterium. It is also recognized that while the inhibitors of the present invention affect the introduction of effector toxins into host cells, and thus block or decrease the virulence or toxicity resulting from infection, the inhibitor compounds are not necessarily bactericidal or effective to inhibit growth or propagation of bacterial cells. For this reason, it will be understood that elimination of the bacterial infection will be accomplished by the host's own immune system or immune effector cells, or by introduction of antibiotic agents. Thus, it is contemplated that the compounds of the present invention will be routinely combined with other active ingredients such as antibiotics, antibodies, antiviral agents, anticancer agents, analgesics (e.g., a nonsteroidal anti-inflammatory drug (NSAID), acetaminophen, opioids, COX-2 inhibitors), immunostimulatory agents (e.g., cytokines or a synthetic immunostimulatory organic molecules), hormones (natural, synthetic, or semi-synthetic), central nervous system (CNS) stimulants, antiemetic agents, anti-histamines, erythropoietin, agents that activate

complement, sedatives, muscle relaxants, anesthetic agents, anticonvulsive agents, antidepressants, antipsychotic agents, and combinations thereof. As used herein, the term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium, manganese (-ic and -ous), potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N,N - dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

The meaning of other terms will be understood by the context as understood by the skilled practitioner in the art, including the fields of organic chemistry, pharmacology, and microbiology.

The present invention provides specific organic compounds that inhibit the T3SS of Pseudomonas aeruginosa. Structural analogs of previously studied T3SS inhibitors were evaluated for inhibition of T3SS-mediated secretion of an effector toxin- β -lactamase fusion protein (ExoS-βΕΑ) using P. aeruginosa strain MDM1746 (PAOl exoS .bloM, Table 3). See, Example 1 below for details of assaying and validating T3SS inhibitors.

In a series of experiments to test the effects of polymerizing phenoxyacetamide T3SS inhibitor analogues, multimeric compounds of Formula I were synthesized and used to perform various activity assays. The results indicated the effects of alternate structures in terms of the linker length, number of phenoxyacetamide units, and the nature of the linker; however, a wide range of linker lengths could be tolerated without adversely affecting and in some cases improving T3SS inhibitory performance. In general, the structure/activity relationships emerging from the experiments were characteristic of discoveries respecting alternative compounds reactive with multiple binding sites within a single target.

From the program of analog synthesis and comparative testing, a number of new multimeric inhibitor compounds were discovered which exhibited improved T3SS inhibitory properties significantly greater than those of the phenoxyacetamide inhibitor compounds that had been described previously. The new T3SS inhibitor compounds are defined by Formulas 1, 11(a), 11(b), III, and IV shown above.

Therefore, the present invention provides novel antibacterial/antivirulence agents with surprisingly high potency against the P. aeruginosa T3SS, including the T3SS in drug- resistant strains. The compounds of the present invention show a level of potency, in comparison to previously reported T3SS inhibitor compounds, that make them promising additions to the developing family of antibacterial/antivirulence agents.

The present invention provides new bacterial type III secretion system (T3SS) inhibitor compounds. Preparation of multimeric forms of aryl/heteroaryl phenoxyacetamide analogues has provided compounds showing a surprising increase in potency as inhibitors of, e.g., the Pseudomonas aeruginosa type III secretion system in comparison to monomeric inhibitors of similar structure. The invention provides additional compounds with T3SS inhibition potencies surprisingly improved by about 100-fold over previous

phenoxyacetamides or other small molecule T3SS inhibitors, and the new compounds have comparable or decreased cytotoxicity.

n is at least 1 and may be up to 20 or more;

each V is, independently, a moiety having the structure of Formula Ila or lib:

wherein

A is independently CH or N;

X is independently selected from hydrogen or halogen;

Z is O, S, NH; or NR³, where R³ is alkyl; R^R¹ , and R¹ are selected independently from: hydrogen, halogen, alkyl, hydroxy, alkoxy, alkylthio, or cyano;

R² is hydrogen or alkyl;

and

and wherein

L is a linker comprising one or more divalent, optionally substituted radicals selected from C1-C6 alkylene, C1-C6 alkenylene, arylene, heteroarylene, ether (-R-0-R-, where R and R' are, independently, C1-C6 alkylene, C1-C6 alkenylene, arylene such as phenylene, or heteroarylene such as 2,5-pyridinylene), thioether (-R-S-R-, where R and R' are, independently, C1-C6 alkylene, C1-C6 alkenylene, arylene such as phenylene, or heteroarylene such as 2,5-pyridinylene), or -C:0-NH- In another embodiment, the present invention provides a family of bacterial type III secretion system (T3SS) inhibitor compounds wherein V may be the same or different moiety having the structure of Formula III:

Formula III wherein

A is CH or N;

X is independently selected from hydrogen or halogen;

R is hydrogen or methyl;

Z is O, S, or NH or NR³; and

W is an aryl or heteroaryl radical forming a five-membered or six-membered ring which may be additionally fused with from 1 to 3 aryl, heteroaryl, cycloalkyl, or

heterocycloalkyl rings, which W radical may be unsubstituted or substituted with up to four substituents selected from halo, hydroxyl, amino, carboxamido, carboxyl, cyano, sulfonamido, sulfonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, and wherein any two substituents together may form an aromatic or non-aromatic ring structure fused with said aryl or heteroaryl radical W, or alternatively wherein substituents on W may be optionally bonded covalently to either Y or R, or both Y and R, to form a heterocyclic or carbocyclic, aromatic or non-aromatic ring systems.

Formula IV wherein A is CH or N;

X is independently selected from hydrogen or halogen;

Y is -CH₂- -CH(CH₃)-, or -C(CH₃)₂-; and

Of particular interest are compounds of the foregoing Formulae I, Ila, lib, III, and IV that are racemic mixtures of the R- and S-isomers or the isolated R-isomer, considering the asymmetric carbon (a carbon). Thus, preferred compounds will be isolated R-isomers denoted by the following compounds:

MBX-4743

MBX-4915

In a preferred embodiment, n can be an integer from 1 to 20 or more. Dimers and trimers are preferred, however, tetramers, decamers, dodecamers, and higher order polymers can also be made and are expected to be effective as T3SS inhibitors. In a preferred embodiment, the multimeric compounds of the present invention comprise the R-isomer in substantially pure form.

The compounds of the invention, or pharmaceutically acceptable salts thereof, of this invention can be combined as the active ingredient in intimate admixture with a

pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g. , oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the invention, and/or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and/or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention can include a

According to another embodiment of the invention, the administration is oral and the compound may be presented, for example, in the form of a tablet or encased in a gelatin capsule or a microcapsule, which simplifies oral application. The production of these forms of administration is within the general knowledge of a technical expert. Multiple routes of administration are envisioned for these drug-like molecules, and highly cost-effective production strategies can be easily achieved.

In a preferred embodiment, the multimeric phenoxyacetamide bacterial T3SS inhibitors of the present invention will specifically target the T3SS needle subunit protein PscF and thus prevent secretion of effector elements into a host cell. In particularly preferred embodiments, the novel T3SS inhibitor compounds of the present invention inhibit T3SS effector transcription by at least 50% at a concentration of 1 μΜ and/or exhibit at least 50% inhibition of effector secretion at a concentration of 1 μΜ or less (ICso <1 μΜ).

In another embodiment, the present invention provides a pharmaceutical composition comprising one or more multimeric bacterial T3SS inhibitor compounds and a

pharmaceutically acceptable carrier or excipient.

In another embodiment, the pharmaceutical composition comprises one or more multimeric T3SS inhibitor compounds as the R-isomer in substantially pure form.

In yet another embodiment, the present invention is directed to the use of a multimeric

T3SS inhibitor compound for the treatment of Gram- negative bacterial infection.

In still another embodiment, the present invention is directed to the use of a multimeric T3SS inhibitor compound to treat or prevent a bacterial infection of Salmonella spp., Shigella flexneri, Pseudomonas spp., Yersinia spp., enteropathogenic and enteroinvasive Escherichia coli, and Chlamydia spp.

In another embodiment, the present invention is directed to the use of a multimeric T3SS inhibitor compound to treat or prevent a bacterial infection by Pseudomonas aeruginosa, Yersinia pestis or Chlamydia trachomatis.

In yet another embodiment, the present invention is directed to the use of a multimeric T3SS inhibitor compound in the manufacture of a medicament for treating or preventing a Gram-negative bacterial infection.

In another embodiment, the present invention is directed to a method for treating an individual infected with or exposed to a Gram-negative bacterium comprising administering to said individual an effective amount a multimeric T3SS inhibitor compounds sufficient to inhibit T3SS-mediated effector secretion of a compound.

In a preferred embodiment, the present invention is directed to a method for treating or preventing a bacterial infection in a mammal comprising administering an effective amount of a multimeric T3SS inhibitor compound of the present invention. In a particularly preferred embodiment, the mammal is a human.

In another embodiment, the method or the present invention for treating or preventing a bacterial infection comprising administering a multimeric T3SS inhibitor compound further comprises the administration of an additional active ingredient selected from the group consisting of an antibiotic, an antibody, an antiviral agent, an anticancer agent, an analgesic, an immunostimulatory agent, a natural, synthetic or semi- synthetic hormone, a central nervous system stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an antipsychotic agent, and combinations thereof.

Of particular interest are compounds that are racemic mixtures of R- and S-isomers or the isolated R-isomer, considering the asymmetric carbon (a carbon). Particularly preferred

MBX-4103

The compounds of the present invention are designed to function by a novel anti- virulence approach of protecting and enabling the host's innate immune system rather than directly killing invading bacteria. While not classic innate immune modulators, the multimeric anti-T3SS agents described herein are believed to act indirectly on host targets by protecting the phagocytes of the innate immune system from most of the acute cytotoxic effects of bacteria having type III secretion systems such as P. aeruginosa. As therapeutic agents, the compounds of the present invention may reduce the frequency of polymicrobial VAP infections, which appear to be due to local innate immune suppression by P. aeruginosa T3SS effector toxins (Diaz, et al., " Pseudomonas aeruginosa induces localized

immunosuppression during pneumonia," Infect. Immun. , 76: 4414-4421 (2008)).

Furthermore, the compounds of the present invention are species-specific and consequently spare normal flora, advantageously aligning this therapeutic approach with an emerging understanding of the protective role of the normal flora in infectious diseases

(Parillo and Dellinger, Critical Care Medicine: Principles of Diagnosis and Management in the Adult, 2^nd ed. (Mosby, New York 2002), pp. 800-802).

If applied in combination with an antibacterial agent, the novel multimeric T3SS inhibitor compounds of the present invention will not contribute to the elimination of normal flora and may permit the use of lower doses of co- administered antibiotics. Finally, the T3SS inhibitor compounds described herein are equally potent against multiple P. aeruginosa strains (including clinical isolates), advantageously are not affected by P. aeruginosa efflux mechanisms, and are expected to exert no selection pressure for the development of resistance outside the body and only relatively weak selection pressure during therapy. This combination of favorable features of the compounds together with the novel mechanism of action provides a new approach to improve the treatment and prevention of acute P.

aeruginosa infections such as VAP and bacteremia.

Preferred multimeric inhibitor compounds of the present invention inhibit T3SS effector transcription by at least 50% at a concentration of 0.1 μΜ using a transcriptional reporter assay or by exhibiting at least 50% inhibition of effector secretion at a concentration of 0.1 μΜ or less (IC50≤0.1 μΜ) in an effector secretion assay.

The inhibitor compounds of the present invention showed T3SS-specific inhibition in Pseudomonas of greater than 50% at 0.1 μΜ using an exoT-lux transcriptional reporter construct transferred into Pseudomonas aeruginosa PAOl (reporter strain MDM852, described herein) and/or showed an IC50 of less than 0.1 μΜ for T3SS as measured in an assay of T3SS-mediated secretion of an effector toxin- -lactamase reporter fusion protein assay described herein using P. aeruginosa strain MDM1746 (PAOl exoSv.blaM) (Table 3). Compounds inhibiting effector transcription by less than 25% at 0.1 μΜ or with an IC50 greater than 1 μΜ are not generally useful as T3SS inhibitors in the compositions and methods described herein.

In particularly preferred embodiments, a multimeric T3SS inhibitor compound useful in the compositions and methods described herein has an IC50 of less than 1 μΜ as measured in a T3SS-mediated effector toxin- -lactamase reporter fusion protein secretion assay described herein (or comparable assay) and also has a relatively low cytotoxicity toward human cells, such as a CC50 value >50-fold the T3SS secretion inhibition IC50, producing a selectivity index (CC50 IC50)≥50, as measured in a standard cytotoxicity assay as described herein or as employed in the pharmaceutical field for antibiotics. Such standard cytotoxicity assays may employ any human cell typically employed in cytotoxicity assays for antibiotics, including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, Hep-2 cells, human embryonic kidney (HEK) 293 cells, 293T cells, and the like. Even more preferably, a multimeric T3SS inhibitor compound described herein has an IC50 value <0.1 μΜ as measured in a T3SS-mediated effector toxin- β-lactamase reporter fusion protein secretion assay as described herein or in a comparable assay. Alternatively, preferred compounds of the present invention exhibit potency (IC50) over 100-fold greater than that of N-(benzo[J|[l,3]dioxol-5-ylmethyl)-2-(2,4-dichlorophenoxy)propanamide, which was an early T3SS inhibitor discovered by high throughput screening.

In yet another embodiment, a T3SS inhibitor compound described herein has a sufficiently high minimal inhibitory concentration (MIC) to indicate that it inhibits T3SS specifically.

The T3SS inhibitor compounds described herein are organic compounds that can also be synthesized to order by commercial suppliers such as ChemBridge Corporation (San Diego, CA, USA), Life Chemicals Inc. (Burlington, ON, Canada), and Timtec LLC (Newark, DE, USA).

Unless otherwise indicated, it is understood that description of the use of a T3SS inhibitor compound in a composition or method also encompasses the embodiment wherein a combination of two or more T3SS inhibitor compounds are employed as the source of T3SS inhibitory activity in a composition or method of the invention.

Pharmaceutical compositions according to the invention comprise a T3SS inhibitor compound as described herein, or a pharmaceutically acceptable salt thereof, as the "active ingredient" and a pharmaceutically acceptable carrier (or "vehicle"), which may be a liquid, solid, or semi-solid compound. By "pharmaceutically acceptable" is meant that a compound or composition is not biologically, chemically, or in any other way, incompatible with body chemistry and metabolism and also does not adversely affect the activity of the T3SS inhibitor or any other component that may be present in a composition in such a way that would compromise the desired therapeutic and/or preventative benefit to a patient.

Pharmaceutically acceptable carriers useful in the invention include those that are known in the art of preparation of pharmaceutical compositions and include, without limitation, water, physiological pH buffers, physiologically compatible salt solutions (e.g., phosphate buffered saline), and isotonic solutions. Pharmaceutical compositions of the invention may also comprise one or more excipients, i.e., compounds or compositions that contribute or enhance a desirable property in a composition other than the active ingredient. Various aspects of formulating pharmaceutical compositions, including examples of various excipients, dosages, dosage forms, modes of administration, and the like are known to those skilled in the art of pharmaceutical compositions and also available in standard pharmaceutical texts, such as Remington's Pharmaceutical Sciences, 18th edition, Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990), Remington: The Science and Practice of Pharmacy, Volumes 1 & 2, 19th edition, Alfonso R. Gennaro, ed., (Mack Publishing Co., Easton, PA 1995), or other standard texts on preparation of pharmaceutical compositions.

Pharmaceutical compositions may be in any of a variety of dosage forms particularly suited for an intended mode of administration. Such dosage forms, include, but are not limited to, aqueous solutions, suspensions, syrups, elixirs, tablets, lozenges, pills, capsules, powders, films, suppositories, and powders, including inhalable formulations. Preferably, the pharmaceutical composition is in a unit dosage form suitable for single administration of a precise dosage, which may be a fraction or a multiple of a dose that is calculated to produce effective inhibition of T3SS.

A composition comprising a T3SS inhibitor compound (or combination of T3SS inhibitors) described herein may optionally possess a second active ingredient (also referred to as "second agent" or "second active agent") that provides one or more other desirable therapeutic or prophylactic activities other than T3SS inhibitory activity. Such a second agent useful in compositions of the invention includes, but is not limited to, an antibiotic, an antibody, an antiviral agent, an anticancer agent, an analgesic (e.g., a non-steroidal antiinflammatory drug (NSAID), acetaminophen, an opioid, a COX-2 inhibitor), an

immunostimulatory agent (e.g., a cytokine or a synthetic immunostimulatory organic molecule), a hormone (natural, synthetic, or semi-synthetic), a central nervous system (CNS) stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an antipsychotic agent, and combinations thereof.

Pharmaceutical compositions as described herein may be administered to humans and other animals in a manner similar to that used for other known therapeutic or prophylactic agents, and particularly as used for therapeutic aromatic or multi-ring antibiotics. The dosage to be administered to an individual and the mode of administration will depend on a variety of factors including age, weight, sex, condition of the patient, and genetic factors, and will ultimately be decided by an attending qualified healthcare provider. Pharmaceutically acceptable salts of T3SS inhibitor compounds described herein include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, malic, palmoic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p- sulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, tannic, carboxymethyl cellulose, polylactic, polyglycolic, and benzenesulfonic acids.

The invention may also envision the "quatemization" of any basic nitrogen-containing groups of a multimeric compound described herein, provided such quatemization does not destroy the ability of the compound to inhibit T3SS. Such quatemization may be especially desirable to enhance solubility. Any basic nitrogen can be quaternized with any of a variety of compounds, including but not limited to, lower (e.g., Ci-C₄) alkyl halides (e.g., methyl, ethyl, propyl and butyl chloride, bromides, and iodides); dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl and diamyl sulfates); long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides); and aralkyl halides (e.g., benzyl and phenethyl bromides).

For solid compositions, conventional nontoxic solid carriers may be used including, but not limited to, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.

Pharmaceutical compositions may be formulated for administration to a patient by any of a variety of parenteral and non-parenteral routes or modes. Such routes include, without limitation, intravenous, intramuscular, intra- articular, intraperitoneal, intracranial, paravertebral, periarticular, periostal, subcutaneous, intracutaneous, intrasynovial, intrasternal, intrathecal, intralesional, intratracheal, sublingual, pulmonary, topical, rectal, nasal, buccal, vaginal, or via an implanted reservoir. Implanted reservoirs may function by mechanical, osmotic, or other means. Generally and particularly when administration is via an intravenous, intra-arterial, or intramuscular route, a pharmaceutical composition may be given as a bolus, as two or more doses separated in time, or as a constant or non-linear flow infusion.

A pharmaceutical composition may be in the form of a sterile injectable preparation, e.g., as a sterile injectable aqueous solution or an oleaginous suspension. Such preparations may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., polyoxyethylene 20 sorbitan monooleate (also referred to as

"polysorbate 80"); TWEEN® 80, ICI Americas, Inc., Bridgewater, New Jersey) and suspending agents. Among the acceptable vehicles and solvents that may be employed for injectable formulations are mannitol, water, Ringer's solution, isotonic sodium chloride solution, and a 1,3-butanediol solution. In addition, sterile, fixed oils may be conventionally employed as a solvent or suspending medium. For this purpose, a bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural

pharmaceutically-acceptable oils, including olive oil or castor oil, especially in their polyoxyethylated versions .

A T3SS inhibitor described herein may be formulated in any of a variety of orally administrable dosage forms including, but not limited to, capsules, tablets, caplets, pills, films, aqueous solutions, oleaginous suspensions, syrups, or elixirs. In the case of tablets for oral use, carriers, which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. Capsules, tablets, pills, films, lozenges, and caplets may be formulated for delayed or sustained release.

Tablets and other solid or semi-solid formulations may be prepared that rapidly disintegrate or dissolve in an individual's mouth. Such rapid disintegration or rapid dissolving formulations may eliminate or greatly reduce the use of exogenous water as a swallowing aid. Furthermore, rapid disintegration or rapid dissolve formulations are also particularly useful in treating individuals with swallowing difficulties. For such

formulations, a small volume of saliva is usually sufficient to result in tablet disintegration in the oral cavity. The active ingredient (a multimeric T3SS inhibitor described herein) can then be absorbed partially or entirely into the circulation from blood vessels underlying the oral mucosa (e.g., sublingual and/or buccal mucosa), or it can be swallowed as a solution to be absorbed from the gastrointestinal tract.

When aqueous suspensions are to be administered orally, whether for absorption by the oral mucosa or absorption via the gut (stomach and intestines), a composition comprising a T3SS inhibitor may be advantageously combined with emulsifying and/or suspending agents. Such compositions may be in the form of a liquid, dissolvable film, dissolvable solid (e.g., lozenge), or semi-solid (chewable and digestible). If desired, such orally administrable compositions may also contain one or more other excipients, such as a sweetener, a flavoring agent, a taste-masking agent, a coloring agent, and combinations thereof.

The pharmaceutical compositions comprising a multimeric T3SS inhibitor as described herein may also be formulated as suppositories for vaginal or rectal administration. Such compositions can be prepared by mixing a T3SS inhibitor compound as described herein with a suitable, non-irritating excipient that is solid at room temperature but liquid at body temperature and, therefore, will melt in the appropriate body space to release the T3SS inhibitor and any other desired component of the composition. Excipients that are particularly useful in such compositions include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

Topical administration of a T3SS inhibitor may be useful when the desired treatment involves areas or organs accessible by topical application, such as the epidermis, surface wounds, or areas made accessible during surgery. Carriers for topical administration of a T3SS inhibitor described herein include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsifying wax, and water. Alternatively, a topical composition comprising a T3SS inhibitor as described herein may be formulated with a suitable lotion or cream that contains the inhibitor suspended or dissolved in a suitable carrier to promote absorption of the inhibitor by the upper dermal layers without significant penetration to the lower dermal layers and underlying vasculature. Carriers that are particularly suited for topical administration include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. A T3SS inhibitor may also be formulated for topical application as a jelly, gel, or emollient. Topical administration may also be accomplished via a dermal patch.

Persons skilled in the field of topical and transdermal formulations are aware that selection and formulation of various ingredients, such as absorption enhancers, emollients, and other agents, can provide a composition that is particularly suited for topical

administration (i.e., staying predominantly on the surface or upper dermal layers with minimal or no absorption by lower dermal layers and underlying vasculature) or transdermal administration (absorption across the upper dermal layers and penetrating to the lower dermal layers and underlying vasculature). Pharmaceutical compositions comprising a T3SS inhibitor as described herein may be formulated for nasal administrations, in which case absorption may occur via the mucous membranes of the nasal passages or the lungs. Such modes of administration typically require that the composition be provided in the form of a powder, solution, or liquid suspension, which is then mixed with a gas (e.g., air, oxygen, nitrogen, or a combination thereof) so as to generate an aerosol or suspension of droplets or particles. Inhalable powder compositions preferably employ a low or non-irritating powder carrier, such as melezitose (melicitose). Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. A

pharmaceutical composition comprising a T3SS inhibitor described herein for administration via the nasal passages or lungs may be particularly effective in treating lung infections, such as hospital-acquired pneumonia (HAP).

Pharmaceutical compositions described herein may be packaged in a variety of ways appropriate to the dosage form and mode of administration. These include but are not limited to vials, bottles, cans, packets, ampoules, cartons, flexible containers, inhalers, and nebulizers. Such compositions may be packaged for single or multiple administrations from the same container. Kits may be provided comprising a composition, preferably as a dry powder or lyophilized form, comprising a T3SS inhibitor and preferably an appropriate diluent, which is combined with the dry or lyophilized composition shortly before administration as explained in the accompanying instructions of use. Pharmaceutical composition may also be packaged in single use pre-filled syringes or in cartridges for auto- injectors and needleless jet injectors. Multi-use packaging may require the addition of antimicrobial agents such as phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzalconium chloride, and benzethonium chloride, at concentrations that will prevent the growth of bacteria, fungi, and the like, but that are non-toxic when administered to a patient.

Consistent with good manufacturing practices, which are in current use in the pharmaceutical industry and which are well known to the skilled practitioner, all components contacting or comprising a pharmaceutical composition must be sterile and periodically tested for sterility in accordance with industry norms. Methods for sterilization include ultrafiltration, autoclaving, dry and wet heating, exposure to gases such as ethylene oxide, exposure to liquids, such as oxidizing agents, including sodium hypochlorite (bleach), exposure to high energy electromagnetic radiation (e.g., ultraviolet light, x-rays, gamma rays, ionizing radiation). Choice of method of sterilization will be made by the skilled practitioner with the goal of effecting the most efficient sterilization that does not significantly alter a desired biological function of the T3SS inhibitor or other component of the composition.

Additional embodiments and features of the invention will be apparent from the following non-limiting examples. Example 1. Materials and Methods for Characterization of T3SS Inhibitors.

Strains, plasmids, and growth media.

Bacterial strains and plasmids used for assays are described in Table 1, below. All P.

aeruginosa strains were derivatives of PAOl (Holloway, et al., 1979, Microbiol. Rev. , 43:73- 102), PAK (Bradley, D. E., 1974, Virology, 58: 149-63), or PA99 (Shaver CM, Hauser AR. 2004. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect Immun 72:6969-6977). E. coli TOP10 (Invitrogen), E. coli DB3.1 (GATEWAY^® host, Invitrogen), E. coli SM10 (de Lorenzo and Timmis, 1994, Methods Enzymol, 235:386-405), and E. coli S17-1 (ATCC 47055) were used as hosts for molecular cloning. Luria-Bertani (LB) medium (liquid and agar) was purchased from Difco. LB was supplemented with 30 μg/ml gentamicin (LBG) with or without 5 mM EGTA (LBG and LBGE, respectively).

Table 1 : Strains and Plasmids

(1) Aiello, et al., 2010, Antimicrob. Agents Chemother. , 54: 1988-99.

(2) Bowlin, et al., 2014, Antimicrob. Agents Chemother., 58:2211-2220.

(3) Shaver and Hauser, 2004. Infect Immun 72:6969-6977. Table 2: Polymerase chain reaction (PCR) primers

Luciferase transcriptional reporter assay.

A transcriptional fusion of the Photorhabdus luminescens lux operon (luxCDABE) to effector gene <?χοΓ (ΡΑ0044) was constructed and used to construct strain MDM852 as described previously (Aiello et al., 2010, op. cit.) For inhibitor IC50 testing, compounds were added to a 96-well microplate in a concentration dilution series. Reporter strain MDM852 was grown at 37° C in LBG to ODeoo -0.025 - 0.05, transferred into the microplate (50 μΐ/well) containing test compounds and EGTA (5 μΐ of 0.1M stock solution), which was covered with a translucent gas-permeable seal (Abgene, Inc., Cat. No. AB-0718). Control wells contained cells with fully induced T3SS (EGTA and DMSO, columns 1 and 2) and uninduced T3SS (DMSO only, columns 11 and 12). Plates were incubated at room temperature for 300 min. Then, luminescence was measured in an Envision Multilabel microplate reader (PerkinElmer). The screening window coefficient, Z' -factor (see Zhang, et al., 1999, /. Biomol. Screen. , 4:67-73), defined as the ratio of the positive and negative control separation band to the signal dynamic range of the assay, averaged 0.7 for the assay. Compounds were confirmed to be >95% pure and to be of the expected mass by LC-MS analysis.

Effector- β-lactamase (βΙΑ) secretion assays.

A gene encoding the exoS promoter/regulatory region and the ExoS- -lactamase (βΙΑ) fusion protein (comprised of the full length P. aeruginosa effector ExoS fused in reading frame to the TEM-1 β-lactamase gene lacking secretion signal codons) was constructed by splicing by overlap extension PCR (SOE-PCR) (Choi and Schweizer, 2005, BMC Microbiol. , 5:30) using primers 1-4 in Table 2 with a secondary PCR using primers 5-6, gel purified, cloned into pDONR (Gateway^®, Invitrogen) sequence confirmed, and then cloned into miniCTX using the terminal BamHl and HmdIII restriction endonuclease sites, and introduced into P. aeruginosa strains to generate strains MDM1746 and MDM1838 as previously described for strain MDM1710 (Bowlin et al., 2014, supra). Secretion of fusion proteins was detected by measuring the hydrolysis of the chromogenic β-lactamase substrate nitrocefin in clear 96-well microplates in a modification of a previously described assay (Lee, et al., 2007, Infect. Immun. ,1 '5: 1089-1098). Cells of strain MDM1746, or negative control MDM1838 (which fails to carry out T3SS secretion due to ApscF) (Table 1), were sub- cultured in the morning from overnight growths in LBG into 0.1 ml of LBGE with or without test compounds and grown for 150 min. Nitrocefin (100 μg/ml final) was added, and A490 measurements taken every minute for 15 min in a Victor³ V 1420 Multilabel HTS Counter (PerkinElmer). Slopes were calculated as a relative measure of the quantity of the effector- LA fusion protein secreted and were absolutely dependent on induction with EGTA, and the presence of a functional pscF gene in the P. aeruginosa cells. Typical signahbackground ratios were 6-10.

Inhibition of P. aeruginosa ExoU-dependent CHO cell killing by T3SS-mediated

translocation.

Rescue of CHO cells from T3SS-mediated cytotoxicity of translocated effector protein ExoU by P. aeruginosa strain MDM1561 (PA99U) (Table 1) was measured using a lactate dehydrogenase (LDH) release assay as previously reported ((Lee, et al., 2005, Infect. Immun. , 73 : 1695-705) except that infection with P. aeruginosa was carried out for 2 hr in the absence of gentamicin. Percent cytotoxicity (% LDH release) was calculated relative to that of the uninfected control, which was set at 0% LDH release, and that of cells infected with P. aeruginosa strain MDM1561 (PA99U) unprotected by test compound (100% LDH release). LDH released from unprotected, infected cells reached at least 80% of the value obtained from complete lysis with 1% Triton X-100 in the 2 hr timeframe of this experiment.

Pseudolipasin, which acts by direct inhibition of the ExoU phospholipase, was used as control inhibitor (Lee, et al., Infect. Immun. , 75: 1089-1098 (2007)).

Minimum Inhibitory Concentration (MIC).

MIC determination was carried out by the broth microdilution method described in the CLSI (formerly NCCLS) guidelines and expressed in μΜ to facilitate comparisons with IC50 and CC50 values. See, NCCLS, Approved standard M7-A4: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, National Committee for Clinical Laboratory Standards, 4th ed., Wayne, PA (1997). Specific T3SS inhibitors do not exhibit detectable MIC values since T3SS is not required for growth in culture in vitro. Determination of Mammalian Cytotoxicity.

The cytotoxic concentration (CC50) of compound versus cultured mammalian cells (HeLa, ATCC CCL-2; American Type Culture Collection, Manassas, VA) was determined as the concentration of compound that inhibits 50% of the conversion of MTS to formazan (Marshall, et al., 1995, "A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function," Growth Regul., 5:69-84). Briefly, 96-well plates were seeded with HeLa cells at a density of 4xl0³ per well in VP-SFM medium without serum (Frazzati-Gallina, et al., 2001, /. Biotechnol. , 92:67-72), in the presence or absence of serial dilutions of a compound dissolved in DMSO. Following incubation for 3 days at 37° C in VP-SFM, cell viability was measured with the vital tetrazolium salt stain 3-(4,5- dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide according to the manufacturer's instructions (Promega, Madison, Wisconsin). Values were determined in duplicate using dilutions of inhibitory compound from 100 μΜ to 0.2 μΜ. Selective T3SS inhibitors typically exhibit selectivity index (CC50/IC50) values >10 and preferably >50.

Example 2. Optimization of multimers

Several dimeric or trimeric analogues of previously tested monomeric inhibitor compounds were synthesized and their level of inhibition of T3SS-mediated secretion, translocation, and cytotoxicity determined.

Synthesis of T3SS inhibitor dimers

Phenoxyacetamides can be synthesized using well-established chemistry from commercially available starting materials.

MBX-4103 The synthesis was initiated with the base -promoted double displacement of both bromines of l-bromo-2-(2-bromoethoxy)ethane with 3-Hydroxybenzonitrile. The resulting 3,3'- ((oxybis(ethane-2, l-diyl))bis(oxy))dibenzonitrile was reduced to (((oxybis(ethane-2, l- diyl))bis(oxy))bis(3, l-phenylene))dimethanamine with the use of Raney Ni under pressurized ¾ atmosphere, which was subsequently peptide coupled to enantiomerically pure (R)-2-

((3,5-dichloropyridin-2-yl)oxy)butanoic acid to yield the target dimer compound MBX-4103.

Synthesis of MBX-4129

The synthesis originated with the base-promoted displacement of bromine of 3- (bromomethyl)benzonitrile with ethyleneglycol. The resulting 3,3'-((ethane- l,2- diylbis(oxy))bis(methylene))dibenzonitrile was reduced to (((ethane- 1 ,2- diylbis(oxy))bis(methylene))bis(3, l-phenylene))dimethanamine with the use of Raney Ni under pressurized ¾ atmosphere, which was subsequently peptide coupled to

enantiomerically pure (R)-2-((3,5-dichloropyridin-2-yl)oxy)butanoic acid to yield the target dimer compound MBX-4129.

Testing of the compounds using the assays set forth in Example 1 confirmed that phenoxyacetamide dimers provide improved inhibitory potency for T3SS-mediated secretion as compared to monomers. The results set forth in Table 3 below indicate that the length of the linker between the two monomer units can be from 11 to 18 carbon bond units between the two nitrogens, but preferably 13- 15 units, and most preferably 14 or 15 units, as shown in data from testing MBX-4129 and MBX-4103, respectively. Even longer linker lengths may be tolerated since the assembled T3SS needle contains many PscF binding sites at different distances from each other; however, compounds with longer linker lengths may prove to have diminished properties important for drug manufacture and administration, such as solubility and bioavailability. The potency of inhibition of T3SS-mediated translocation of effector toxin ExoU into mammalian cells by dimers was less predictable, but dimers MBX-4129 and MBX-4103 exhibited over 100-fold increased potency as compared to the known inhibitor, MBX-1641. Trimer MBX-4137 was not very potent, but trimer activity may be critically dependent on the precise position of the monomer units relative to each other. Polyether linkers were effective, but interruptions of the polyether by aromatic groups as in MBX-4138 were also tolerated.

Clearly, alterations of the structure had the ability to drastically change the T3SS inhibitory performance of the compounds, particularly the linker length and composition, which led to a marked improvement in potency over reference compounds such as MBX- 1641.

Table 3 Structure and Activity of Select Compounds

Compound

Structure MW Identifier

MBX-4908 880.59

MBX-4915 776.49

Table 3 Continued: Activity of Select Compounds

Compound Ave. Secretion Trans-location Selectivity

Dimer Length Identifier ICso (μΜ) ICso (μΜ) (CCso/ICso)

MBX-4128 0.4 >50 >125 13 length units

MBX-4108 0.3 + 0.2 8.5 >33 14 length units

MBX-4129 0.02 + 0.02 0.10 >500 14 length units

MBX-3997 8.9 + 1.3 n.d. n.d. 14 length units

MBX-4079 0.6 + 0.3 >50 >80 15 length units

MBX-4103 0.02 + 0.003 0.10 >500 15 length units

MBX-4126 0.2 >25 >125 15 length units

MBX-4137 40.0 >50 n.d. 15 length units

MBX-4138 2.4 + 0.3 >50 >20 15 length units

MBX-4139 17 + 1.4 >25 n.d. 17 length units

MBX-4127 0.17 + 0.01 >25 >150 18 length units Compound Ave. Secretion Trans-location Selectivity

Dimer Length Identifier ICso (μΜ) ICso (μΜ) (CCso/ICso)

MBX-4743 0.01 0.2 >100 15 length units

MBX-4905 0.027 12.5 >4 15 length units

MBX-4906 0.05 >50 n.d. 15 length units

MBX-4908 0.80 >50 n.d. 14 length units

MBX-4915 0.08 4.5 >44.4 15 length units

The results of these studies underscored the unpredictability of the effect of alterations of length and linker composition to dimers of the phenoxyacetamide scaffold.

Consideration of the foregoing data defined a new group of compounds of related structure that are useful as T3SS inhibitor compounds and have potency and/or toxicity profiles that make them candidates for use as therapeutic agents. The new family of inhibitor compounds are as set forth in Formulas I, Ila, lib, III, and IV described above. Example 3. Determination of active and inactive isomers.

The compounds in Table 3 were evaluated for their potency of inhibition of T3SS- mediated secretion and translocation and for their cytotoxicity by using the assays described in Example 1. Preferably, inhibitors display low IC50 values for inhibition of the T3SS- mediated secretion and translocation assays but exhibit minimal cytotoxicity (high CC50 values). Nearly 100-fold increases in potency were observed between the phenoxyacetamide monomer MBX-1641 and the optimized analog MBX-2359 when compared to the dimers MBX-4103 and MBX-4129, illustrating the marked and surprising benefit of dimers for inhibiting T3SS (Table 3). Two results indicate that the dimers bind to the same target site as do the monomers, but do so with more avidity. First, dimers of R-isomers are much more potent than dimers of mixed isomeric forms (compare MBX-4108 with MBX-4129 and MBX-4079 with MBX-4103 in Table 3). Second, as illustrated in Fig. 1, the T3SS of P. aeruginosa strain MDM2446 carrying a phenoxyacetamide-resistant pscF allele, pscF(R75H), displays resistance to both the monomer MBX-2359 and the dimer MBX-4103. However, the dimer is so much more potent than the monomer that its IC50 vs. the resistant mutant is comparable to the IC50 of the monomer vs. the wild-type strain MDM1746.

Consistent with their mechanism as virulence factor inhibitors, neither the monomers nor the dimers display significant MIC values vs. wild-type or efflux-deficient strains of P.

aeruginosa.

All publications, patent applications, patents, and other documents cited herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Obvious variations to the disclosed compounds and alternative embodiments of the invention will be apparent to those skilled in the art in view of the foregoing disclosure. All such obvious variants and alternatives are considered to be within the scope of the invention as described herein.

Claims

1. A bacterial type III secretion system (T3SS) inhibitor compound of Formula I:

(V— L)_n— V (I),

n is at least 1 and may be up to 20 or more;

each V is, independently, a moiety having the structure of Formula Ila or lib:

Formula Ila

Formula lib wherein

A is independently CH or N;

X is independently selected from hydrogen or halogen;

Z is O, S, NH; or NR³, where R³ is alkyl;

R^R¹ , and R^1" are selected independently from: hydrogen, halogen, alkyl, hydroxy, alkoxy, alkylthio, or cyano;

R² is hydrogen or alkyl;

Y is a divalent straight-chain, branched, or cyclic alkyl, alkenyl or alkynyl radical of from 1 to 6 carbon atoms, which Y radical may be unsubstituted or substituted with up to four substituents selected from halo, cyano, hydroxyl, amino, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy; and W is an aryl or heteroaryl radical forming a five-membered or six-membered ring which may be additionally fused with from 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, which W radical may be unsubstituted or substituted with up to four substituents selected from halo, hydroxyl, amino, carboxamido, carboxyl, cyano, sulfonamido, sulfonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, and wherein any two substituents together may form an aromatic or non-aromatic ring structure fused with said aryl or heteroaryl radical W, or alternatively wherein substituents on W may be optionally bonded covalently to either Y or R², or both Y and R², to form a heterocyclic or carbocyclic, aromatic or non-aromatic ring systems;

and wherein

L is a linker comprising one or more divalent, optionally substituted radicals selected from C1-C6 alkylene, C1-C6 alkenylene, arylene, heteroarylene, ether (-R-0-R-, where R and R' are independently selected from C1-C6 alkylene, Cl- C6 alkenylene, arylene , or heteroarylene), thioether (-R-S-R-, where R and R' are independently selected from C1-C6 alkylene, C1-C6 alkenylene, arylene, or heteroarylene) , or -C:0-NH-.

2. The T3SS inhibitor compound according to Claim 1 having the Formula III:

Formula III wherein

A is CH or N;

X is independently selected from hydrogen or halogen;

R is hydrogen or methyl;

Z is O, S, or NH or NR³; and

W is an aryl or heteroaryl radical forming a five-membered or six-membered ring which may be additionally fused with from 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, which W radical may be unsubstituted or substituted with up to four substituents selected from halo, hydroxyl, amino, carboxamido, carboxyl, cyano, sulfonamido, sulfonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, and wherein any two substituents together may form an aromatic or non-aromatic ring structure fused with said aryl or heteroaryl radical W, or alternatively wherein substituents on W may be optionally bonded covalently to either Y or R, or both Y and R, to form a heterocyclic or carbocyclic, aromatic or non-aromatic ring systems.

3. The T3SS inhibitor compound according to Claim 1 having the Formula IV:

Formula IV wherein

A is CH or N;

X is independently selected from hydrogen or halogen;

Y is -CH2-, -CH(CH₃)-, or -C(CH₃)₂-; and

W is an aryl or heteroaryl radical forming a five-membered or six-membered ring which may be additionally fused with from 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, which W radical may be unsubstituted or substituted with up to four substituents selected from halo, hydroxyl, amino, carboxamido, carboxyl, cyano, sulfonamido, sulfonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, and wherein any two substituents together may form an aromatic or non-aromatic ring structure fused with said aryl or heteroaryl radical W, or alternatively substituents on W may be optionally bonded covalently to either Y or N, or both Y and N, to form a heterocyclic or carbocyclic, aromatic or non-aromatic ring systems.

4. The compound according to Claim 1 , selected from the group consisting of:

57

MBX-4915

and particular isomers of any of the foregoing compounds.

The compound according to Claim 1 comprising the R-isomer in substantially pure form.

A pharmaceutical composition, or pharmaceutically acceptable salt thereof, comprising one or more bacterial T3SS inhibitor compounds according to any one of Claims 1-5 and a pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition according to Claim 6, wherein said one or more T3SS inhibitor compounds is the R-isomer in substantially pure form.

Use of a compound according to any one of Claims 1-7 for the treatment of Gram- negative bacterial infection.

9. The use according to Claim 8, wherein said bacterial infection is an infection of Salmonella spp., Shigella flexneri, Pseudomonas spp., Yersinia spp.,

enteropathogenic and enteroinvasive Escherichia coli, and Chlamydia spp.

10. The use according to Claim 9, wherein said bacterial infection is an infection by Pseudomonas aeruginosa, Yersinia pestis or Chlamydia trachomatis.

11. Use of a compound according to any one of Claims 1-5 for the manufacture of a medicament for treating Gram-negative bacterial infection.

12. A method for treating a mammal infected with or exposed to a Gram-negative bacterium comprising administering to said individual an effective amount to inhibit T3SS-mediated effector secretion of a compound according to any one of Claims 1-5.

13. The method according to Claim 12, wherein said mammal is a human.

14. The method according to Claim 12, wherein said Gram-negative bacterium is of the genus Pseudomonas, Salmonella, Yersinia, or Chlamydia.

15. The method according to Claim 14, wherein said Gram-negative bacterium is Pseudomonas aeruginosa, Yersinia pestis or Chlamydia trachomatis.

16. The method according to Claim 15, wherein said Gram-negative bacterium is

Pseudomonas aeruginosa.

17. The method according to Claim 12, further comprising administering an additional active ingredient selected from the group consisting of an antibiotic, an antibody, an antiviral agent, an anticancer agent, an analgesic, an immunostimulatory agent, a natural, synthetic or semi-synthetic hormone, a central nervous system stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an antipsychotic agent, and combinations thereof.