Chimeric ABC transporters and screening methods

Abstract

The present disclosure relates to chimeric ABC transporter proteins and methods for using chimeric ABC transporters to screen for molecules that bind to the periplasmic, extracellular, and / or luminal surfaces of ABC transporter proteins. For example, in some embodiments, the screening method involves providing a chimeric ABC transporter in which one or more regions of the periplasmic, extracellular, and / or luminal surfaces of the ABC transporter are replaced with one or more equivalent regions of the periplasmic, extracellular, and / or luminal surfaces of a different ABC transporter, and screening for molecules that bind to the ABC transporter but not the chimeric ABC transporter. The present disclosure also relates to molecules that bind to the periplasmic, extracellular, and / or luminal surfaces of ABC transporter proteins, for example, identified by such screening.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 315,255, filed March 1, 2022, U.S. Provisional Application No. 63 / 213,652, filed June 22, 2021, and U.S. Provisional Application No. 63 / 181,118, filed April 28, 2021, which are hereby incorporated by reference in their entireties for any purpose.

[0002] The present disclosure relates to chimeric ABC transporter proteins and methods of using chimeric ABC transporters to screen for molecules that bind to the periplasmic, extracellular, and / or luminal surfaces of ABC transporter proteins. For example, in some embodiments, the screening method involves providing a chimeric ABC transporter in which one or more regions of the periplasmic, extracellular, and / or luminal surfaces of the ABC transporter are replaced with one or more equivalent regions of the periplasmic, extracellular, and / or luminal surfaces of a different ABC transporter, and screening for molecules that bind to the ABC transporter but not to the chimeric ABC transporter. The present disclosure also relates to molecules that bind to the periplasmic, extracellular, and / or luminal surfaces of the ABC transporter, for example, identified by such screening. [Background technology]

[0003] ATP-binding cassette ("ABC") transporters constitute a superfamily of integral membrane proteins found in prokaryotes and eukaryotes, as well as in eukaryotic organelles that are involved in the ATP-powered translocation of many types of substrates across the membrane. ABC transporters have a transmembrane pore that is either accessible from the inner surface of the membrane (inward-facing conformation) or from the outer surface of the membrane (outward-facing conformation). Hydrolysis of ATP drives a conformational change in the protein, resulting in the "flipping" of the transmembrane pore from the inside to the outside of the membrane, or vice versa, to transport the substrate across the membrane. See, for example, FIG. 6.

[0004] Gram-positive bacteria have a single cytoplasmic membrane. Gram-negative bacteria have an outer membrane and an inner / cytoplasmic membrane. The space between the outer and inner membranes is the periplasm. For example, in Gram-negative bacterial cells, ABC transporters can be used to flip certain substrates from the outer membrane to the inner membrane and vice versa, such as the transport of lipopolysaccharide (LPS) between the outer and inner (cytoplasmic) membranes. In contrast, eukaryotic cells have a single membrane that separates the exterior of the cell from the cytosol. Eukaryotic organelles generally have a single membrane that separates the cytosol from the organelle lumen.

[0005] It may be desirable to inhibit the activity of ABC transporters for a variety of reasons. For example, molecules that can inhibit certain ABC transporters in bacteria may be effective antibiotics. One possible antibiotic target is MsbA, a highly conserved ABC transporter in Enterobacteriaceae (Gram-negative) that transports lipopolysaccharide (LPS) from the cytoplasm across the inner / cytoplasmic membrane to the periplasm for incorporation into the outer membrane. MsbA is a major component of the outer membrane and is essential for the growth of Gram-negative bacteria; depletion of MsbA disrupts LPS trafficking and causes cell lysis, and blocking MsbA ATPase activity inhibits bacterial growth. MsbA may be an antibiotic target because it has low identity to human ABC transporters, e.g., human P-gp is only about 30% identical to MsbA.

[0006] Previous high-throughput screening efforts in Gram-negative bacteria to identify molecules that bind to and inhibit the activity of ABC transporters such as MsbA utilized mutant cell types with a more permeable outer membrane, e.g., the imp E. coli strain (Figure 3E). These screens tended to be biased toward identifying molecules that bind to the inward conformation of bacterial ABC transporters. This is problematic because, in order to bind to the inward conformation in Gram-negative cells, a molecule must first cross both the outer membrane and the inner / cytoplasmic membrane, and most molecules identified were unable to do so and were therefore ineffective at inhibiting the activity of ABC transporters in wild-type cells that lack the atypically permeable outer membrane (Figure 6). Similarly, molecules that bind to the inward conformation in Gram-positive bacterial cells or to eukaryotic cells must cross the cytoplasmic membrane before binding to their target. There is a need for methods of drug discovery that can be adapted to high throughput screening techniques to select for molecules that bind to the solvent accessible regions on ABC transporters that are exposed when in the outward conformation: the periplasmic, extracellular and / or luminal faces, e.g., the periplasmic, extracellular and / or luminal clefts found within the periplasmic, extracellular and / or luminal faces of the protein. Molecules that bind to this part of the ABC transporter do not need to cross many membranes to bind to their target and, in some cases, can still act as inhibitors of the ABC transporter, e.g., by competing for binding with the transporter's normal substrate or by blocking access to the normal substrate binding sites on the outer face of the protein. Summary of the Invention

[0007] To screen for molecules that bind to the periplasmic, extracellular, and / or luminal surfaces of ABC transporters, the present inventors have developed a screening strategy using chimeric ABC transporter proteins in which one or more regions of the periplasmic, extracellular, and / or luminal surfaces of an ABC transporter are replaced with one or more equivalent regions of the periplasmic, extracellular, and / or luminal surfaces of a different ABC transporter. Such molecules can be used, for example, in counterselection screens in which molecules that bind to the ABC transporter but not to the chimeric ABC transporter under screening conditions are identified as molecules that bind to the periplasmic, extracellular, and / or luminal surfaces of the ABC transporter. Described herein are chimeric ABC transporters and methods of using them to identify test molecules that bind to the periplasmic, extracellular, and / or luminal regions of the ABC transporter, as well as related binding molecules, molecular complexes, kits, and methods of using the identified molecules.

[0008] The present disclosure includes, for example, any one or combination of the following embodiments.

[0009] Embodiment 1. A method of determining whether a test molecule binds to the periplasmic, extracellular, and / or luminal surface of a parent ABC transporter comprising: a) providing a chimeric ABC transporter in which one or more regions of the periplasmic, extracellular, and / or luminal surface of the parent ABC transporter are replaced with one or more equivalent regions of the periplasmic, extracellular, and / or luminal surface of a different ABC transporter; and b) contacting the chimeric ABC transporter with a test molecule that binds to the parent ABC transporter in an outward conformation, where if the test molecule does not bind to the chimeric ABC transporter, the test molecule is determined to bind to the periplasmic, extracellular, and / or luminal surface of the parent ABC transporter.

[0010] Embodiment 2. A method for determining whether a test molecule binds to the periplasmic, extracellular, and / or luminal face of a parent ABC transporter, comprising: a) trapping the parent ABC transporter in an outward conformation; b) selecting a test molecule that binds to the parent ABC transporter in the outward conformation; c) providing a chimeric ABC transporter in which one or more regions of the periplasmic, extracellular, and / or luminal face of the parent ABC transporter are replaced with one or more equivalent regions of the periplasmic, extracellular, and / or luminal face of a different ABC transporter; and d) contacting the chimeric ABC transporter with a test molecule of (b) that binds to the parent ABC transporter in the outward conformation, wherein if the test molecule does not bind to the chimeric ABC transporter, the test molecule is determined to bind to the periplasmic, extracellular, and / or luminal face of the parent ABC transporter.

[0011] Embodiment 3. The method of embodiment 1 or 2, further comprising trapping the chimeric ABC transporter in an outward conformation prior to contacting the chimeric ABC transporter with the test molecule.

[0012] Embodiment 4. The parent and / or chimeric ABC transporter is 2+ 4. The method of any one of embodiments 2 to 3, wherein the parent and / or chimeric ABC transporter is trapped in an outward conformation by treatment with ATP and vanadate.

[0013] Embodiment 5 The method of any one of embodiments 1 to 4, wherein the parent ABC transporter is a type IV or type V ABC transporter.

[0014] Embodiment 6 The method of any one of embodiments 1 to 5, wherein the parent ABC transporter is a type IV ABC transporter.

[0015] Embodiment 7 The method of any one of embodiments 1 to 6, wherein the parent ABC transporter is derived from a Gram-negative bacterium.

[0016] Embodiment 8. The method of embodiment 7, wherein the gram-negative bacteria is selected from Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas psychrotolerans, Candidatus acumulibacter, Janthinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira species strain-OH-2.

[0017] Embodiment 9 The method of embodiment 8, wherein the parent ABC transporter is derived from E. coli.

[0018] Embodiment 10 The method of any one of embodiments 7 to 9, wherein the parent ABC transporter is MsbA.

[0019] Embodiment 11 The method of any one of embodiments 7 to 10, wherein the different ABC transporters are derived from Gram-negative bacteria.

[0020] Embodiment 12. The method of embodiment 11, wherein the gram-negative bacteria is selected from Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerans, Candidatus acumulibacter, Jansinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira species strain-OH-2.

[0021] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the chimeric ABC transporter differs from the parent ABC transporter in that at least one periplasmic, extracellular, or luminal loop and up to 50% of the transmembrane segments on either side of the loop have been replaced with an equivalent region of a different ABC transporter.

[0022] Embodiment 14. The method of embodiment 13, wherein the chimeric ABC transporter differs from the parent ABC transporter in that at least one periplasmic, extracellular, or lumen-facing loop and 50% of the transmembrane segments on either side of the loop have been replaced with the equivalent region of a different ABC transporter.

[0023] Embodiment 15. The method of embodiment 13, wherein the chimeric ABC transporter differs from the parent ABC transporter in that at least one periplasmic, extracellular, or lumen-facing loop and up to 25% of the transmembrane segments on either side of the loop have been replaced with the equivalent region of a different ABC transporter.

[0024] Embodiment 16. The method of embodiment 13, wherein the chimeric ABC transporter differs from the parent ABC transporter in that at least one periplasmic, extracellular, or lumen-facing loop and up to 10% of the transmembrane segments on either side of the loop have been replaced with an equivalent region of a different ABC transporter.

[0025] Embodiment 17. The method of embodiment 13, wherein the chimeric ABC transporter differs from the parent ABC transporter in that at least two, at least three, or all of the periplasmic, extracellular, or lumen-facing loops and up to 50% of the transmembrane segments on either side of each loop have been replaced with equivalent regions from a different ABC transporter.

[0026] Embodiment 18 The method of any one of embodiments 1 to 17, wherein the parent ABC transporter and the different ABC transporter are encoded by homologous genes from two different species, respectively.

[0027] Embodiment 19. The method of any one of embodiments 1 to 12, wherein the parent ABC transporter is Escherichia coli MsbA (EcMsbA) and the chimeric ABC transporter is EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) have been replaced with the equivalent regions of Pseudomonas psychrotolerans (PpMsbA).

[0028] Embodiment 20. The method of any one of embodiments 1 to 12, wherein the parent ABC transporter is EcMsbA and the chimeric ABC transporter is EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) have been replaced with the equivalent regions of Candidatus accumulibacter (CaMsbA).

[0029] Embodiment 21. The method of any one of embodiments 1 to 12, wherein the parent ABC transporter is EcMsbA and the chimeric ABC transporter is EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) have been replaced with the equivalent regions of Jansinobacterium agaricidamnosum (JaMsbA).

[0030] Embodiment 22. The method of any one of embodiments 1 to 12, wherein the parent ABC transporter is EcMsbA and the chimeric ABC transporter is EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) have been replaced with the equivalent regions of Thiomicrospira cyclica (TcMsbA).

[0031] Embodiment 23. The method of any one of embodiments 1 to 12, wherein the parent ABC transporter is EcMsbA and the chimeric ABC transporter is EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) have been replaced with the equivalent regions of Magnetospira strain-OH-2 (MqMsbA).

[0032] Embodiment 24 The method of any one of embodiments 1 to 23, wherein the molecule binds to the periplasmic, extracellular, or luminal cleft of the parent ABC transporter.

[0033] Embodiment 25 The method of any one of embodiments 1 to 24, further comprising performing an ATPase assay of the parent ABC transporter in the presence of the molecule.

[0034] Embodiment 26 The method of any one of embodiments 1 to 25, further comprising performing a parent ABC transporter cell viability or proliferation assay and / or an ABC transporter functional assay in the presence of the molecule.

[0035] 27. K of 20 μM or less D 27. A molecule identified by the method of any one of embodiments 1 to 26, which binds to a parent ABC transporter at

[0036] 28. K of 10 μM or less D 28. The molecule of embodiment 27, which binds to the parent ABC transporter at

[0037] Embodiment 29. A K of 20 nM or less. D 28. The molecule of embodiment 27, which binds to the parent ABC transporter at

[0038] Embodiment 30. A K of 500 nM or less D 28. The molecule of embodiment 27, which binds to the parent ABC transporter at

[0039] Embodiment 31. K of 1 nM or less D 28. The molecule of embodiment 27, which binds to the parent ABC transporter at

[0040] Embodiment 32. K of 1 to 20 μM D 28. The molecule of embodiment 27, which binds to the parent ABC transporter at

[0041] Embodiment 33. K of 10 to 20 μM D 28. The molecule of embodiment 27, which binds to the parent ABC transporter at

[0042] Embodiment 34. K of 1 nM to 20 μM D 28. The molecule of embodiment 27, which binds to the parent ABC transporter at

[0043] Embodiment 35. K between 1 nM and 500 nM D 28. The molecule of embodiment 27, which binds to the parent ABC transporter at

[0044] Embodiment 36. A molecule identified by the method of any one of embodiments 1 to 26, which is a peptide.

[0045] Embodiment 37. A molecule identified by the method of any one of embodiments 1 to 26, which is a small molecule.

[0046] Embodiment 38. A molecule identified by the method of any one of embodiments 1 to 26, which is an antibody.

[0047] Embodiment 39. A molecule identified by the method of any one of embodiments 1 to 26, which is a binding fragment of a peptide, a small molecule, or an antibody.

[0048] Embodiment 40 The peptide of embodiment 36, which is a macrocycle.

[0049] Embodiment 41. The macrocycle of embodiment 40, which is a 6-14-mer, 6-10-mer, 6-8-mer, or 8-10-mer macrocycle.

[0050] Embodiment 42. The macrocycle of embodiment 40 or 41 having at least one lipophilic side chain and at least one positively charged side chain.

[0051] Embodiment 43 A molecule according to any one of embodiments 27 to 42, which binds to the periplasmic, extracellular, or luminal cleft of a parent ABC transporter.

[0052] Embodiment 44. A macrocyclic peptide G1118, G1119, or G1122.

[0053] Embodiment 45. Macrocyclic peptide G1365.

[0054] Embodiment 46. Mg 2+ , molecules that compete with the macrocycles G1118, G1119, and / or G1122 for binding to the periplasmic face of the parent ABC transporter when the parent ABC transporter is trapped in an outward conformation by treatment with ATP and vanadate.

[0055] Embodiment 47. The molecule of embodiment 46, which inhibits binding of G1118, G1119, and / or G1122 to the parent ABC transporter by at least 50, 60, 70, 80, 90 or 100% in a competitive assay.

[0056] Embodiment 48. Mg 2+ , a molecule that competes with the macrocycle G1365 for binding to the periplasmic face of the parent ABC transporter when the parent ABC transporter is trapped in an outward-facing conformation by treatment with ATP and vanadate.

[0057] Embodiment 49. The molecule of embodiment 48, which inhibits binding of G1365 to the parent ABC transporter by at least 50, 60, 70, 80, 90 or 100% in a competitive assay.

[0058] Embodiment 50. A method of making a chimeric ABC transporter comprising replacing up to 50% of at least one periplasmic, extracellular, or lumen-facing loop and transmembrane segments on either side of the loop of a parent ABC transporter with the equivalent region of a different ABC transporter.

[0059] Embodiment 51. The method of embodiment 50, wherein at least one periplasmic, extracellular, or lumen-facing loop and 50% of the transmembrane segments on either side of the loop of the parent ABC transporter are replaced with the equivalent region of a different ABC transporter.

[0060] Embodiment 52. The method of embodiment 50, wherein at least one periplasmic, extracellular, or lumen-facing loop and up to 25% of the transmembrane segments on either side of the loop of the parent ABC transporter are replaced with the equivalent region of a different ABC transporter.

[0061] Embodiment 53. The method of embodiment 50, wherein at least one periplasmic, extracellular, or lumen-facing loop and up to 10% of the transmembrane segments on either side of the loop of the parent ABC transporter are replaced with the equivalent region of a different ABC transporter.

[0062] Embodiment 54. The method of embodiment 50, wherein at least two, at least three, or all of the periplasmic, extracellular, or lumen-facing loops of the parent ABC transporter and up to 50% of the transmembrane segments on either side of each loop are replaced with equivalent regions of a different ABC transporter.

[0063] Embodiment 55 The method of any one of embodiments 50 to 54, wherein the parent ABC transporter and the different ABC transporter are type IV or type V ABC transporters, respectively.

[0064] Embodiment 56 The method of any one of embodiments 50 to 55, wherein the parent ABC transporter and the different ABC transporter are each a type IV ABC transporter.

[0065] Embodiment 57 The method of any one of embodiments 50 to 56, wherein the parent ABC transporter is derived from a Gram-negative bacterium.

[0066] Embodiment 58. The method of embodiment 57, wherein the gram-negative bacteria is selected from Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerans, Candidatus acumulibacter, Jansinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

[0067] Embodiment 59. The method of embodiment 58, wherein the gram-negative bacterium is Escherichia coli.

[0068] Embodiment 60 The method of any one of embodiments 57 to 59, wherein the parent ABC transporter is MsbA.

[0069] Embodiment 61 The method of any one of embodiments 57 to 60, wherein the different ABC transporters are derived from Gram-negative bacteria.

[0070] Embodiment 62. The method of embodiment 61, wherein the different ABC transporters are derived from a bacterium selected from Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerans, Candidatus acumulibacter, Jansinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

[0071] Embodiment 63 The method of any one of embodiments 50 to 62, wherein the parent ABC transporter and the different ABC transporter are encoded by homologous genes from two different species, respectively.

[0072] Embodiment 64 The method of embodiment 63, wherein the parent ABC transporter and the different ABC transporter are MsbA transporters derived from two different Gram-negative bacterial species.

[0073] Embodiment 65. A chimeric ABC transporter in which at least one periplasmic, extracellular, or lumen-facing loop and up to 50% of the transmembrane segments on either side of the loop of a parent ABC transporter are replaced with the equivalent region of a different ABC transporter.

[0074] Embodiment 66. A chimeric ABC transporter according to embodiment 65, in which at least one periplasmic, extracellular, or lumen-facing loop and 50% of the transmembrane segments on either side of the loop of the parent ABC transporter are replaced with the equivalent region of a different ABC transporter.

[0075] Embodiment 67. The chimeric ABC transporter of embodiment 65, wherein at least one periplasmic, extracellular, or lumen-facing loop and up to 25% of the transmembrane segments on either side of the loop of the parent ABC transporter are replaced with the equivalent region of a different ABC transporter.

[0076] Embodiment 68. The chimeric ABC transporter of embodiment 65, wherein at least one periplasmic, extracellular, or lumen-facing loop and up to 10% of the transmembrane segments on either side of the loop of the parent ABC transporter are replaced with the equivalent region of a different ABC transporter.

[0077] Embodiment 69. The chimeric ABC transporter of embodiment 65, wherein at least two, at least three, or all of the periplasmic, extracellular, or lumen-facing loops of the parent ABC transporter and up to 50% of the transmembrane segments on either side of each loop are replaced with the equivalent regions of a different ABC transporter.

[0078] Embodiment 70 The chimeric ABC transporter of any one of embodiments 65 to 69, wherein the parent ABC transporter is derived from a Gram-negative bacterium.

[0079] Embodiment 71. The chimeric ABC transporter of embodiment 70, wherein the Gram-negative bacterium is selected from Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerans, Candidatus acumulibacter, Jansinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

[0080] Embodiment 72 The chimeric ABC transporter of embodiment 71, wherein the parent ABC transporter is derived from E. coli.

[0081] Embodiment 73 The chimeric ABC transporter of any one of embodiments 70 to 72, wherein the parent ABC transporter is MsbA.

[0082] Embodiment 74 The chimeric ABC transporter of any one of embodiments 70 to 73, wherein the different ABC transporters are derived from Gram-negative bacteria.

[0083] Embodiment 75. The chimeric ABC transporter according to embodiment 74, wherein the Gram-negative bacterium is selected from Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerans, Candidatus acumulibacter, Jansinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

[0084] Embodiment 76 The chimeric ABC transporter of any one of embodiments 70 to 75, wherein the different ABC transporter is MsbA.

[0085] Embodiment 77. The chimeric ABC transporter of any one of embodiments 65 to 76, wherein the parent ABC transporter and the different ABC transporter are encoded by homologous genes from two different species, respectively.

[0086] Embodiment 78. The chimeric ABC transporter of embodiment 77, wherein the parent ABC transporter and the different ABC transporter are MsbA transporters derived from two different Gram-negative bacterial species.

[0087] Embodiment 79. A chimeric ABC transporter comprising the parent ABC transporter Escherichia coli MsbA (EcMsbA) in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) are replaced with the equivalent regions from Pseudomonas psychrotolerans (PpMsbA).

[0088] Embodiment 80. A chimeric ABC transporter comprising EcMsbA in which periplasmic loop 1 (L1, EcMsbA residues Leu47 to Pro68), periplasmic loop 3 (L3, EcMsbA residues Met159 to Leu171), and periplasmic loop 5 (L5, EcMsbA residues Ala262 to Ile292) have been replaced with the equivalent regions from Candidatus accumulibacter (CaMsbA).

[0089] Embodiment 81. A chimeric ABC transporter comprising EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) are replaced with the equivalent regions from Jansinobacterium agaricidamnosum (JaMsbA).

[0090] Embodiment 82. A chimeric ABC transporter comprising EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) are replaced with the equivalent regions of Thiomicrospira cyclica (TcMsbA).

[0091] Embodiment 83. A chimeric ABC transporter comprising EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) are replaced with the equivalent regions (MqMsbA) of Magnetospira strain-OH-2.

[0092] Embodiment 84. A molecular complex comprising the chimeric ABC transporter of any one of embodiments 65 to 83 bound to a peptide, a small molecule, an antibody, a binding fragment of a peptide, a binding fragment of a small molecule, or a binding fragment of an antibody.

[0093] Embodiment 85. The conjugate of embodiment 84, wherein the peptide is a macrocycle.

[0094] Embodiment 86. The conjugate of embodiment 85, wherein the macrocycle is a 6-14-mer macrocycle.

[0095] Embodiment 87 A molecular complex comprising a parent ABC transporter and a molecule according to any one of embodiments 27 to 49.

[0096] Embodiment 88. The conjugate of embodiment 87, wherein the peptide is a macrocycle.

[0097] Embodiment 89. The conjugate of embodiment 88, wherein the macrocycle is a 6-14-mer macrocycle.

[0098] Embodiment 90. The conjugate of embodiment 89, wherein the macrocycle is G1118, G1119, G1122, or G1365.

[0099] Embodiment 91. A kit comprising the chimeric ABC transporter of any one of embodiments 65 to 83 and reagents for carrying out the method of any one of embodiments 1 to 26, optionally wherein the chimeric ABC transporter is attached to a matrix or beads, and optionally wherein the kit comprises: a. A parent ABC transporter and / or a different ABC transporter from which the chimeric ABC transporter is engineered; b. A matrix or beads for attaching the ABC transporter, optionally streptavidin-, avidin-, or deglycosylated avidin-coated beads, or magnetic beads; c. one or more detergents to solubilize the ABC transporter on the matrix or beads; d. at least one wash buffer; d. at least one elution buffer; f. at least one positive or negative control molecule The kit further comprises one or more of:

[0100] Embodiment 92 The kit of embodiment 91, further comprising instructions for use.

[0101] Embodiment 93. A method of treating a bacterial infection in an individual, comprising administering to the individual an effective amount of a molecule of any one of embodiments 27 to 49.

[0102] Embodiment 94 Use of a molecule according to any one of embodiments 27 to 49 for treating a bacterial infection in a subject.

[0103] EMBODIMENT 95. Sequence: A peptide comprising ClacF-X1-X2-L-X3-X4-D-X5-X6-X7-X8-MeF-VC, wherein i. X1 is W, V, or Y; ii. X2 is W or Y; iii. X3 is W or Y; iv. X4 is S, D, V, or H; v. X5 is N, where N is selected from any natural amino acid other than C or an unnatural amino acid selected from Bph ((S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid), Dopa (L-3,4-dihydroxyphenylalanine), MeF (N-methyl-L-phenylalanine), and MeG (N-methyl-L-glycine); vi. X6 is Y, K, A, S, D, R, or V; vii. X7 is W, Y, or Bph; viii. X8 is W or Y; Optionally, the peptide further comprises a G residue following the C residue at the C-terminus, wherein ClacF is N-chloroacetyl L-phenylalanine, Bph is (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid, Dopa is L-3,4-dihydroxyphenylalanine, MeF is N-methyl-L-phenylalanine, and MeG is N-methyl-L-glycine.

[0104] Embodiment 96 The peptide of embodiment 95, wherein X1 is W or Y.

[0105] Embodiment 97. The peptide of embodiment 96, wherein X1 is W.

[0106] Embodiment 98. A peptide according to any one of embodiments 95 to 97, wherein X2 is W.

[0107] Embodiment 99. A peptide according to any one of embodiments 95 to 98, wherein X3 is W.

[0108] Embodiment 100. A peptide according to any one of embodiments 95 to 99, wherein X4 is S, D, V, or H.

[0109] Embodiment 101. The peptide of embodiment 100, wherein X4 is D.

[0110] Embodiment 102. A peptide according to any one of embodiments 95 to 101, wherein X5 is V, D, H, G, or Y.

[0111] Embodiment 103. The peptide of embodiment 102, wherein X5 is V or H.

[0112] Embodiment 104. The peptide of embodiment 103, wherein X5 is V.

[0113] Embodiment 105. A peptide according to any one of embodiments 95 to 104, wherein X6 is D or S.

[0114] Embodiment 106 The peptide of embodiment 105, wherein X6 is S.

[0115] Embodiment 107. A peptide according to any one of embodiments 95 to 106, wherein X7 is W.

[0116] Embodiment 108. A peptide according to any one of embodiments 95 to 107, wherein X8 is W.

[0117] Embodiment 109. A macrocycle formed from a peptide according to any one of embodiments 95 to 108, which is cyclized due to a thioether bond between the N-terminal chloroacetyl group of ClacF and the sulfhydryl group of the C residue.

[0118] Embodiment 110. The amino acid sequence: A peptide comprising ClacF-X1-Y-Bph-MeF-X2-VC, wherein i.X1 is V, S, Y, W, Dopa, L, V, A, R, K, or D; ii. X2 is R, V, Dopa, or Y; A peptide in which ClacF is N-chloroacetyl L-phenylalanine, Bph is (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid, Dopa is L-3,4-dihydroxyphenylalanine, and MeF is N-methyl-L-phenylalanine.

[0119] Embodiment 111. The peptide of embodiment 110, wherein X1 is S, Y, L, V, A, R, K, or D.

[0120] Embodiment 112. The peptide of embodiment 111, wherein X1 is V or Y.

[0121] Embodiment 113. A peptide according to any one of embodiments 110 to 112, wherein X1 is V.

[0122] Embodiment 114. A peptide according to any one of embodiments 110 to 113, wherein X2 is R or Y.

[0123] Embodiment 115. The peptide of embodiment 114, wherein X2 is R.

[0124] Embodiment 116. A macrocycle formed from a peptide according to any one of embodiments 110 to 115, which is cyclized due to a thioether bond between the N-terminal chloroacetyl group of ClacF and the sulfhydryl group of the C residue.

[0125] Embodiment 117. A peptide or macrocycle according to any one of embodiments 95 to 116, wherein the peptide or macrocycle is conjugated to another molecule, such as an antibiotic or antibacterial agent, optionally wherein the conjugation is at the C-terminal amino acid residue of the sequence.

[0126] Embodiment 118. The peptide or macrocycle of embodiment 117, wherein the antibiotic or antibacterial agent is a polymyxin, e.g., polymyxin B or polymyxin E. [Brief description of the drawings]

[0127] [Figure 1A] A diagram of the protein structure of an ABC transporter and how the protein is arranged in the membrane relative to the periplasmic / extracellular space and the cytoplasm / cytosol is shown. Each individual amino acid is represented by a circle surrounding the amino acid, which is identified by a single letter code. Also, some amino acid positions on the chain are numbered, for example, positions 50 and 60. Figure 1A shows the MsbA ABC transporter from E. coli. Periplasmic or extracellular loop regions that can be replaced with equivalent regions of different ABC transporters to create chimeric ABC transporters are shown and labeled. Regions of the protein that cross the membrane ("transmembrane segments" or "TM segments") that can be replaced with equivalent regions of ABC transporters from different ABC transporters are also shown. Rectangular boxes indicate the regions that can be replaced in each protein. [Figure 1B] Diagrams of the protein structures of different ABC transporters and how the proteins are arranged in the membrane relative to the periplasmic / extracellular space and the cytoplasm / cytosol are shown. Each individual amino acid is represented by a circle surrounding the amino acid, which is identified by a single letter code. Also, some amino acid positions on the chain are numbered, for example, position 50 and position 60. Figure 1B shows the human ABC transporter ABCD4, which has about 25-30% sequence identity with MsbA. Periplasmic or extracellular loop regions that can be replaced with equivalent regions of different ABC transporters to create chimeric ABC transporters are shown and labeled. Regions of the protein that cross the membrane ("transmembrane segments" or "TM segments") that can be replaced with equivalent regions of ABC transporters from different ABC transporters are also shown. Rectangular boxes indicate the regions that can be replaced in each protein. [Figure 1C-1]Diagrams of the protein structures of different ABC transporters and how the proteins are arranged in the membrane relative to the periplasmic / extracellular space and the cytoplasm / cytosol are shown. Each individual amino acid is represented by a circle surrounding the amino acid, which is identified by a single letter code. Also, some amino acid positions on the chain are numbered, for example, position 50 and position 60. Figure 1C shows the human ABC transporter ABCC1, which has about 25-30% sequence identity with MsbA. Periplasmic or extracellular loop regions that can be replaced with equivalent regions of different ABC transporters to create chimeric ABC transporters are shown and labeled. Regions of the protein that cross the membrane ("transmembrane segments" or "TM segments") that can be replaced with equivalent regions of ABC transporters from different ABC transporters are also shown. Rectangular boxes indicate the regions that can be replaced in each protein. [Figure 1C-2] Diagrams of the protein structures of different ABC transporters and how the proteins are arranged in the membrane relative to the periplasmic / extracellular space and the cytoplasm / cytosol are shown. Each individual amino acid is represented by a circle surrounding the amino acid, which is identified by a single letter code. Also, some amino acid positions on the chain are numbered, for example, position 50 and position 60. Figure 1C shows the human ABC transporter ABCC1, which has about 25-30% sequence identity with MsbA. Periplasmic or extracellular loop regions that can be replaced with equivalent regions of different ABC transporters to create chimeric ABC transporters are shown and labeled. Regions of the protein that cross the membrane ("transmembrane segments" or "TM segments") that can be replaced with equivalent regions of ABC transporters from different ABC transporters are also shown. Rectangular boxes indicate the regions that can be replaced in each protein. [Figure 2A]Three-dimensional schematics of ABC transporters and chimeric ABC transporters and how they are positioned within the membrane relative to the periplasm / extracellular space and the cytoplasm / cytosol are shown. The left panel of FIG. 2A shows a three-dimensional schematic of Gram-negative bacterial MsbA positioned within the cell membrane (dark bands intersecting the structure). The center panel of FIG. 2A shows a three-dimensional schematic of the macrocycle-MsbA complex, in which the macrocycle is bound to the periplasmic face of the protein, specifically to the periplasmic cleft. The right panel of FIG. 2A shows a chimeric MsbA (MsbA-chimera 5), ​​in which the darker filled amino acids at the top of the ribbon diagram are taken from the equivalent region in a different ABC transporter. Such chimeras can be used, for example, to help select molecules that bind to the periplasmic face, including the periplasmic cleft, of MsbA, such as the macrocycle shown in the center panel. [Figure 2B] 3D schematic diagrams of ABC transporters and chimeric ABC transporters and how they are positioned within the membrane relative to the periplasm / extracellular space and the cytoplasm / cytosol. The left panel of FIG. 2A shows a 3D schematic diagram of the Gram-negative bacterial MsbA positioned within the cell membrane (dark bands intersecting the structure). The center panel of FIG. 2B shows a chimeric human ABCD4 transporter (PDB:6JBJ) where the filled amino acids of the protein are taken from the equivalent regions in different ABC transporters. The left panel of FIG. 2B also shows the chimeric MsbA protein, showing the similarities in the architecture of the two proteins. The right panel of FIG. 2B shows a chimeric human ABCC1 transporter (PDB:6BHU) where the filled amino acids at the top of the ribbon diagram are taken from the equivalent regions in different ABC transporters. [Figure 2C]Three-dimensional schematic diagrams of ABC transporters and chimeric ABC transporters and how they are positioned within the membrane relative to the periplasmic / extracellular space and the cytoplasm / cytosol. The left panel of FIG. 2A shows a three-dimensional schematic diagram of the Gram-negative bacterial MsbA positioned within the cell membrane (dark bands intersecting the structure). FIG. 2C shows a schematic diagram of each of the three chimeric proteins from a periplasmic or extracellular perspective. Each periplasmic or extracellular loop is labeled. The filled-in amino acids in the ribbon diagrams are from the equivalent regions in the different ABC transporters. Each extracellular loop is also labeled. [Figure 3A] Figure 3 shows a strategy for characterizing macrocyclic MsbA inhibitors. Figure 3A shows a schematic of the INSITE screening strategy to identify macrocycles that target the periplasmic face of EcMsbA. [Figure 3B] Strategies for characterizing macrocyclic MsbA inhibitors are shown. Figure 3B shows exemplary macrocycle inhibitors G1118 and G1365. These inhibitors contain thioether bonds as indicated (unnatural amino acids: Clac-F, N-chloroacetyl-L-phenylalanine; MeF, N-methyl-L-phenylalanine; Bph, (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid). [Figure 3C-D] Strategy for characterizing macrocyclic MsbA inhibitors. Figures 3C-D show dose-response curves of G1118 (Figure 3C) and G1365 (Figure 3D) against WT E. coli MsbA and MsbA-chim5. IC50 values ​​were determined by fitting a nonlinear 4-parameter inhibition model (see Example 3) and data are means ± sem from three independent experiments. [Figure 3E]Strategy for characterizing macrocyclic MsbA inhibitors is shown. Figure 3E shows representative electron micrographs comparing msbA+, msbA- and inhibitor-exposed E. coli CFT073 UPEC lptD(imp4213) cells (Table 1). G907 is a quinoline inhibitor of MsbA described in Figure 11D (Alexander et al., 2018; Ho et al., 2018). Inner membrane elaborations are marked with arrows. Images represent >10 isolated cells for each condition. Scale bars are 0.1 μm. [Figure 3F] Strategy for characterizing macrocyclic MsbA inhibitors is shown. Figure 3F shows electron micrographs comparing E. coli imp strain with G1118 (right panel; arrow indicates inner membrane elaboration) with E. coli imp strain without G1118 (left panel). The IC50 of G1118 was determined to be 5 nM. [Figure 3G-L] Strategy for characterizing macrocyclic MsbA inhibitors. Figure 3G-L show dose-response curves for G1325 (Figure 3G; IC50 = 1.2 μM; EC50 = 10 μM), G1330 (Figure 3H; IC50 = 13 μM; EC50 = 30 μM), G1365 (Figure 3I; IC50 = 300 nM; EC50 = 20 μM), G1349 (Figure 3J; IC50 = 50 μM; EC50 = 30 μM), G1323 (Figure 3K; IC50 = 2.8 μM), and G1320 (Figure 3L; IC50 = 185 μM) against E. coli strains impMsbAWT, impMsbAHigh, and UPEC WT. [Figure 3M] Strategy for characterizing macrocyclic MsbA inhibitors. Figure 3M shows a potential binding site for G092 quinoline. [Figure 3N] Strategy for characterizing macrocyclic MsbA inhibitors Figure 3N shows a potential binding site for G1118 (Figure 3N). [Figure 4A-I]Figure 4A shows a cryo-EM map of the G1118-EcMsbA complex. G1118 and LPS are indicated on the map. Figure 4B shows a diagram of G1118 binding in the periplasmic cleft of MsbA. The GKK-tail has been omitted for clarity. Figure 4C-D show a close-up view of selected interactions between G1118 and MsbA. Figure 4E shows a frequency plot of observed macrocyclic cDNAs in the G1118 family during sequencing after the final round of enrichment (unnatural amino acids are MeF=F*, N-methyl-L-phenylalanine; MeG=G*, N-methyl-L-glycine). Figure 4F shows a cryo-EM map of the G1365-EcMsbA complex. G1365 is indicated. Figure 4G-H shows a close-up view of the select interactions between G1365 and MsbA. Figure 4I shows a frequency plot of observed macrocyclic cDNAs in the G1365 family during sequencing after the final round of enrichment (unnatural amino acids are Bph=B*, (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid; MeF=F*, N-methyl-L-phenylalanine). [Figure 4J] Figure 4J shows that G1365 binds near the membrane-exposed region of the outward cleft. [Figure 4K-N] Figure 4K-N shows that G1365 binds near the membrane-exposed region of the outward cleft. [Figure 5A-B] Figure 5A shows the peripheral LPS binding sites of MsbA on the inner leaflet of the inner membrane. Figure 5B shows a close-up of the LPS binding site highlighting the interaction between LPS and MsbA. The 2'-hydroxymyristate (2'-C14) and 2''-laurate acyl (2''-C12) chains are labeled. Kdo residues have been omitted for clarity. [Figure 5C]Figure 5C shows LPS bound to the surrounding binding sites essential in the outward conformation. Figure 5C shows growth curves of E. coli MG1655 msbA-cKO lptD(imp4213) expressing the indicated WT or mutant alleles of E. coli MsbA from a low-copy plasmid (Table 1). Data are means ± sem from three independent experiments. [Figure 5D] Figure 5D shows LPS bound to surrounding binding sites essential in the outward conformation. Figure 5D shows representative thin-section electron micrographs comparing cells expressing msbA+, msbA-, and the indicated msbA mutant constructs in E. coli CFT073 UPEC lptD(imp4213) strain. Inner membrane elaborations are indicated with arrows. Images represent >10 isolated cells for each variant. Scale bar is 0.1 µm. [Figure 5E] Figure 5D shows LPS bound to the surrounding binding sites essential for the outward conformation. Figure 5E shows the amino acids that, when mutated, cause growth defects in E. coli. [Figure 6] A model for the selective recognition and transport of LPS by MsbA is shown. Panel 1 - In an outward conformation, LPS can bind to peripheral binding sites along the inner membrane. Mature Kdo2-lipid A preferentially binds positively charged aromatic residues on the 4'-phosphate side of LPS. Panel 2 - MsbA returns to an inward conformation and LPS is locally concentrated upon release from peripheral binding sites (PDB:6BL6). Facilitated diffusion of LPS into the central cavity occurs along a ridge of positively charged residues in MsbA. Panel 3 - LPS becomes enclosed within the central space within the portal as previously described (PDB:5TV4). Panel 4 - ATP binding triggers a large conformational change resulting in an outward state that releases LPS into the outer leaflet of the inner membrane. LPS diffuses away from MsbA for subsequent processing and transport to the outer membrane. [Figure 7] Crystallography data collection and refinement statistics are shown. [Figure 8] An overview of cryo-EM data acquisition parameters and model refinement statistics is shown. [Figure 9A]Multiple sequence alignment of selected MsbA homologs is shown. In FIG. 9A, the E. coli MsbA sequence is shown as a reference, and putative homologs from other species selected from the chimeric transporter design are included. In FIG. 9A, SEQ ID NO: 4 is the E. coli MsbA protein sequence (line 1), SEQ ID NO: 5 is the MsbA protein sequence of Pseudomonas cyclotolerans (line 2), SEQ ID NO: 6 is the MsbA protein sequence of Candidatus accumulibacter (line 3), SEQ ID NO: 7 is the MsbA protein sequence of Jansinobacterium agaricidamnosum (line 4), SEQ ID NO: 8 is the MsbA protein sequence of Thiomicrospira cyclica (line 5), and SEQ ID NO: 9 is the MsbA protein sequence of Magnetospira OH-2 (line 6). [Figure 9B] Figure 9B shows the overall sequence identity for the MsbA homologs in Figure 9A. [Figure 10A] Constructs of chimeric MsbA transporters are shown. Figure 10A shows an exemplary sequence of an E. coli MsbA-based chimera. The regions marked "Loop 1", "Loop 2", and "Loop 3" indicate the periplasmic regions of the transmembrane helices and loops that were replaced in the generation of the engineered chimeric transporter proteins. In Figure 10A, SEQ ID NO: 4 is the E. coli MsbA protein sequence (line 1), SEQ ID NO: 12 is the E. coli MsbA-chimera 1 protein sequence (line 2), SEQ ID NO: 13 is the E. coli MsbA-chimera 2 protein sequence, SEQ ID NO: 14 is the E. coli MsbA-chimera 3 protein sequence (line 3), SEQ ID NO: 15 is the E. coli MsbA-chimera 4 protein sequence (line 4), and SEQ ID NO: 16 is the E. coli MsbA-chimera 5 protein sequence (line 5). [Figure 10B] Constructs of chimeric MsbA transporters. Figure 10B shows the outward facing E. coli MsbA and the MsbA-chimera. A side view is shown in the upper panel, and a top view is shown in the lower panel. In the MsbA-chimera, regions with amino acid substitutions in loops 1-3 (as shown in Figure 10A) are indicated. [Figure 11A-B] Purification and characterization of EcMsbA chimeras. Figure 11A shows SDS-PAGE of purified EcMsbA chimeras visualized with Coomassie Brilliant Blue staining. Figure 11B shows the overlay of the size-exclusion chromatography profile with the indicated UV (280 nm) curve. [Figure 11C] (C) Comparison of the ATPase activity of decreasing concentrations of the EcMsbA chimera compared to the WT protein. Data are means ± sem from independent experiments. [Figure 11D] Purification and evaluation of EcMsbA chimeras. Figure 11D shows exemplary chemical structures of quinoline and benzophenone inhibitors (Alexander et al., 2018; Ho et al., 2018). Data are means ± sem from independent experiments. [Figure 11E] Purification and evaluation of EcMsbA chimeras are shown. Figure 11E shows the dose-response curves of compounds for purified EcMsbA and EcMsbA-chim5. Data are means ± sem from three independent experiments. IC50 values ​​(in brackets in Figure 11E) were determined by fitting the inhibitory dose-response curves to a nonlinear four-parameter inhibition model (Example 3). [Figure 12A] The crystal structure of MsbA-chimera5 reveals the putative benzophenone receptor site. Figure 12A shows the overall structure of MsbA-chim5 in an inward conformation. LPS is shown as a sphere. The assigned benzophenone G758 is shown as a stick. The Fo-Fc map (1.5σ mesh) is calculated before G758 is included in the model and refinement. [Figure 12B]The crystal structure of MsbA-chimera 5 reveals the putative benzophenone receptor site. Figure 12B shows a close-up of the putative benzophenone binding site on MsbA, along with the Fo-Fc map from Figure 12A and the 2Fo-Fc map (1.5σ mesh). G758 putatively binds in a shallow hydrophobic pocket around residues identified through frequency of resistance mapping studies (data not shown). [Figure 13A-B] Evaluation of biochemical and phenotypic activity of MsbA macrocycle inhibitors. Figure 13A shows dose-response curves of G1118 against purified and amphipol-renatured MsbA homologs from E. coli, Enterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosa. Figure 13B shows dose-response curves of G1118 against E. coli CFT073 (WT) and CFT073 lptD(imp4213)(imp) strains. Data are means ± sem from independent experiments. IC50 values ​​(in brackets) were determined by fitting the dose-response curves to a nonlinear four-parameter inhibition model (Example 3). [Figure 13C-D] Evaluation of biochemical and phenotypic activity of MsbA macrocycle inhibitors. Figure 13C shows dose-response curves of G1365 against purified and amphipol-renatured MsbA homologs from E. coli, Enterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosa. Figure 13D shows dose-response curves of G1118 against E. coli CFT073 (WT) and CFT073 lptD(imp4213)(imp) strains. Data are means ± sem from three independent experiments. IC50 values ​​(in brackets) were determined by fitting the dose-response curves with a nonlinear four-parameter inhibition model (Example 3). [Figure 14A] Biochemical evaluation of the conjugates used in the structural studies. Figure 14A shows the chemical structures of the G1118 and G1365 derivatives used in the isolation of the PD-1365 resistant mutant (G1365·I2,Dopa3) in the structural studies. Regions of the derivatives that differ from the G1118 or G1365 parent macrocycles, i.e., KK, R2, I2,Dopa3, and L2G4S2GEE, are shown circled. [Fig. 14B-G] Biochemical evaluation of the complexes used in the structural studies is shown. Figures 14B-F show dose-response curves of parent and derivative macrocycles of G1118 and G1365 against purified EcMsbA. Figure 14G shows a comparison of the ATPase activity of increasing concentrations of EcMsbA in the presence or absence of Fab 12G7. Data are means ± sem from three independent experiments (Figures 14B-G). IC50 values ​​(in brackets in Figures 14B-F) were determined by fitting the inhibitory dose-response curves with a nonlinear four-parameter inhibition model (Example 3). [Figure 15A] Cryo-EM data processing for MsbA in complex with G1118. Figure 15A shows the processing pipeline. [Figure 15B-C] Cryo-EM data processing for MsbA in complex with G1118. Figure 15B shows the local resolution plot calculated in Relion. Figure 15C shows the orientation distribution of all particles from the final round of 3D refinement. [Figure 15D] Cryo-EM data processing for MsbA in complex with G1118 is shown. Figure 15D shows the FSC plot from the refinement in cisTEM. The maximum resolution used for the alignment was 4.5 Å. [Figure 15E] Cryo-EM data processing for MsbA in complex with G1118 is shown. Figure 15E shows selected densities from the cryo-EM map. [Figure 16A-B] Macrocycle binding in the periplasmic cleft of MsbA. In Figure 16A-B, the electrostatic surface of MsbA bound to G1118 highlights the complex chemical environment of the macrocycle binding site. The approximate membrane boundary and the non-enforced C2 symmetry axis are labeled. [Fig. 16C-D] 16C-D show the electrostatic surface of MsbA bound to G1365. [Figure 17A]Cryo-EM data processing for MsbA in complex with G1365. Figure 17A shows the processing pipeline. [Fig. 17B-C] Cryo-EM data processing for MsbA in complex with G1365 is shown. Figure 17B shows the orientation distribution of all particles from the final round of 3D refinement. Figure 17C shows the FSC plot from refinement in cisTEM. The maximum resolution used for the alignment was 4.5 Å. [Figure 17D] Cryo-EM data processing for MsbA in complex with G1365 is shown. Figure 17D shows the local resolution plot calculated with RELION. [Figure 17E] Cryo-EM data processing for MsbA in complex with G1365 is shown. Figure 17E shows selected densities from the cryo-EM map. [Figure 18A-B] The conserved surrounding LPS binding sites are shown. Figure 18A shows the MsbA-G1118-LPS complex with symmetrically related LPS sites. Figure 18B shows a sequence conservation analysis of EcMsbA. [Fig. 18C-D] The conserved surrounding LPS binding site is shown. Figure 18C shows the electrostatic potentials on the surface of MsbA in the inward-facing LPS-bound conformation (PDB:5TV4). A positively charged running line runs from the LPS-binding site toward the central cavity. The missing side chain atoms were added through Maestro's Protein Preparation Wizard. The binding position of LPS in the outward-facing G1118-bound structure is shown. In Figure 18D, the structure of MsbA-G1118-LPS (blue) is aligned with the inward-facing MsbA (white, PDB:5TV4). Residues within 15 Å of LPS were used for the alignment. [Figure 19A]Figure 19A shows the evaluation of cell growth phenotypes of MsbA surrounding LPS binding site mutants. Figure 19A shows α-FLAG-MsbA and α-GroEL Western blots of solubilized extracts from E. coli MG1655 msbA-cKO lptD(imp4213) expressing WT or the indicated mutants of E. coli MsbA from the pLMG18 vector in the presence of 2% arabinose (untagged WT EcMsbA is also expressed under these conditions). Blots are representative of n=2 independent experiments. [Fig. 19B-E] Figure 19B-E show the evaluation of cell growth phenotypes of MsbA surrounding LPS binding site mutants. Figure 19B-E show growth curves of E. coli MG1655 msbA-cKO lptD(imp4213) expressing the indicated WT or mutant alleles of E. coli MsbA, or a plasmid-free control, in the presence or absence of arabinose. 2% arabinose was used to induce expression of WT E. coli MsbA from the chromosome, whereas in the absence of arabinose, the only source of MsbA is constitutive expression from pLMG18 vectors expressing WT or the indicated mutants of E. coli MsbA. Data are means ± sem from three independent experiments. [Fig. 19F-I] Figure 19F-I shows the evaluation of cell growth phenotypes of MsbA surrounding LPS binding site mutants. Growth curves of E. coli MG1655 msbA-cKO lptD(imp4213) expressing the indicated WT or mutant alleles of E. coli MsbA, or a plasmid-free control, in the presence or absence of arabinose. 2% arabinose was used to induce expression of WT E. coli MsbA from the chromosome, whereas in the absence of arabinose, the only source of MsbA is constitutive expression from pLMG18 vectors expressing WT or the indicated mutants of E. coli MsbA. [Figure 19J] Figure 19 shows evaluation of the cell growth phenotype of MsbA peri-LPS binding site mutants. Figure 19J shows a comparison of the ATPase activity of decreasing concentrations of EcMsbA mutants compared to the WT protein. Data are means ± sem from three independent experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0128] I. Definition Unless otherwise specified, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. As utilized in accordance with the present disclosure, the following terms shall be understood to have the following meanings, unless otherwise specified:

[0129] In this application, the use of "or" means "and / or" unless otherwise stated. In the context of multiple dependent claims, the use of "or" refers to the preceding multiple independent or dependent claims as an alternative only. Also, terms such as "element" or "component" include both elements and components that are comprised of a single unit and elements and components that are comprised of multiple subunits, unless otherwise stated.

[0130] As used herein, the transitional term "consisting essentially of," when referring to steps of a claimed method, means that the method does not contain additional steps beyond the specified steps that materially affect the basic and novel properties of the method. As used herein, the transitional term "consisting essentially of," when referring to a composition or article of manufacture, such as a kit, means that it does not contain additional components beyond those specified that materially affect its basic and novel properties.

[0131] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an ABC transporter protein" includes a plurality of such transporter proteins, reference to "the cell" includes reference to one or more cell (or cells) and equivalents thereof known to those skilled in the art, and so forth.

[0132] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include every integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

[0133] Units, prefixes, and symbols are shown in the International System of Units (SI) accepted format. Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of the disclosure, which can be achieved by reference to the entire specification. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0134] "ABC transporter" as used herein refers to ATP-binding cassette ("ABC") transporter. ABC transporters constitute a superfamily of integral membrane proteins found in prokaryotes and eukaryotes that are involved in the ATP-powered translocation of many substrates across membranes, ranging from inorganic and organic small molecules, such as amino acids, sugars, nucleosides, vitamins, and metal clusters, to larger organic compounds, including peptides, lipid molecules, oligonucleotides, and polysaccharides. ABC transporters can generally be grouped into "exporters" and "importers", although some ABC transporters may be classified into another group, non-transporter ABC proteins.

[0135] ABC transporters have a characteristic architecture that generally includes at least four domains: two transmembrane domains (TMDs) embedded in the membrane and two nucleotide-binding domains (NBDs). Hydrolysis of ATP on the NBDs drives conformational changes in the TMDs, resulting in alternating access of substrates from the inside and outside of the membrane to transport them one-way across the membrane. Each TMD is composed of transmembrane alpha helices, each TMD typically has 6-10 transmembrane alpha helices, with most exporters having 6 transmembrane alpha helices per TMD. The NBDs are highly conserved. In contrast, the TMDs that create the translocation pathway are more variable, depending, for example, on the substrate the ABC transporter is transporting.

[0136] One classification system described in Thomas et al., "Structural and functional diversity calls for a new classification of ABC transporters," 594 FEBS Letters 3767-3775 (2020) (incorporated by reference in its entirety for its description of the ABC transporter classification system) groups ABC transporters into seven separate types, I-VII, based on their TMD fold. This nomenclature is universally applicable and is based on ABC transporter structural information determined by sequence analysis, homology modeling, X-ray crystallography and single particle cryo-electron microscopy, among other sources. Type I transporters have the following transmembrane helix configuration: (5-6)+(5-6 / 8). Type II transporters have the following transmembrane helix configuration: 10+10. Type III transporters have the following transmembrane helix configuration: 4-8(T)+6-7(S). Type IV transporters have the following transmembrane helix configuration: 6+6. Type V transporters also have a 6+6 transmembrane helix configuration and are further defined as being of the ABCG / ABCA / Wzm type based on sequence similarity and known substrate specificity. Type VI transporters also have a 6+6 transmembrane helix configuration and are further defined as being of the LptB2FG type based on distinct structural features. Type VII transporters have the following transmembrane helix configuration: 4+4. Thus, as used herein, a "type IV" ABC transporter refers to, for example, an ABC transporter classified as type IV under this classification system, "type V" refers to a type V transporter classified under this system, and so forth.

[0137] The terms "outward conformation" and "inward conformation" as used herein refer to the conformation of the alpha-helices of the two transmembrane domains or regions (TMDs) of an ABC transporter, which are packed in such a way that they form a transmembrane pore that is either accessible from the inner region of the membrane (inward facing) or from the outside of the membrane (outward facing). For example, in Gram-negative bacterial cells, ABC transporters integrated into the inner membrane have an inward conformation in which the pore opens toward the cytoplasm and an outward conformation in which the pore opens toward the periplasm (the space between the inner and outer membranes). In Gram-positive bacterial cells or eukaryotic cells, ABC transporters integrated into the cell membrane have an inward conformation in which the pore opens toward the cytoplasm and an outward conformation in which the pore opens toward the outside of the cell. In ABC transporters integrated into the membrane of an intracellular organelle, such as a mitochondrion, the ABC transporter has an outward conformation in which the pore opens toward the lumen of the intracellular organelle and an inward conformation in which the pore opens toward the cytoplasm / cytosol. The outward conformation is therefore one in which the pore opens into the lumen of the organelle.

[0138] The term "periplasmic, extracellular, and / or luminal face" of an ABC transporter refers to a face or region of the ABC transporter that is accessible when the transporter is in an outward conformation, depending on the membrane in which the transporter is located. "Periplasmic, extracellular, and / or luminal cleft" refers to a pocket or pore formed by packing of alpha-helices that are accessible in the outward conformation of the protein. In some cases, for example, in ABC transporters of gram-negative bacteria, the part that is on the outside of the ABC transporter faces the periplasm, while in other cases it faces the outside of the cell (e.g., extracellular) or it faces the lumen, where the ABC transporter is within an intracellular organelle (e.g., lumen). Thus, as used herein, "periplasmic cleft" refers to a pocket or pore in an ABC transporter formed by packing of alpha-helices of the transmembrane domain that is accessible to the periplasm. As used herein, "extracellular cleft" refers to a pocket or pore in an ABC transporter formed by the packing of the alpha-helices of the transmembrane domain, which is accessible to the environment outside the cell. As used herein, "luminal cleft" refers to a pocket or pore in an ABC transporter formed by the packing of the alpha-helices of the transmembrane domain, which is accessible to the lumen of an intracellular organelle, such as, for example, mitochondria, the endoplasmic reticulum, or the Golgi apparatus.

[0139] As used herein, a "chimeric ABC transporter" refers to an ABC transporter in which one or more of the periplasmic, extracellular, or lumen-facing loops and up to 50% of the transmembrane segments on either side of the loop(s) are replaced with the equivalent region of a different ABC transporter. As an example, the drawings in Figures 1A-B show three periplasmic (or extracellular or lumen-facing) loops, each of which connects two alpha helices across the membrane. As used herein, an "equivalent region" (also called a "corresponding region") that is inserted from a different ABC transporter is a region that is in the same position in the protein when the residues are removed from the parent ABC transporter and folded back. In some cases, the region in the ABC transporter that is removed and replaced with the region from the chimeric ABC transporter can be determined using sequence alignment and structural information on the two proteins.

[0140] In some cases, chimeric ABC transporters are formed from a parent ABC transporter and a different ABC transporter derived from a "homologous gene from a different species." As used herein, this phrase means that the two genes are members of the same gene family and are capable of transporting the same molecule, such as the representative MsbA protein from two different bacterial species, such as E. coli and another Gram-negative bacterial species.

[0141] The term "gram-negative bacteria" as used herein refers to bacteria that do not retain crystal violet dye when the gram staining method is used. Gram staining is a common method for identification of general bacteria. In the gram staining method, bacteria can be heat fixed on a glass slide, stained with crystal violet dye, washed with iodine, decolorized with alcohol or another organic solvent, and then counterstained with safranin. The gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria. Gram-positive bacteria remain purple because they have a single thick cell wall that the solvent cannot easily penetrate. Gram-negative bacteria are decolorized because they have a cell wall with a very thin layer that allows removal of the dye by the solvent. In the final step, safranin stains the gram-negative cells red.

[0142] The term "gram-positive bacteria" as used herein refers to bacteria that retain the crystal violet dye when the gram staining method is used. Gram staining is a common method for identification of common bacteria. In the gram staining method, bacteria can be heat-fixed on a glass slide, stained with crystal violet dye, washed with iodine, decolorized with alcohol or another organic solvent, and then counterstained with safranin. The gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria. Gram-positive bacteria remain purple because they have a single thick cell wall that the solvent cannot easily penetrate. Gram-negative bacteria are decolorized because they have a cell wall with a very thin layer that allows removal of the dye by the solvent. In the final step, safranin stains the gram-negative cells red.

[0143] The term "peptide" as used herein refers to a chain of 50 or fewer amino acids linked by peptide bonds, including amino acid chains of 2-50, 2-15, 2-10, 2-8, or 6-14 amino acids.

[0144] The term "small molecule" as used herein refers to an organic molecule having a molecular weight between 50 and 2500 Daltons.

[0145] As used herein, "macrocycle" or "macrocyclic molecule" refers to a cyclic polymer or a polymeric ring portion of a polymer. Macrocycles range in size from 500 Daltons to 2000 Daltons. In some cases herein, macrocycles are cyclic peptides or peptide derivatives.

[0146] As used herein, the term "binding fragment" refers to a portion of a larger molecule, such as a small molecule, peptide, or antibody, that is expected to directly contact an ABC transporter. Binding fragments can be used in high throughput screening.

[0147] In this disclosure, "bind" or "binding" or "specific binding" and similar terms, when referring to a molecule that "binds" to an ABC transporter protein or chimeric ABC transporter protein, means, for example, that the binding affinity is strong enough that the interaction between the members of the binding pair cannot be due to random molecular association ("non-specific binding"). Thus, the binding is selective or specific. Such binding typically has a dissociation constant (K D ) (i.e., corresponding to an affinity of 100 μM or greater), and often requires a K of 20 μM or less, 10 μM or less, 1 μM or less, or 500 nM or less. D may be accompanied by.

[0148] As used herein, the term "competitive assay" refers to an assay in which a molecule being tested interferes with or inhibits the specific binding of a reference molecule to a common target.

[0149] The term "ATPase assay" refers to an assay used to measure the extent of conversion of ATP to ADP by a protein, such as an ABC transporter protein.

[0150] The term "treat" as well as words derived therefrom, as used herein, does not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment, including, for example, reducing at least one symptom or condition, and in some cases, for example, about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. Moreover, treatment also includes preventing, improving, or inhibiting one or more conditions or symptoms of a disorder, as well as delaying the onset of a disorder, or a symptom thereof.

[0151] The term "effective amount" or "therapeutically effective amount," as used herein, refers to a sufficient amount of a molecule disclosed herein being administered to relieve to some extent one or more of the symptoms of the disease or condition being treated, e.g., an infection.

[0152] Where appropriate, other definitions are included in the sections below.

[0153] II. Chimeric ABC transporters In some embodiments, the present invention comprises chimeric ABC transporters.

[0154] In some embodiments, to generate a chimeric ABC transporter, the starting material is a specific ABC transporter (the "parent" ABC transporter). In some embodiments, the parent ABC transporter is derived from a eukaryotic cell. In some embodiments, the parent ABC transporter is a human ABC transporter. In some embodiments, the parent ABC transporter is derived from a Gram-positive bacterium. In some embodiments, the parent ABC transporter is derived from a Gram-negative bacterium. In some embodiments, the parent ABC transporter is one that can be embedded in an intracellular organelle membrane. In some embodiments, the parent ABC transporter is a transporter found in a specific bacterial species selected from Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerans, Candidatus acumulibacter, Jansinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2. In some embodiments, the parent ABC transporter is MsbA, e.g., E. coli MsbA.

[0155] In some embodiments, after a parent ABC transporter is selected, an equivalent but different ABC transporter, in some embodiments from a different organism or species, is identified and an amino acid sequence or region is taken from it to form a chimera with the parent transporter. In some cases, the goal is to identify a different ABC transporter that is sufficiently different in sequence that a test molecule that binds to the parent ABC transporter does not bind to the different ABC transporter in an equivalent region, but a region that is sufficiently similar except that the chimera folds properly into its precise architecture. In some embodiments, the different ABC transporter is identified by searching for different ABC transporters that share 20-99% sequence identity with the parent ABC transporter. In some embodiments, the different ABC transporter is from a gene homologous to the one being tested, but from a different, optionally related, species or organism. For example, in some cases, chimeras may be constructed from homologous genes of ABC transporters from two different bacterial species or genera, or species such as human and mouse, or human and primate, e.g., MsbA proteins from two different bacterial species. In some embodiments, the three-dimensional structures of the parent ABC transporter, if known, are compared to the three-dimensional structures of the different ABC transporters.

[0156] In some embodiments, when the parent ABC transporter is a human protein, the different ABC transporters are derived from homologous genes from different eukaryotic species. In some embodiments, the different ABC transporters are derived from homologous genes from different mammalian species. In some embodiments, when the parent ABC transporter is derived from a Gram-positive bacterium, the different ABC transporter is derived from another Gram-positive bacterium, optionally from a homologous gene from another species. In some embodiments, when the parent ABC transporter is derived from a Gram-negative bacterium, the different ABC transporter is derived from a Gram-negative bacterium, optionally from a homologous gene from another species. In some embodiments, the parent ABC transporter and the different ABC transporter are selected from two different species selected from Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerans, Candidatus acumulibacter, Jansinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2. In some embodiments, the chimeric ABC transporter comprises a region of a parent ABC transporter selected from a eukaryotic cell, a Gram-positive bacterium, a Gram-negative bacterium, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerans, Candidatus acumulibacter, Jansinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2, and / or MsbA, and a region of a different ABC transporter selected from a eukaryotic cell, a Gram-positive bacterium, a Gram-negative bacterium, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerans, Candidatus acumulibacter, Jansinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2, and / or MsbA.

[0157] In some embodiments, it may be advantageous to select a different ABC transporter that is the same type of ABC transporter as the parent ABC transporter, as defined in the classification system of Thomas et al., "Structural and functional diversity calls for a new classification of ABC transporters," 594 FEBS Letters 3767-3775 (2020) (incorporated by reference in its entirety for its description of the ABC transporter classification system), which groups ABC transporters into seven separate types, I-VII, based on their transmembrane domain (TMD) folds. For example, in some embodiments, the parent ABC transporter is a type I ABC transporter and the different ABC transporter is a type I ABC transporter. In some embodiments, the parent ABC transporter is a type II ABC transporter and the different ABC transporter is a type II ABC transporter. In some embodiments, the parent ABC transporter is a type III ABC transporter and the different ABC transporter is a type III ABC transporter. In some embodiments, the parent ABC transporter is a type IV ABC transporter and the different ABC transporter is a type IV ABC transporter. In some embodiments, the parent ABC transporter is a type V ABC transporter and the different ABC transporter is a type V ABC transporter. In some embodiments, the parent ABC transporter is a type VI ABC transporter and the different ABC transporter is a type VI ABC transporter. In some embodiments, the parent ABC transporter is a type VII ABC transporter and the different ABC transporter is a type VII ABC transporter.

[0158] ABC transporters have a characteristic architecture that generally includes at least four domains: two transmembrane domains (TMDs) embedded in the membrane (the transmembrane segments can be part of the TMDs) and two nucleotide-binding domains (NBDs). Each TMD is composed of a transmembrane alpha helix, with each TMD typically having 6-10 transmembrane alpha helices, with most exporters having 6 transmembrane alpha helices per TMD. In some embodiments, ABC transporters have loops that face the periplasm, extracellular, or luminal space when the molecule is in an outward conformation, and transmembrane segments that connect to both sides of such loops. (See Figures 1A-1C.) In some embodiments, the transporter has three such loops. In some embodiments, the transporter has six such loops.

[0159] Figure 1A shows a schematic diagram of an ABC transporter from E. coli. The TMDs are parts of the amino acid chain that cross the membrane region; they are typically alpha-helices. The six TMDs in the schematic diagram are labeled 1-6. The schematic diagram also shows a loop on the periplasmic side of the membrane and a loop on the cytoplasmic side of the membrane. The loops each have a first end that connects to the TMD, a second end that connects to the TMD, and a portion that contacts the periplasm or cytoplasm. The ABC transporter shown has three loops that face the periplasm (e.g., loops 1, 2, and 3, which correspond to loops 1, 3, and 5 of MsbA (loops 2, 4, and 6 of MsbA face the cytoplasm). The rectangular boxes indicate regions that can be replaced with equivalent regions from different ABC transporters, i.e., at least one of the loops that face the periplasm, or all three of the loops, and up to 50% of the TMD segment.

[0160] FIG. 1B shows a schematic of an ABC transporter derived from the human ABC transporter ABCD4. The six TMDs in the schematic are labeled 1 through 6. The schematic also shows the loops on the extracellular side of the membrane and the loops on the cytosolic side of the membrane. In the schematic, the extracellular loops are labeled 1 through 3. The rectangular boxes indicate regions that may be replaced with equivalent regions from different ABC transporters.

[0161] Figure 1C shows a schematic diagram of an ABC transporter derived from the human ABC transporter ABCC1. This ABC transporter is structurally different from the ABC transporters derived from E. coli and human ABCD4, also shown in Figures 1A and 1B, but many of the same identifying characteristics are present. For example, there are extracellular loops, but instead of three as in ABCD4, there are six extracellular loops, which are labeled 1 through 6 in the schematic. Similarly, there are 12 TMDs, which are labeled 1 through 12 in the schematic. Rectangular boxes indicate regions that may be replaced with equivalent regions from different ABC transporters.

[0162] In each of the three schematics in Figures 1A-C, rectangular boxes indicate regions that may be replaced with equivalent regions from a different ABC transporter. In some embodiments, up to 50%, 0-50%, 10-50%, 25-50%, 0-10%, 0-25%, or 10-25% of the TMDs on either side of the loop replaced in the chimera are also replaced. In some embodiments, the disclosure includes chimeric ABC transporters in which at least one periplasmic, extracellular, or lumen-facing loop and up to 50% (i.e., 50%, 0-50%, 10-50%, 25-50%, 0-10%, 0-25%, or 10-25%) of the transmembrane segments on either side of the loop are replaced with equivalent regions from a different ABC transporter. In some embodiments, the invention includes chimeric ABC transporters in which at least one periplasmic, extracellular, or lumen-facing loop and 50% of the transmembrane segments on either side of the loop are replaced with the equivalent region of a different ABC transporter. In some embodiments, the invention includes chimeric ABC transporters in which at least one periplasmic, extracellular, or lumen-facing loop and up to 25% of the transmembrane segments on either side of the loop are replaced with the equivalent region of a different ABC transporter. In some embodiments, the invention includes chimeric ABC transporters in which at least one periplasmic, extracellular, or lumen-facing loop and up to 10% of the transmembrane segments on either side of the loop are replaced with the equivalent region of a different ABC transporter. In some embodiments, the invention includes chimeric ABC transporters in which two or more, or all, of the periplasmic, extracellular, or lumen-facing loops and up to 50% (i.e., 50%, 0-50%, 10-50%, 25-50%, 0-10%, 0-25%, or 10-25%) of the transmembrane segments on either side of each loop are replaced with the equivalent regions from a different ABC transporter.

[0163] In some embodiments, prediction of the positions (i.e., boundaries) of loops and transmembrane segments in the parent and distinct ABC transporters can be made based on available experimental structure templates in the Protein Data Bank (PDB), if available, or alternatively, by using the closest available PDB structure template and standard homology modeling techniques and software (i.e., Swiss-Modell, Phyre2, MOE). In some embodiments, in the absence of a suitable PDB template, prediction of the positions (i.e., boundaries) of loops and transmembrane segments in the ABC transporter and distinct ABC transporters can also be made using standard databases or algorithms (i.e., Uniprot, TMHMM server, etc.).

[0164] In some embodiments, once the location of the ABC transporter and the different ABC transporter and at least one loop and / or transmembrane segment in each have been identified, a region of the ABC transporter can be replaced with a region from the different ABC transporter to create a chimeric ABC transporter. In some embodiments, for example, if the region from an ABC transporter is loop 1, the equivalent region from the different ABC transporter is also loop 1. In some embodiments, for example, if the region from an ABC transporter is 25% of the transmembrane segment / TMD1, the equivalent region from the different ABC transporter is also 25% of the transmembrane segment / TMD1.

[0165] In some embodiments, where the ABC transporter is MsbA, the chimeric ABC transporter comprises EcMsbA (amino acid sequence available at Uniprot P60752) in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) have been replaced with the equivalent regions of Pseudomonas psychrotolerans (PpMsbA; Uniprot A0A1G5PEL0). (The term loop herein refers to both periplasmic and cytoplasmic loops, with L1, L3, and L5 facing the periplasm and L2, L4, and L6 facing the cytoplasm. Thus, L1, L3, and L5 of EcMsbA are equivalent to loops 1, 2, and 3 shown in FIG. 1A.) The sequence is based on the amino acid sequence of the E. coli protein, which has accession number Uniprot P60752 (see www(dot)uniprot(dot)org entry P60752, incorporated herein by reference). See also the sequence listing herein (Table 7).

[0166] In some embodiments, the chimeric ABC transporter comprises EcMsbA in which periplasmic loop 1 (L1, EcMsbA residues Leu47 to Pro68, periplasmic loop 3 (L3, EcMsbA residues Met159 to Leu171, and periplasmic loop 5 (L5, EcMsbA residues Ala262 to Ile292) have been replaced with the equivalent regions of Candidatus accumulatoribacter species SK-12 (CaMsbA; Uniprot A0A011NLL4).

[0167] In some embodiments, the chimeric ABC transporter comprises EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) are replaced with the equivalent regions from Jansinobacterium agaricidamnosum (JaMsbA; Uniprot A0A3G2E7N4).

[0168] In some embodiments, the chimeric ABC transporter comprises EcMsbA in which one or more of EcMsbA residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) are replaced with the equivalent regions of Thiomicrospira cyclica from DSM 14477 (TcMsbA; Uniprot F6DCY0).

[0169] In some embodiments, the chimeric ABC transporter comprises EcMsbA in which residues Leu47 to Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159 to Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262 to Ile292 in periplasmic loop 5 (L5) are replaced with the equivalent regions of Magnetospira species strain-OH-2 (MqMsbA; Uniprot W6KCN7).

[0170] III. Screening Methods Using Chimeric ABC Transporters The disclosure particularly encompasses methods of identifying molecules that bind to the periplasmic, extracellular, and / or luminal faces of particular ABC transporter proteins using chimeric versions of such proteins, in some cases the methods identify molecules that bind to the periplasmic, extracellular, and / or luminal clefts of the proteins.

[0171] In some embodiments, the method comprises determining whether a test molecule binds to the periplasmic, extracellular, and / or luminal face of a parent ABC transporter, and comprises (a) providing a chimeric ABC transporter in which one or more regions of the periplasmic, extracellular, and / or luminal face of the parent ABC transporter are replaced with one or more equivalent regions of the periplasmic, extracellular, and / or luminal face of a different ABC transporter, as described above; and (b) contacting the chimeric ABC transporter with a test molecule that binds to the parent ABC transporter in an outward conformation, wherein if the test molecule does not bind to the chimeric ABC transporter, it is determined that the test molecule binds to the periplasmic, extracellular, and / or luminal face of the parent ABC transporter.

[0172] In some cases, a molecule is first tested to determine whether it binds to a parent ABC transporter in an outward conformation, for example, by (a) trapping the parent ABC transporter in an outward conformation, and then (b) selecting a test molecule that binds to the parent ABC transporter in the outward conformation before completing the above steps. Thus, after this selection, the method includes (c) providing a chimeric ABC transporter in which one or more regions of the periplasmic, extracellular, and / or luminal surface of the parent ABC transporter are replaced with one or more equivalent regions of the periplasmic, extracellular, and / or luminal surface of a different ABC transporter, as described above, and (d) contacting the chimeric ABC transporter with a test molecule of (b) that binds to the parent ABC transporter in the outward conformation, and if the test molecule does not bind to the chimeric ABC transporter, it is determined that the test molecule binds to the periplasmic, extracellular, and / or luminal surface of the ABC transporter. In any of these methods, in some embodiments, the method further comprises trapping the chimeric ABC transporter in an outward conformation prior to contacting the chimeric ABC transporter with the test molecule. 2+ , ATP, or vanadate (e.g., vanadate or orthovanadate anions) can be trapped in an outward conformation.

[0173] These fundamental steps are also illustrated, for example, in Figure 6. As depicted in Figure 6, ABC transporters have both inward and outward conformations. Previously identified ABC transporter binders, such as those in the quinoline class, bind inward conformations, as shown in the second panel on the left of Figure 6. As described herein, transporters can be used to bind Mg 2+The ABC transporter can be trapped in an outward conformation by treatment with agents such as a combination of , ADP, and vanadate, thus exposing the periplasmic, extracellular, or luminal faces of the protein and the associated periplasmic, extracellular, or luminal clefts (Figure 6). To selectively identify molecules that bind to the periplasmic, extracellular, and / or luminal faces or their associated clefts, counter-selections can be performed using appropriate chimeric ABC transporters as described above (Figures 1A-1C, 2A-2C, and 6). Molecules that bind to the parent ABC transporter but not to the chimera under appropriate assay conditions can be selected, e.g., chimeric null binders.

[0174] In some embodiments, the method is performed with either the test molecule or the ABC transporter immobilized, such as on a bead or matrix platform. In some cases, the parent ABC transporter and / or the chimeric ABC transporter are immobilized on a bead or matrix platform, for example, using biotin / streptavidin or a similar set of reagents. When beads are used for immobilization, they can have any shape, such as, for example, flakes or chips, spheres, pellets, etc. The matrix may be composed of beads or smaller particles, for example, a slurry or gel, which can then be placed on a plate or chip, such as a microwell plate. In some embodiments, the matrix or beads are coated with streptavidin, avidin, or deglycosylated avidin. In some embodiments, the beads are magnetic beads, for example, to facilitate collection of the beads during the assay, by use of a magnetic instrument. For example, in some cases, the protein may be biotinylated and then exposed to a matrix or beads coated with streptavidin, which allows the protein to attach to the matrix or beads.

[0175] In certain assays herein, such as those in which parent and / or chimeric ABC transporters are immobilized while the test molecule is in solution, molecules determined to bind to a particular transporter can be identified as those that remain bound to the immobilized transporter under assay conditions following incubation and washing of the immobilized transporter, and thus are eluted from the protein binding matrix or beads upon addition of an elution buffer. Molecules that do not bind to a particular transporter protein in the assay can be identified as those that are not eluted (e.g., above trace levels) upon addition of an elution buffer, and thus are removed from the immobilized protein upon washing of the protein binding matrix or beads.

[0176] In some embodiments, the parent or chimeric ABC transporter is solubilized in a detergent or similar molecule that mimics a biological membrane so that it maintains the proper fold and ability to form the correct outward conformation. Exemplary detergents or related molecules or systems for solubilizing ABC transporters and / or chimeric ABC transporters include lauryl maltose neopentyl glycol (LMNG), in some embodiments 0.02% LMNG, as well as dodecyl-β-D-maltoside (DDM), brij-35, glycol-diosgenin, digitonin, amphiphors, such as amphiphor A8-35, and lipid nanodiscs. For example, detergents can solubilize the transporter and stabilize it, while amphiphors can effectively encapsulate the hydrophobic parts of proteins and stabilize them, and proteins can also be stabilized within a type of lipid bilayer created by lipid nanodiscs.

[0177] In some embodiments, a library of test molecules is screened. The library is contacted with the chimeric ABC transporter on a matrix or bead, and then washed at least once with a washing buffer to remove unbound molecules. The bound test molecules are then eluted and analyzed. Molecules that preferentially bind to the ABC transporter in its outward conformation compared to the related chimeric ABC transporter in its outward conformation can be identified as binding to the periplasmic, extracellular, and / or luminal surface of the ABC transporter, because those regions are mutated in the chimeric ABC transporter.

[0178] In some embodiments, further experiments are carried out on the molecules selected in the above screening, for example, to determine their binding affinity to parent ABC transporter and chimeric ABC transporter, respectively, and to determine how they affect the function of ABC transporter. Thus, for example, in some embodiments, ATPase assay is carried out to determine the ATP vs. ADP activity of ABC transporter in the presence of identified molecules. In some embodiments, identified molecules that bind to the periplasmic, extracellular, and / or luminal surface of ABC transporter can act as inhibitors of the ATPase activity of ABC transporter. ATPase assays are known in the art, and various commercial kits are available, such as Tanscreener ADP2 Assay (BellBrook Labs, Cat.#3010-1K) and Molecular Probes ATP Determination Kit (Thermofisher, Cat.#A22066).

[0179] In some embodiments, the binding affinity of the identified molecules to the parent and / or chimeric ABC transporter can be determined. In some cases, this can be done with an ELISA assay similar to that used in the initial screen, e.g., IC 50 A value is obtained. In some cases, for example, a competitive ELISA assay may be performed using a parent ABC transporter bound to a matrix or beads and a chimeric ABC transporter free in solution (or vice versa). Molecules that bind to a parent ABC transporter preferentially over the corresponding chimeric ABC transporter should bind to the parent ABC transporter to approximately the same extent in the presence and absence of the chimeric ABC transporter. Such an assay may be performed, for example, to confirm that a particular test molecule or molecules identified in a previous screen are selective for the periplasmic, extracellular, and / or luminal surface of the transporter. In some embodiments, the binding assay may be performed in cell culture to test for binding to the parent ABC transporter in the plasma membrane of the cells or otherwise in their normal cellular state. Other types of binding assays are known in the art.

[0180] In some embodiments, one or more functional assays may be performed to test the effect of the molecules identified in the screen on the function of the parent ABC transporter. For example, in some cases, molecules that bind to the periplasmic, extracellular, and / or luminal surfaces of the ABC transporter act as inhibitors of the transporter. For example, if inhibition of ABC transporter activity is expected to result in, for example, loss of cell viability or reduced cell growth, cell viability or growth assays may be performed to determine whether the presence of the identified molecule affects these parameters. In some cases, other assays may be performed to determine the effect of the molecule on the function of the transporter. In some cases, transport of the transporter's substrate in the presence and absence of the molecule may be tested. For example, in the case of MsbA, which is tested in the examples below, certain binding molecules were found to slow down LPS transport by the protein based on electron microscopy (EM) analysis. The lack of LPS transport causes stacking of inner and outer membranes at the surface of the bacterial cell, clearly visible by EM. See, for example, FIG. 3E. Suitable functional assays for the ABC transporter are known or can be readily developed based on knowledge in the art.

[0181] IV. Test Molecules for Screening Methods In some embodiments, the disclosure includes screening methods that test for specific types of molecules, as well as molecules identified by any of the screening methods described herein as binding to the periplasmic, extracellular, and / or luminal face or cleft of a particular ABC transporter. In some cases, the identified molecule does not bind to the chimeric ABC transporter used in the screening method, but does bind to the parent ABC transporter on which the chimera is based. In some cases, the identified molecule binds to the parent ABC transporter with at least 10 times tighter affinity than the chimeric ABC transporter used for screening. In some cases, the identified molecule binds to the parent ABC transporter with at least 100 times tighter affinity than the chimeric ABC transporter used for screening. In some cases, the identified molecule binds to the parent ABC transporter with at least 1000 times tighter affinity than the chimeric ABC transporter used for screening.

[0182] In some embodiments, the molecule being tested is a peptide. In some embodiments, the peptide is a 6-14-mer peptide, e.g., a 6-12-mer, a 6-10-mer, a 6-8-mer, an 8-12-mer, an 8-10-mer, etc. In some embodiments, the peptide is a 14-mer. See Table 3 and Figure 3F. In some embodiments, the peptide is a 6-10-mer. In some embodiments, the peptide is an 8-10-mer. In some embodiments, the peptide is a 6-8-mer. In some embodiments, the peptide is an 8-mer. See Figures 3E and 3G-L, and Table 5. In some embodiments, the peptide is a 3-40-mer, 3-20-mer, 4-16-mer, 4-14-mer or 6-14-mer, e.g., a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer, 35-mer, 36-mer, 37-mer, 38-mer, 39-mer, or 40-mer.

[0183] In some embodiments, the peptide is a macrocycle. In some embodiments, the macrocycle is a 6-14-mer macrocycle, e.g., a 6-12-mer, a 6-10-mer, a 6-8-mer, an 8-12-mer, an 8-10-mer, etc. In some embodiments, the macrocycle is a 14-mer macrocycle. See Table 3 and Figure 3F. In some embodiments, the macrocycle is a 6-10-mer macrocycle. In some embodiments, the macrocycle is an 8-10-mer macrocycle. In some embodiments, the macrocycle is a 6-8-mer macrocycle. In some embodiments, the macrocycle is an 8-mer macrocycle. See Figures 3E and 3G-L, and Table 5. In some embodiments, the macrocycle is a 3-40-mer, 3-20-mer, 4-16-mer, 4-14-mer, or 6-14-mer, e.g., a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer, 35-mer, 36-mer, 37-mer, 38-mer, 39-mer, or 40-mer macrocycle. In some embodiments, the macrocycle has at least one lipophilic side chain and at least one positively charged side chain.

[0184] In some embodiments, the molecules tested in the screening herein are small molecules. In some embodiments, the molecules tested are antibodies, which may include full-length antibodies of any of IgG, IgM, IgA, IgD, and IgE, as well as antigen-binding fragments of antibodies such as Fv, Fab', (Fab')2, scFv, nanobodies, single-chain antibodies, bispecific or multispecific antibodies.

[0185] In some embodiments, the molecule being tested is a binding fragment of a peptide, a binding fragment of a small molecule, or a binding fragment of an antibody (eg, an antigen-binding fragment).

[0186] Also encompassed herein are specific macrocycles identified in screens for binders of E. coli MsbA. In some embodiments, the macrocycle is G1118, a 14-mer macrocycle. See Figures 3F, 4A-D, and 6-. G1118 binds E. coli MsbA with an IC of 5 nM, e.g., to MsbA in an imp E. coli strain (wherein the outer membrane is permeable). 50 See Figure 3F. G1118 has the sequence ClAc-FWWLWDDVSWWMeFVCNH2 or ClAc-FWWLWDDVSWWMeFVCGKKNH2. In some embodiments, the macrocycle is G1365, which is an 8-mer macrocycle. See Figures 3E, 3G-L, and 4J-N. G1365 has the sequence ClAc-FVYBphMeFRVCNH2. G1365 binds to imp E. coli MsbA with a biochemical IC of, for example, 300 nM. 50 coli MsbA, which has an affinity for E. coli MsbA that is at least 10-fold tighter than its affinity for MsbA from Acinetobacter baumannii (A. bau.) when tested in imp strains that have a permeable outer membrane (see Figure 3G-L and Table 5 below).

[0187] The macrocyclic peptide activators tested included the 14-mer G1118 (SEQ ID NO: 1 or 2), as well as the macrocyclic peptides G1119 (ClAc-FWWLWSDMeGDWWMeFVC-NH2; SEQ ID NO: 10) and G1122 (ClAc-FRYLWMeAWGLVWDNC-NH2; SEQ ID NO: 11), and the 8-mer G1365 (SEQ ID NO: 3).

[0188] In some embodiments, the molecules identified in the screens herein have a K of 20 μM or less for ABC transporters. DIn some embodiments, the molecule binds to the ABC transporter with a K of 10 μM or less. D In some embodiments, the molecule binds to the ABC transporter with a K of 20 nM or less. D In some embodiments, the molecule binds to the ABC transporter with a K of 500 nM or less. D In some embodiments, the molecule binds to the ABC transporter with a K of 1 nM or less. D In some embodiments, the molecule binds to the ABC transporter with a K of 1-20 μM. D In some embodiments, the molecule binds to the ABC transporter with a K of 10-20 μM. D In some embodiments, the molecule binds to the ABC transporter with a K between 1 nM and 20 μM. D In some embodiments, the molecule binds to the ABC transporter with a K between 1 nM and 500 nM. D In some cases, the identified molecule does not bind to the chimeric ABC transporter used in the screening method, but does bind to the parent ABC transporter on which the chimera is based. In some cases, the identified molecule binds to the parent ABC transporter with an affinity that is at least 10 times tighter than the chimeric ABC transporter used in the screen. In some cases, the identified molecule binds to the parent ABC transporter with an affinity that is at least 10 times tighter than the chimeric ABC transporter used in the screen. In some cases, the identified molecule binds to the parent ABC transporter with an affinity that is at least 100 times tighter than the chimeric ABC transporter used in the screen. In some cases, the identified molecule binds to the parent ABC transporter with an affinity that is at least 1000 times tighter than the chimeric ABC transporter used in the screen. Binding affinity can be determined by methods known in the art.

[0189] In some embodiments, a molecule identified as binding to a particular ABC transporter can be used as a positive control or competitor in an assay used to screen other test molecules. For example, in some methods, a molecule is identified as binding to the periplasmic, extracellular, or luminal surface or cleft of an ABC transporter by determining that it competes with a previously known molecule to bind to the periplasmic, extracellular, or luminal surface or cleft of the same ABC transporter. For example, in the case of E. coli MsbA, the macrocycles G1118 and G1365, as well as G1119 and G1122, can be used as competitors or positive controls in an assay to look for additional binders. In some such embodiments, the screen is performed to determine whether E. coli MsbA is capable of binding to Mg. 2+ The screening may identify molecules that compete with G1118, G1119, and / or G1122 for binding to the periplasmic face of E. coli MsbA when trapped in an outward conformation, such as by treatment with ATP and vanadate. In some embodiments, the molecule inhibits binding of G1118, G1119, and / or G1122 to the ABC transporter by at least 50, 60, 70, 80, 90, or 100% in a competition assay. In some such embodiments, the ... 2+ Molecules may be identified that compete with G1365 for binding to the periplasmic face of E. coli MsbA when trapped in an outward conformation, such as by treatment with ATP and vanadate, hi some embodiments, the molecule inhibits binding of G1365 to the ABC transporter by at least 50, 60, 70, 80, 90 or 100% in a competition assay.

[0190] In some embodiments, the 14-mer macrocyclic peptide may comprise the following sequence: ClacF-X1-X2-L-X3-X4-D-X5-X6-X7-X8-MeF-VC, where X1 is W, V, or Y; X2 is W or Y; X3 is W or Y; X4 is S, D, V, or H; and X5 is N, where N is any naturally occurring amino acid except C, or any of the amino acids selected from the group consisting of Bph ((S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid), Dopa (L-3,4-dihydroxyphenylalanine), MeF (N-methyl-L-phenylalanine), and MeG ( X is selected from unnatural amino acids selected from N-methyl-L-glycine; X is Y, K, A, S, D, R, or V; X is W, Y, or Bph; X is W or Y; optionally, the peptide further comprises a G residue following the C residue at the C-terminus, ClacF is N-chloroacetyl L-phenylalanine, Bph is (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid, Dopa is L-3,4-dihydroxyphenylalanine, MeF is N-methyl-L-phenylalanine, and MeG is N-methyl-L-glycine. In some cases, X1 is W or Y. In some cases, X1 is W. In some cases, X2 is W. In some cases, X3 is W. In some cases, X4 is S, D, V, or H. In some cases, X4 is D. In some cases, X5 is V, D, H, G, or Y. In some cases, X5 is V or H. In some cases, X5 is V. In some cases, X6 is D or S. In some cases, X6 is S. In some cases, X7 is W. In some cases, X8 is W. In some of the above embodiments, the macrocycle is cyclized due to a thioether bond between the chloroacetyl group at the N-terminus of ClacF and the sulfhydryl group of the C residue.

[0191] In some embodiments, the 8-mer macrocyclic peptide may comprise the following sequence: ClacF-X1-Y-Bph-MeF-X2-VC, where X1 is V, S, Y, W, Dopa, L, V, A, R, K, or D, X2 is R, V, Dopa, or Y, ClacF is N-chloroacetyl L-phenylalanine, Bph is (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid, Dopa is L-3,4-dihydroxyphenylalanine, and MeF is N-methyl-L-phenylalanine. In some cases, X1 is S, Y, L, V, A, R, K, or D. In some cases, X1 is V or Y. In some cases, X1 is V. In some cases, X2 is R or Y. In some cases, X2 is R. In some embodiments, the macrocycle is cyclized due to a thioether bond between the chloroacetyl group at the N-terminus of ClacF and the sulfhydryl group of the C residue.

[0192] In other cases, the macrocycle has the sequence G1118, G1119, or G1122 (SEQ ID NO: 1 or 2 (for G1118), SEQ ID NO: 10 (for G1119), or SEQ ID NO: 11 (for G1122)). In still other cases, the macrocycle has the sequence G1365 (SEQ ID NO: 3).

[0193] In some embodiments, the peptide or macrocycle is conjugated to another molecule, such as an antibiotic or antibacterial, optionally at the C-terminal amino acid residue of the sequence. In some embodiments, the antibiotic or antibacterial is a polymyxin, e.g., polymyxin B or polymyxin E.

[0194] V. Molecular complexes In some embodiments, the disclosure includes a molecular complex comprising an ABC transporter described herein bound to a molecule such as a peptide, a small molecule, or an antibody, a binding fragment of a peptide, a small molecule, or an antibody. In some embodiments, the disclosure includes a molecular complex comprising an ABC transporter and a macrocycle, which in some embodiments is a 6-14-mer, a 6-10-mer, a 6-8-mer, or an 8-10-mer macrocycle. In some embodiments, the molecule binds to the ABC transporter with a KD of 20 μM or less, 10 μM or less, 20 nM or less, 500 nM or less, 1 nM or less, 1-20 μM, 10-20 μM, 1 nM-20 μM, and / or 1 nM-500 nM. In some embodiments, the disclosure includes a molecular complex comprising a chimeric ABC transporter described herein bound to a molecule such as a peptide, a small molecule, an antibody, or a binding fragment of a peptide, a small molecule, or an antibody. In some embodiments, the invention includes a molecular complex comprising a chimeric ABC transporter and a macrocycle, which in some embodiments is an 8-10-mer macrocycle.

[0195] In some embodiments, the molecule is a peptide. In some embodiments, the peptide is a 6-14-mer peptide, e.g., a 6-12-mer, a 6-10-mer, a 6-8-mer, an 8-12-mer, an 8-10-mer, etc. In some embodiments, the peptide is a 14-mer. See Table 3 and Figure 3F. In some embodiments, the peptide is a 6-10-mer. In some embodiments, the peptide is an 8-10-mer. In some embodiments, the peptide is a 6-8-mer. In some embodiments, the peptide is an 8-mer. See Figures 3E and 3G-L, and Table 5. In some embodiments, the peptide is a 3-40-mer, 3-20-mer, 4-16-mer, 4-14-mer or 6-14-mer, e.g., a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer, 35-mer, 36-mer, 37-mer, 38-mer, 39-mer, or 40-mer.

[0196] In some embodiments, the peptide is a macrocycle. In some embodiments, the macrocycle is a 6-14-mer macrocycle, e.g., a 6-12-mer, a 6-10-mer, a 6-8-mer, an 8-12-mer, an 8-10-mer, etc. In some embodiments, the macrocycle is a 14-mer macrocycle. See Table 3 and Figure 3F. In some embodiments, the macrocycle is a 6-10-mer macrocycle. In some embodiments, the macrocycle is an 8-10-mer macrocycle. In some embodiments, the macrocycle is a 6-8-mer macrocycle. In some embodiments, the macrocycle is an 8-mer macrocycle. See Figures 3E and 3G-L, and Table 5. In some embodiments, the macrocycle is a 3-40-mer, 3-20-mer, 4-16-mer, 4-14-mer, or 6-14-mer, e.g., a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer, 35-mer, 36-mer, 37-mer, 38-mer, 39-mer, or 40-mer macrocycle. In some embodiments, the macrocycle has at least one lipophilic side chain and at least one positively charged side chain.

[0197] In some embodiments, the molecule in the complex is a small molecule. In some embodiments, the molecule is an antibody, which can include any full-length antibody, such as IgG, IgM, IgA, IgD, and IgE, as well as antigen-binding fragments of antibodies, such as Fv, Fab', (Fab')2, scFv, nanobodies, single chain antibodies, bispecific or multispecific antibodies.

[0198] In some embodiments, the molecule is a binding fragment of a peptide, a binding fragment of a small molecule, or a binding fragment of an antibody (eg, an antigen-binding fragment).

[0199] In some embodiments, the molecule in the complex is G1118, G1365, G1119, or G1122. In some embodiments, the molecule in the complex is, for example, an ABC transporter that transports Mg 2+ When trapped in an outward conformation by treatment with ATP and vanadate, it competes with the macrocycles G1118, G1119, and / or G1122 for binding to the periplasmic face of an ABC transporter, such as E. coli MsbA. In some embodiments, the molecule inhibits binding of G1118, G1119, and / or G1122 to the ABC transporter by at least 50, 60, 70, 80, 90, or 100% in a competition assay ... 2+ , ATP, and vanadate treatment to trap the ABC transporter in an outward conformation. In some embodiments, the molecule inhibits binding of G1365 to the ABC transporter by at least 50, 60, 70, 80, 90, or 100% in a competition assay.

[0200] VI.Use In some embodiments, the ABC transporter is a bacterial ABC transporter. For example, in some cases, bacterial ABC transporters are potential targets for antibiotics, for example, due to their relatively low sequence homology to mammalian ABC transporters. In some cases, the bacterial ABC transporter is an MsbA transporter from a bacterial species or pathogen. In some cases, the screening methods herein may be used to identify molecules that inhibit bacterial ABC transporters. Such molecules may have antibiotic activity. For example, the macrocycles G1118 and G1365 described herein were each found to inhibit the LPS transport activity of E. coli MsbA and also inhibit cell growth. Thus, the present disclosure also encompasses the use of molecules identified in the screenings herein against bacterial ABC transporters in treating infections in subjects, such as bacterial infections.

[0201] VII. Kit The present disclosure also includes kits that include reagents related to the screening methods herein. In some cases, the kits include a chimeric ABC transporter. In some cases, the kits include reagents used in the screening methods herein with or without a particular chimeric ABC transporter. In some cases, the kits include a parent (non-chimeric) ABC transporter.

[0202] In some embodiments, the kits herein may include a parent or chimeric ABC transporter attached to a matrix. In some embodiments, the kits herein may include an ABC transporter attached to a matrix particle, such as a bead. Such beads may have any shape, such as, for example, a flake or chip, a sphere, a pellet, etc. In some embodiments, such beads are streptavidin-coated beads, avidin-coated beads, or deglycosylated avidin-coated beads. In some embodiments, such beads are magnetic beads. The ABC transporter may or may not be pre-attached to a matrix. In some embodiments, a reagent is included to facilitate attachment of the ABC transporter to the bead or matrix, such as through a biotin-streptavidin or similar system.

[0203] In some embodiments, the kit may include reagents related to the screening methods herein. In some embodiments, the kit may include some or all of the reagents necessary to determine whether a test molecule binds to the periplasmic, extracellular, and / or luminal surface of an ABC transporter. The kit may include, for example, one or more detergents for solubilizing the ABC transporter and / or chimeric ABC transporter. Exemplary detergents or related molecules or systems for solubilizing the ABC transporter and / or chimeric ABC transporter include lauryl maltose neopentyl glycol (LMNG), in some embodiments, 0.02% LMNG, as well as dodecyl-β-D-maltoside (DDM), brij-35, glycol-diosgenin, digitonin, amphiphors, such as amphiphor A8-35, and lipid nanodiscs. The kit may include, for example, ATP, Mg2+, vanadate, and / or sodium orthovanadate, or other reagents that trap the ABC transporter in an outward conformation. The kit may include, for example, one or more wash buffers. In some embodiments, the wash buffer is Tris, MgCl 2 , LMNG, ATP, DTT, and / or sodium orthovanadate. In some embodiments, the kit may include one or more elution buffers. In some embodiments, the kit may include reagents for quantitative PCR. In some embodiments, the kit may include reagents for performing an ATPase assay on the ABC transporter in the presence or absence of a test molecule.

[0204] In some embodiments, the kit may include a test molecule or library of test molecules, e.g., peptides, small molecules, and / or antibodies. In some embodiments, the peptides in the kit may be macrocycles. In some embodiments, the kit may include test molecules that are binding fragments of peptides, small molecules, or antibodies. The kit may also include control molecules, such as positive controls that are known to bind to the periplasmic, extracellular, and / or luminal faces of a particular ABC transporter, or negative controls that do not bind at that location or that bind to the chimeric ABC transporter but not its parent ABC transporter.

[0205] The kit may include, for example, detection reagents for detecting binding. The kit may also include a control molecule and reagents for use with the control molecule.

[0206] In some embodiments, the kit may also include instructions for use. EXAMPLES

[0207] Below are examples of the methods and compositions of the present disclosure. These examples are not intended to limit the disclosure, but are for the purpose of illustrating it only, and it will be understood that various other embodiments may be practiced in light of the general description above.

[0208] Introduction MsbA is an essential ATP-binding cassette (ABC) transporter in Gram-negative bacteria involved in flipping lipopolysaccharide (LPS) for transport across the inner membrane (IM) and subsequently to the cell surface. Structural studies have revealed an alternate access mechanism for LPS transport by MsbA, but how substrate selectivity is achieved was unclear. Previous structures of apo-MsbA revealed an inward-facing conformation with LPS bound in a central space within the entrance, shielded from the large phospholipid bilayer. In this enclosed location, LPS is coordinated by a ring of conserved basic residues with all acyl chains trapped in a hydrophobic cavity.

[0209] An unbiased high-throughput screen was performed on purified WT EcMsbA. By monitoring MsbA ATPase activity, quinoline and benzophenone class inhibitors were found to trap the inward-facing conformation by targeting membrane-exposed binding sites. Although these efforts validated MsbA as a potential antibacterial target, small molecule series could not be advanced due to its high lipophilicity and poor physiochemical properties. Notably, all small molecule modulators of ABC transporters structurally characterized thus far also bind to the inward-facing state, raising the possibility that traditional drug discovery approaches are inherently biased towards these inward-facing states. Thus, whether any state of the transport cycle of MsbA is permissible for drug discovery remains a fundamental open question.

[0210] To overcome the challenges associated with targeting the hydrophobic membrane-embedded binding site in MsbA, a new inhibitor discovery strategy was devised. To discover inhibitors with improved physiochemical properties, a solvent-accessible region on the periplasmic face of MsbA was targeted. In particular, a key aspect of the MsbA transport cycle is the pronounced shift from an inward- to an outward-facing state, which exposes a large, solvent-accessible cleft to the periplasm (panel 1 of Figure 6). Under biochemical conditions, MsbA can be trapped in an outward-facing conformation that stabilizes this periplasmic cleft for potential inhibitor discovery. Id and Figure 3A. To bias selection toward the solvent-exposed periplasmic cleft, we utilized a counterselection strategy using MsbA chimeras in which the periplasmic faces of the transmembrane segments have been replaced en bloc with sequences derived from distantly related ABC transporters (Figures 3A and 9A-12B, as well as Example 7). After multiple rounds of screening with enrichment, we identified G1118 and G1365 as two macrocycles that bind MsbA. Both G1118 and G1365 were selective and state-dependent in their binding of MsbA and demonstrated potent biochemical and phenotypic activity. The high-resolution structures of these macrocycles in complex with MsbA reveal the molecular basis for their state-dependent inhibition and represent the first known antagonists targeting the outward cleft in ABC transporters. Thus, our strategy presents a template for the development of selective modulators of MsbA and, by extension, other challenging drug targets.

[0211] Example 1: Materials and methods for preparation and expression of chimeric MsbA transporter proteins a. Chimeric protein design The wild-type MsbA transporter sequence from Escherichia coli (E. coli) MsbA (EcMsbA; UniProtKB:P60752) was used in a BLAST search to identify homologous proteins in other Gram-negative bacterial species. By performing repeated BLAST searches, candidate MsbA transporters with an overall sequence identity to EcMsbA of approximately 40%, including within the periplasmic loop region, were selected for further consideration. Multiple sequence alignments and structural homology models (SWISSPROT) of candidate MsbA homologs were then manually analyzed to select candidates for subsequent chimeric construct engineering. Based on these analyses, candidate MsbA transporters derived from the following Gram-negative bacterial strains: Pseudomonas cyclotolerans (PpMsbA), Candidatus accumulibacter (CaMsbA), Jansinobacterium agaricidamnosum (JaMsbA), Thiomicrospira cyclica (TcMsbA), and Magnetospira strain-OH-2 (MqMsbA) were selected for chimerization against EcMsbA. To generate the chimeras, the corresponding sequences of periplasmic loop 1 (L1, EcMsbA residues Leu47 to Pro68), periplasmic loop 3 (L3, EcMsbA residues Met159 to Leu171), and periplasmic loop 5 (L5, EcMsbA residues Ala262 to Ile292) were simultaneously replaced with the equivalent regions of candidate MsbA homologs to generate chimeric constructs (Figure 10A).

[0212] Five chimeric constructs were synthesized (Genscript) and cloned into the pET-52b expression vector (EMD Millipore) modified for restriction-independent cloning. The chimeras were then overexpressed by auto-induced fermentation at 17 °C for 64 h using the E. coli host Rosetta2 (DE3; EMD Millipore). Cells were harvested and chimeras were purified as previously described for wild-type E. coli MsbA (EcMsbA; Ho et al., Nature 557(7704):196-201 (2018)). Candidate CaMsbA (MsbA Chim2 ), TcMsbA(MsbA Chim4 ), and MqMsbA (MsbAChim5 Chimeras generated using periplasmic sequences from MsbA could be readily expressed and purified from E. coli with recovery yields similar to WT MsbA. The final buffer for purified MsbA chimeric proteins was 20 mM Tris (pH 8.0), 100 mM NaCl, and 0.005% lauryl maltose neopentyl glycol (LMNG; wt / v). Protein aliquots were flash frozen and stored at -80°C.

[0213] b. Protein Expression and Purification Wild-type (WT) MsbA transporter from Escherichia coli (E. coli; EcMsbA), as well as periplasmic chimeras, were expressed and purified as previously described (Ho et al., 2018) using n-dodecyl-α-D-maltoside (αDDM, for cryo-EM structural studies and some biochemical assays as indicated; Anatrace), lauryl maltose neopentyl glycol (LMNG, for biochemical assays; Anatrace), or 3α-hydroxy-7α,12α-di-((O-β-D-maltosyl)-2-hydroxyethoxy)-cholane (FA3, for crystallography studies) as solubilizing detergents. WT MsbA proteins from E. cloacae (EnMsbA), K. pneumoniae (KpMsbA), and P. aeruginosa (PaMsbA) were similarly expressed and purified using LMNG as the solubilizing detergent. The final buffer for purified wild-type MsbA chimeric proteins was 20 mM Tris (pH 8.0), 100 mM NaCl, and 0.03% (wt / v) DDM or 0.02% LMNG (wt / v). Protein aliquots were flash frozen and stored at -80°C.

[0214] In some cases, a recognition motif (GLNDIFEAQKIEWHE) for the E. coli BirA biotin ligase was included between the amino-terminal FLAG affinity tag sequences, allowing efficient site-specific biotinylation (avidity) of purified MsbA. Evaluation of the ATPase activity and pharmacology of the chimeras revealed that these large sequence changes could be tolerated without significant loss of biochemical activity or obvious structural changes at the known antagonist receptor site. In particular, MsbA, which replaced the periplasmic face of EcMsbA with sequences from a candidate MsbA homolog derived from Magnetospira spirillum strain-OH-2, Chim5 still remained sensitive to both the quinoline and benzophenone classes of previously identified small molecule MsbA inhibitors.

[0215] For biochemical evaluation of E. coli MsbA point mutants, WT EcMsbA and Tyr87Ala / Trp91Ala (EcMsbA Y87A / W91A) and Lys95Ala / Arg238Ala (EcMsbA K95A / R238A) mutants were synthesized (Genscript) and cloned into a modified p15A plasmid (pLMG18) ( Storek et al., 2018 ) downstream of the Ptac promoter.

[0216] MsbA was overexpressed using the E. coli host BL21, and induction was performed with 1 mM IPTG at 37° C. for 3 hours.

[0217] Cells expressing MsbA were harvested and resuspended in 50 mM Tris (pH 8.0), 500 mM NaCl (Buffer A) supplemented with cOmplete™ protease inhibitor (Roche), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2 units / mL Benzonase nuclease (Sigma-Aldrich). After cell lysis by microfluidization, protein solubilization was performed by adding LMNG to 1% (wt / v) and gentle agitation overnight at 4°C. After centrifugation at 185,000 × g for 1 h, the clarified supernatant was gently mixed with anti-FLAG M2-agarose resin (Sigma) pre-equilibrated with buffer B (Buffer A supplemented with 0.005% LMNG (wt / v)) for 1-2 h at 4°C. The FLAG resin was collected by gravity flow, washed twice with 10 column volumes of buffer B, and eluted twice with 3 column volumes of buffer B supplemented with 0.2 mg / mL of FLAG peptide. Each wild-type or mutant MsbA protein was passed over a Superdex® 200 column (GE Healthcare) or a Superose 6 Increase column (Cytiva) in 20 mM Tris (pH 8.0), 100 mM NaCl, and 0.005% LMNG (wt / v). Peak fractions containing MsbA were pooled and concentrated to 5-10 mg / mL using a Vivaspin® centrifuge device (50K molecular weight cutoff). Protein aliquots were flash frozen and stored at -80°C.

[0218] c. Reconstitution into amphipol Where indicated for use with amphipol-integrated MsbA, purified MsbA in detergent was adjusted to 1 mg / mL and reconstituted in A8-35 amphipol (Zoonens and Popot, 2014) (Anatrace) as previously described (Ho et al., 2018). The final buffer for purified amphipol-integrated MsbA protein was 20 mM Tris (pH 8.0) and 100 mM NaCl. Protein aliquots were flash frozen and stored at -80 °C.

[0219] d.Sequence conservation analysis An MsbA sequence (taxid:543) from the Enterobacteriaceae family of Gram-negative bacteria was identified using a BLAST search against the refseq_select database using EcMsbA as the query sequence (Uniprot ID:P60752). A total of 118 sequences from this search annotated as MsbA homologs were aligned using the Constraint-based Multiple Alignment Tool (COBALT) (Papadopoulos and Agarwala, 2007). The multiple sequence alignment was loaded into ChimeraX and sequence conservation was determined using the sum of pairs method in AL2CO (Pei and Grishin, 2001).

[0220] Example 2: Screening materials and methods to identify MsbA-binding macrocycles a. Macrocyclic peptide library design Counterselection with chimeric MsbA proteins was used to test peptide macrocycles and identify molecules that bind to the periplasmic face of EcMsbA (Figure 3A). A thioether-macrocyclic peptide library was constructed by using N-chloroacetyl L-phenylalanine (ClAc-F) as an initiator in a genetically reprogrammed in vitro translation system (Kashiwagi et al., 2013). The genetic code was engineered by adding N-methyl-L-phenylalanine (MeF) and (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid (Bph) in addition to all 20 natural amino acids except cysteine. After in vitro translation, a thioether bond spontaneously formed between the N-terminal ClAc group of the initiator L-phenylalanine residue and the sulfhydryl group of the downstream cysteine ​​residue to generate macrocyclic peptides.

[0221] b. Selection of periplasmic MsbA-binding molecules Affinity selection of macrocyclic peptides that bind to MsbA was performed using site-specific biotinylated EcMsbA solubilized in 0.02% LMNG and trapped in an outward conformation by including 50 μM ATP, 10 mM Mg2+, and 200 μM sodium orthovanadate throughout the selection. Briefly, 10 μM of the mRNA library was hybridized with the peptide-linker (11 μM) for 3 min at room temperature (RT). The mRNA library was translated for 30 min at 37°C in a genetically reprogrammed in vitro translation system to generate the peptide-mRNA fusion library (Goto et al., 2011; Kawakami et al., 2013). Each reaction contained 2 μM of mRNA-peptide-linker conjugate, 12.5 μM of initiator tRNA (tRNAfMet aminoacylated with ClAc-L-Phe), and 25 μM of each elongator tRNA aminoacylated with the specified non-canonical / canonical amino acid. In the first round of selection, translation was carried out at a 20 μL scale. After translation, the reaction was quenched with 17 mM EDTA. The product was subsequently reverse transcribed for 30 min at 42 °C using RNase H-free reverse transcriptase (Promega), and the buffer was changed to vanadate buffer: 50 mM Tris (pH 7.5), 10 mM MgCl 2 , 0.02% LMNG (wt / v), 50 uM ATP, 1 mM DTT and 200 uM sodium orthovanadate.

[0222] For affinity selection, the peptide-mRNA / cDNA solution was incubated with 250 nM biotinylated EcMsbA and 500 nM EcMsbAChim5 as a sink for 60 min at 4°C. Streptavidin-coated beads (Dynabeads™ M-280 Streptavidin, ThermoFisher Scientific) were further added and incubated for 10 min to isolate macrocycles that bind to the periplasmic face of EcMsbA. The cDNA was eluted from the beads by washing them three times with cold vanadate buffer and heating to 95°C for 5 min, and fractionated recovery from the affinity selection step was assessed by quantitative PCR using Sybr Green I on a LightCycler™ thermal cycler (Roche). After six rounds of affinity maturation, off-rate selection was further repeated two times by increasing the washing stringency before elution to identify high affinity binders. The final enriched cDNA was sequenced using a MiSeq next-generation sequencer (Illumina).

[0223] c.Mutation scan of the G1118 and G1365 macrocycles For the mutation scans of the G1118 (Table 4 below) and G1365 (Table 6 below) macrocycles, site-saturated DNA libraries were constructed based on these parental sequences by pooling multiple DNA templates, each containing a single "NNU" degenerate codon at a different position, such that after translation, every single amino acid change relative to the parental sequence was sampled. The genetic code was designed by adding Pro, Lys, Ala, Dopa (L-3,4-dihydroxyphenylalanine), and MeG (N-methyl-L-glycine) in addition to the amino acids used in the parent peptides. After in vitro translation, one round of affinity selection was independently performed using the two macrocycle libraries as described above. The input and recovered output DNA pools were subjected to deep sequencing by NGS. The enrichment factor for each single variant was calculated as follows: NGS frequency in the output pool divided by NGS frequency in the input pool, normalized by the parental value.

[0224] Example 3: Materials and methods for characterization of MsbA enzyme variants and strains A plasmid The following plasmids (Table 1) were used for E. coli phenotypic studies. TIFF2024519413000002.tif75170

[0225] b. MsbA ATPase assay The ATPase activity of MsbA was measured using the Transcreener ADP2 FP Assay (BellBrook Labs). IC 50To determine the ATPase activity, compounds were incubated with 2x MsbA enzyme solution for 10 minutes, then 2x ATP solution was added to initiate the ATPase reaction. The final conditions for the reaction were MsbA enzyme, 50 μM ATP in 50 mM Tris (pH 7.5), 10 mM MgCl2, 1% glycerol, 0.1% bovine gamma globulin, 1 mM dithiothreitol (DTT), 0.007% Brij-35, and 0.5% dimethyl sulfoxide (DMSO). The following concentrations were used for each MsbA variant: 5 nM EcMsbA, EnMsbA, KpMsbA and 80 nM PaMsbA refolded in amphipol, 15 nM EcMsbA and EcMsbA-chim5 in LMNG detergent, 22 nM EcMsbA and 27 nM EcMsbA-12G7 complex in DDM detergent. Enzyme reactions were incubated for 1 h at room temperature and then quenched by adding Transcreener Detection buffer. IC 50 was determined by fitting the inhibition dose-response curves with a nonlinear four-parameter inhibition model (GraphPad Prism).

[0226] c. Intracellular IC 50 Decision Intracellular IC of various strains 50 (Table 2) was determined. TIFF2024519413000003.tif72170

[0227] Aliquots of 200 nL of eight two-fold dilutions of each compound were dispensed in duplicate into 384-well plates using an Echo Liquid Handler (Labcyte). Strains were grown overnight for 16 h in cation-adjusted Mueller-Hinton liquid medium containing 0.002% Tween, washed with PBS, and cultured at OD in cation-adjusted Mueller-Hinton liquid medium containing 0.002% Tween and 10% glycerol. 600Cells were resuspended at 0.05% CO2 = 1 and frozen in 100 ml aliquots. For each experiment, an aliquot was thawed on ice and diluted 1:1000 in cation-adjusted Mueller-Hinton liquid medium containing 0.002% Tween. 10 μL cells / well were added to 384-well plates containing compounds. Plates were incubated at 37°C for 6 hours, then 10 μL of BacTiter-Glo reagent (Promega) was added to each well and luminescence was measured. IC was determined using Genedata Screener software. 50 was calculated.

[0228] d. Time course of growth of E. coli complemented with the MsbA allele Cultures of E. coli MG1655 msbA-cKO lptD(imp4213) carrying pLMG18 vectors expressing WT or mutant alleles of E. coli MsbA were grown overnight at 37°C in 5 mL of LB containing 10 μg / ml chloramphenicol and 2% arabinose to induce expression of WT E. coli MsbA from the chromosome. The overnight cultures were pelleted, washed with 5 ml of phosphate-buffered saline, and incubated in LB at OD 600 The cultures were then resuspended at OD = 1. Each culture was then diluted 1:100 into 5 mL LB, 5 mL LB + 2% arabinose. All cultures were grown on a roller drum at 37°C and OD600 was measured every hour. Growth curves were performed in triplicate on different days.

[0229] e. Western blot Cultures of E. coli MG1655 msbA-cKO lptD(imp4213) carrying pLMG18 vectors expressing WT or mutant alleles of E. coli MsbA were grown overnight at 37°C in 5 mL of LB containing 10 μg / ml chloramphenicol and 2% arabinose to induce expression of WT E. coli MsbA from the chromosome. A control strain containing no plasmid was grown under the same conditions but without chloramphenicol. Overnight cultures were diluted 1:100 in 15 mL of the same medium in a 50 mL conical tube and grown with shaking at 37°C for 3 hours. These cultures were pelleted, washed with 15 mL of PBS, and diluted to OD in LB. 600 The cultures were then resuspended at OD=1. Each culture was then diluted 1:100 in 20 mL of LB for strains expressing the E. coli MsbA allele or strains without the plasmid control, and in 20 mL of LB + 1 mM IPTG for strains expressing the A. baumannii MSbA allele. 7.5 mL of each of the cells at OD=1 were pelleted and lysed as 2% arabinose samples. The diluted cultures were grown at 37°C for 3 hours and then pelleted and lysed.

[0230] To generate lysates, each sample was resuspended in 500 μl of MSD lysis buffer (150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1% Triton X-100) supplemented with 15 mg / mL lysozyme and incubated at 37 °C for 30 min. Lysates were pelleted at top speed in a microcentrifuge for 10 min at 4 °C and the supernatant transferred to a clean tube. Samples were lysed in MES buffer on 20-well 4-12% Bis-Tris midi gels (BioRad) and loading was assessed at OD using GroEL as a loading control. 600Western blots were transferred to nitrocellulose using the iBlot system (Thermo Fisher Scientific). Membranes were blocked with Odyssey TBS blocking buffer (Li-Cor, #927-50000) and detected with anti-FLAG M2 (Sigma, #F3165) at 1:10000 plus anti-GroEL (Enzo, #ADI-SPS-875) at 1:10000, followed by IRDye 800CW anti-mouse and IRDye 680RD anti-rabbit at 1:10000 each (Li-Cor, #925-32210 and #926-68071).

[0231] f.MIC assay MICs of cultures were determined in cation-adjusted Mueller-Hinton liquid medium containing 0.002% Tween-80 as previously described ( Ho et al., 2018 ).

[0232] g. Isolation of PD-1365-resistant mutants MG1655 lptD (imp4213) was cultured at an OD of 0.005 in a 96-well plate containing 100 μL / well of Mueller-Hinton liquid medium + 0.002% Tween-80 + 50 μM G1365·I2,Dopa3 (4× MIC). 600 The resistant strain was inoculated with 100 μM MsbA and grown at 37°C for 2 days. G1365·I2,Dopa3 is a close analog of G1365 with two amino acid substitutions (Val2Ile and Tyr3Dopa; Figure 14A). Cultures from two wells that showed growth were streaked onto LB agar plates containing 50 μM G1365·I2,Dopa3, and only one culture produced colonies on the selection plate. The MsbA ORF from this resistant strain was PCR amplified and sequenced, revealing a point mutation resulting in an amino acid change from aspartic acid to asparagine at residue 252. MIC assays against the parental strain and the msbA(D252N) mutant confirmed that the MIC of G1365·I2,Dopa3 increased from 12.5 μM in the parental strain to 100 μM in MG1655 lptD(imp4213)msbA(D252N).

[0233] Example 4: Electron Microscopy Materials and Methods Electron microscopy of E. coli CFT073 lptD(imp4213) or CFT073 lptD(imp4213)msbA-cond-ko cells rescued with pLMG18 plasmids expressing the indicated E. coli MsbA mutants was performed as previously described (HO et al., 2018). Where indicated, the use of macrocyclic or small molecule inhibitors of MsbA was used, overnight cultures of CFT073 lptD(imp4213) grown in LB were diluted 1:100 in LB and grown to log phase for 2.5 h at 37 °C, then pelleted, washed twice with PBS, and incubated at OD in LB. 600 = 0.1. 50 μl of a 10 mM stock of the indicated inhibitor was then added to 5 mL of resuspended CFT073 lptD(imp4213) and the culture was incubated for 3 h at 37 °C. Cells were pelleted, washed, and fixed and processed for transmission electron microscopy as previously described (HO et al., 2018).

[0234] Example 5: Synthesis of macrocyclic compounds a. Synthesis of benzophenone (G758) TIFF2024519413000004.tif97170

[0235] Step 1: tert-Butyl 2-(4-methylpiperidin-1-yl)acetate.

[0236] A mixture of 4-methylpiperidine (25.0 g, 252.1 mmol), potassium carbonate (104.5 g, 756.3 mmol) and tert-butyl bromoacetate (49.2 g, 252.1 mmol) in acetonitrile (400 mL) was stirred at 25° C. for 12 h and concentrated under reduced pressure. The residue was purified by column chromatography eluting with 0% to 30% ethyl acetate in petroleum ether to give tert-butyl 2-(4-methyl-1-piperidyl)acetate (45.0 g, 189.9 mmol, 75.3% yield) as a pale yellow solid. TIFF2024519413000005.tif14170

[0237] Step 2: 2-(4-Methylpiperidin-1-yl)acetic acid.

[0238] A solution of tert-butyl 2-(4-methyl-1-piperidyl)acetate (45.0 g, 211.0 mmol) and hydrochloric acid (4N, 369.2 mL, 1476.7 mmol) in acetic acid (150 mL) was stirred at 25° C. for 14 h and concentrated under reduced pressure to give the crude product, which was used directly without further purification. 1H NMR (400 MHz, DMSO-d6): δ 4.07 ppm (s, 2H), 3.42 (br. s., 2H), 3.02 (br. s., 2H), 1.77 (d, J = 12.8 Hz, 2H), 1.59 (br. s., 1H), 1.51 - 1.36 (m, 2H), 0.92 (d, J = 6.8 Hz, 3H). TIFF2024519413000006.tif14170

[0239] Step 3: 2-(4-Methylpiperidin-1-yl)acetyl chloride.

[0240] To a mixture of 2-(4-methyl-1-piperidyl)acetic acid (1.0 g, 6.7 mmol) in dichloromethane (20 mL) was added oxalyl chloride (1.4 mL, 15.9 mmol) and 2 drops of N-N-dimethylformamide. The resulting mixture was stirred at 20° C. for 1.5 h and concentrated under reduced pressure to give crude 2-(4-methyl-1-piperidyl)acetyl chloride (1.0 g, 5.7 mmol, 89.5% yield). The crude was used directly in the next step without further purification. TIFF2024519413000007.tif22170

[0241] Step 4: tert-Butyl (2-bromo-6-chloropyridin-3-yl)carbamate.

[0242] A solution of 2-bromo-6-chloro-pyridin-3-amine (2.5 g, 12.1 mmol), di-tert-butyl dicarbonate (2.6 g, 12.1 mmol), 4-dimethylaminopyridine (294.4 mg, 2.4 mmol) and triethylamine (2438.78 mg, 24.1 mmol) in dichloromethane (20 mL) was stirred at 30 °C for 12 h and diluted with dichloromethane (50 mL). The solution was washed with water (2 x 15 mL), brine (15 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography eluting with 0% to 30% ethyl acetate in petroleum ether to give tert-butyl N-(2-bromo-6-chloro-3-pyridyl)carbamate (1.3 g, 4.2 mmol, 35.1% yield) as a white solid. TIFF2024519413000008.tif35170

[0243] Step 5: tert-Butyl (6-chloro-2-((3-fluoropyridin-2-yl)(hydroxy)methyl)pyridin-3-yl)carbamate.

[0244] To a solution of tert-butyl N-(2-bromo-6-chloro-3-pyridyl)carbamate (1.0 g, 3.25 mmol) in tetrahydrofuran (20 mL) was added butyllithium (2.5 M in hexanes, 1.6 mL, 3.90 mmol) at −78° C. The mixture was stirred at −78° C. for 1 h, and then 3-fluoro-2-formylpyridine (610.1 mg, 4.88 mmol) was added. The resulting mixture was stirred at −78° C. for 4 h and quenched by adding saturated ammonium chloride (10 mL). The solution was diluted with ethyl acetate (50 mL), washed with water (2×10 mL), brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography eluting with 0% to 40% ethyl acetate in petroleum ether to give tert-butyl N-[6-chloro-2- [(3-fluoro-2-pyridyl)-hydroxy-methyl]-3-pyridyl]carbamate (550.0 mg, 1.55 mmol, 47.8% yield) was obtained as a yellow oil. TIFF2024519413000009.tif36170

[0245] Step 6: tert-Butyl (6-chloro-2-(3-fluoropicolinoyl)pyridin-3-yl)carbamate

[0246] tert-Butyl N-[6-chloro-2-[(3-fluoro-2-pyridyl)-hydroxy-methyl]-3-pyridyl] in dichloromethane (15 mL) A mixture of carbamate (460.0 mg, 1.30 mmol) and Dess-Martin periodinane (2.2 g, 5.20 mmol) was stirred at 20° C. for 3 h. The reaction mixture was diluted with dichloromethane (20 mL), washed with water (2×10 mL), brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography eluting with 0% to 30% ethyl acetate in petroleum ether to give tert-butyl N-[6-chloro-2-(3-fluoropyridine-2-carbonyl)-3-pyridyl]carbamate (300.0 mg, 0.85 mmol, 65.6% yield) as a white solid. TIFF2024519413000010.tif33170

[0247] Step 7: tert-Butyl (6-cyano-2-(3-fluoropicolinoyl)pyridin-3-yl)carbamate

[0248] A mixture of tert-butyl N-[6-chloro-2-(3-fluoropyridine-2-carbonyl)-3-pyridyl]carbamate (230.0 mg, 0.65 mmol), tris(dibenzylideneacetone)dipalladium (59.9 mg, 0.07 mmol), zinc cyanide (383.9 mg, 3.27 mmol) and cyclopentyl(diphenyl)phosphane iron (72.5 mg, 0.13 mmol) in N,N-dimethylacetamide (10 mL) was heated at 90 °C for 2 h under microwave conditions and concentrated under reduced pressure. The residue was purified by column chromatography eluting with 0% to 50% ethyl acetate in petroleum ether to give tert-butyl N-[6-cyano-2-(3-fluoropyridine-2-carbonyl)-3-pyridyl]carbamate (150.0 mg, 0.44 mmol, 67.0% yield) as a white solid. TIFF2024519413000011.tif34170

[0249] Step 8: 5-amino-6-(3-fluoropicolinoyl)picolinonitrile trifluoroacetate

[0250] A solution of tert-butyl N-[6-cyano-2-(3-fluoropyridine-2-carbonyl)-3-pyridyl]carbamate (120.0 mg, 0.35 mmol) and trifluoroacetic acid (4.0 mL, 51.22 mmol) in dichloromethane (16 mL) was stirred at 15° C. for 1 h and concentrated under reduced pressure to give crude 5-amino-6-(3-fluoropyridine-2-carbonyl)pyridine-2-carbonitrile trifluoroacetate (84.0 mg, 0.25 mmol, 71.4% yield) as a yellow oil. TIFF2024519413000012.tif34170

[0251] Step 9: N-(6-cyano-2-(3-fluoropicolinoyl)pyridin-3-yl)-2-(4-methylpiperidin-1-yl)acetamide

[0252] A mixture of 2-(4-methyl-1-piperidyl)acetyl chloride (121.8 mg, 0.69 mmol) and 5-amino-6-(3-fluoropyridine-2-carbonyl)pyridine-2-carbonitrile (84.0 mg, 0.35 mmol) in tetrahydrofuran (5 mL) was stirred at 60° C. for 2 h and concentrated under reduced pressure. The residue was purified by reverse phase chromatography (28-58 acetonitrile in water / 0.05% NH4OH) to give N-[6-cyano-2-(3-fluoropyridine-2-carbonyl)-3-pyridyl]-2-(4-methyl-1-piperidyl)acetamide (9.40 mg, 0.02 mmol, 6.8% yield) as a white solid. 1H NMR (400 MHz, methanol-d4): δ 9.34 ppm (d, J = 8.8 Hz, 1H), 8.53 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 4.8 Hz, 1H), 7.85 - 7.78 (m, 1H), 7.72 - 7.63 (m, 1H), 3.23 (s, 2H), 2.95 - 2.85 (m, 2H), 2.35 - 2.23 (m, 2H), 1.68 - 1.49 (m, 4H), 1.45 - 1.36 (m, 1H), 0.96 (d, J = 6.4 Hz, 3H). LCMS (m / z): [M+H]+ Calculated value: C20H20FN5O2, 381.16; Found value: 382.1.

[0253] b. Synthesis of G1118, G1365, and derivatives The thioether macrocyclic peptides were synthesized using standard Fmoc solid-phase peptide synthesis (SPPS). After coupling of all amino acids, the deprotected N-terminus was chloroacetylated on-resin, followed by global deprotection using a trifluoroacetic acid (TFA) deprotection cocktail. The peptides were then precipitated from the deprotection solution by adding a greater than 10-fold excess of diethyl ether. The crude peptide pellet was then dissolved and re-pelletized three times using diethyl ether. After the final wash, the pellet was left to dry, then resuspended in DMSO, followed by the addition of triethylamine for intramolecular cyclization via the formation of a thioether bond between the cysteine ​​thiol and the N-terminal chloroacetyl group. Upon completion of cyclization, the reaction was quenched with AcOH and the cyclic peptides were purified using standard reversed-phase HPLC methods. The molecular masses were confirmed by single quadrupole LC / MS (LCMS-2020 system, Shimadzu).

[0254] c. Synthesis of other 14-mer peptides Additional 14-mer peptides were designed according to the following formula (see, e.g., FIG. 4E for a diagrammatic representation of one particular variant): ClacF-X1-X2-L-X3-X4-D-X5-X6-X7-X8-MeF-VC, where i. X1 is W, V, or Y; ii. X2 is W or Y; iii. X3 is W or Y; iv. X4 is S, D, V, or H; v. X5 is N, where N is selected from any natural amino acid other than C or an unnatural amino acid selected from Bph ((S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid), Dopa (L-3,4-dihydroxyphenylalanine), MeF (N-methyl-L-phenylalanine), and MeG (N-methyl-L-glycine); vi. X6 is Y, K, A, S, D, R, or V; vii. X7 is W, Y, or Bph; viii. X8 is W or Y; Optionally, the peptide further comprises a G residue following the C residue at the C-terminus, where ClacF is N-chloroacetyl-L-phenylalanine, Bph is (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid, Dopa is L-3,4-dihydroxyphenylalanine, MeF is N-methyl-L-phenylalanine and MeG is N-methyl-L-glycine. Eleven different molecules according to this formula were also tested for their inhibitory effect on MsbA ATPase activity in the assay described in Example 3 above, with IC's ranging from 0.2 to 0.8 μM. 50 The minimum inhibitory concentration (MIC) for inhibition of E. coli MsbA (EcMsbA) was found to be greater than 100 μM, and the EC 50 ranged from about 2 to about 8 μM for all but one of the molecules tested (exceeding 100 μM for the last molecule tested). Overall, these data indicate that the macrocyclic peptide G1118 and related peptides according to the formula above are generally capable of inhibiting MsbA and cellular activities associated with MsbA.

[0255] Example 6: Materials and methods for protein structure determination A. Protein crystallization The inhibitor G758 was added to MsbA-chim5 at 10 mg ml / L, purified in FA3 to a final concentration of 1 mM, and incubated on ice for 1 h. No exogenous LPS was added at any time. The G758-LPS-MsbA-chim5 complex was crystallized at 19°C using sitting-drop vapor diffusion by mixing the complex with mother liquor (150 mM NaF, 13% PEG 3350, 100 mM HEPES, pH 7.0) in a 1:1 (v / v) ratio. After reaching full size for 1 week, the crystals were dehydrated by first soaking in 10% ethylene glycol, 15% PEG 3350, 100 mM HEPES (pH 7.0) for 30 min, then in 20% ethylene glycol, 20% PEG 3350, 100 mM HEPES (pH 7.0) for an additional 30 min, before being flash-frozen in liquid nitrogen to improve diffraction.

[0256] b. Structure determination, refinement and analysis Diffraction data were collected at 100K using beamline 17ID at the Advanced Light Source. X-ray diffraction data were integrated and scaled using autoPROC (Vonrhein et al., 2011) with anisotropy correction performed with STARANISO (Tickle et al. (2020) Global Phasing Ltd; staraniso.globalphasing.org / cgi-bin / staraniso.cgi). The structure was determined by molecular replacement with PHENIX (Adams et al., 2010) using one MsbA subunit from PDB 6BPL as a search model. Manual adjustments to the initial solution were performed through rigid-body motion in Coot (Emsley et al., 2010). Iterative rounds of refinement and model building in Coot were guided by inspection of omit maps. In combination with the use of omit maps in the early stages of refinement, the chimera 5 sequence was replaced in the model with the WT E. coli MsbA sequence. Strict shape and secondary structure restraints were applied throughout the refinement to maintain strict stereochemistry, but non-crystallographic symmetry restraints were not applied until the end. Strong omit densities were evident very early for G758, LPS (central space within the entrance), and unassigned surfactant-like densities were modeled only very late in the refinement. The geometry of the final G758-LPS-MsbA-chim5 model was assessed using MolProbity (Chen et al., 2010). All structural diagrams were generated using PyMol software (The PyMOL Molecular Graphics System v.1.8 (Schrodinger, LLC., 2015)).

[0257] c. Cryo-electron microscopy (cryo-EM) sample preparation and data collection Purified MsbA was mixed 1:1 (w / w) with Fab 12G7. The MsbA-12G7 complex was separated from free Fab by size-exclusion chromatography using a running buffer consisting of 20 mM Tris (pH 8), 100 mM NaCl, and 0.03% αDDM. The MsbA-12G7 complex was purified by HPLC using 20 mM MgCl 2 The vanadate-trapped samples were incubated with 15 mM ATP and 3.3 mM sodium vanadate for 1 h on ice. The vanadate-trapped samples were resuspended in 20 mM Tris (pH 8), 100 mM NaCl, 0.03% αDDM, 20 mM MgCl 2 The eluates were further purified by size-exclusion chromatography with a running buffer of 15 mM ATP and 1 mM sodium vanadate. Peak fractions were concentrated to 2 mg / mL and incubated with 37 μM G1118 or 67 μM G1365 derivatives (Figure 14A) and 0.007% Brij-35. After 30 min incubation with the ligands on ice, 3.5 μL of each sample was frozen on glow-discharged Ultrafoil 2 / 2 200 mesh grids using a Vitrobot at 4 °C and 100% humidity. Cryo-EM data were collected on a Titan Krios (ThermoFisher Scientific, Waltham, MA) operated at 300 kV equipped with a BioQuantum energy filter with a slit width of 20 eV and a K2 Summit electron direct detection camera (Gatan, Inc, Pleasanton, CA). Images were recorded at a nominal magnification of 165,000x with a physical pixel size of 0.849 Å. Each image stack had a frame time of 0.25 s and a resolution of approximately 40 e - / Å 2 The data were collected at a fixed defocus range of 1.0-2.0 µm.

[0258] d. Cryo-EM data processing for G1118 Cryo-EM data for G1118 were processed using cisTEM (Grant et al., 2018) (Figure 15A-E). A total of 9,999 movies were full-frame motion corrected using cisTEM and binned to a pixel size of 1.3 Å. Contrast transfer function parameters were fitted with CTFFIND-4 using the 30-4.0 Å bands of the spectrum, and 5,984 micrographs with CTE resolution better than 8 Å were selected for further processing. A total of 1,206,819 particles were picked using cisTEM. An ab initio model was generated using cisTEM with well-aligned particles in the 2D class average. The complete data set was refined against an ab initio model with C1 symmetry. Angle assignments from the consensus refinement were used for alignment-free 3D classification. 410,386 particles from the best class were further refined manually by iteratively increasing the resolution limit used for refinement. Densities for surfactant micelles and Fabs were filtered to 20 Å by using a mask around MsbA. With a maximum frequency of 4 Å used for alignment, refinement converged to a resolution of 3.0 Å (Fourier shell correlation (FSC) = 0.143 determined in cisTEM). For model building and figure production, maps were sharpened in cisTEM with the following parameters: flattening from a resolution of 10 Å, application of a pre-cutoff B-factor of -90 Å2 from the origin of reciprocal space, and application of a figure of merit filter.

[0259] Cryo-EM data processing for e.G1365 Cryo-EM data were processed using a combination of cisTEM (Grant et al., 2018) and RELION (Scheres, 2012) (Figure 17A-E). A total of 10,273 micrographs were collected for G6671 and 17,575 for G3081. Each dataset was full-frame motion corrected using cisTEM and binned to a pixel size of 1.132 Å. Contrast transfer function parameters were fitted with CTFFIND-4 using the 30-4.0 Å band of the spectrum. A total of 937,971 particles for G6671 and 993,151 particles for G3081 were picked using cisTEM. Each dataset was subjected individually to a series of 2D and 3D classifications in relion. Precise placement of the macrocycles was not possible due to the limited resolution when processing each dataset individually. To increase the average power, 218,877 particles from the G3081 data set and 222,975 particles from the G6671 data set were merged and the refinement was used for a series of automatic refinements in cisTEM. The density outside the MsbA mask was filtered to 20 Å. For model building of MsbA, an additional round of local refinement was performed using a high resolution limit of 4 Å, yielding a 3.1 Å map (Fourier shell correlation (FSC) = 0.143 determined in cisTEM). An additional focused refinement was performed using a mask containing only the TMD domain of MsbA with a high resolution limit of 4.5 Å to aid in macrocycle model building, yielding a map of 3.2 Å with good density for G1365. For model building and figure production, maps were sharpened in cisTEM with the following parameters: smoothing from 8 Å resolution, application of a pre-cutoff B-factor of either -90 Å2 for MsbA maps or -120 Å2 for TMD-focused refinement, and application of a figure of merit filter. Electrostatic maps were calculated in PyMol using APBS.

[0260] f. Model refinement A homology model of E. coli MsbA was generated based on the AMPPNP-bound structure of S. typhimurium MsbA (PDB: 3B60) using SWISS-MODEL (Waterhouse et al., 2018). The resulting model was fitted as a rigid body to the G1118 cryo-EM map. The model was refined through manual tuning in Coot (Emsley and Cowtan, 2004) and ChimeraX (Goddard et al., 2018; Pettersen et al., 2021) with ISOLDE (Croll, 2018), followed by real-space refinement in phenix.real_space_refinement (Afonine et al., 2018). The macrocycle was parameterized using Corina (Molecular Networks GmbH) and manually fitted to the density. MsbA coordinates from the G1118 structure were then used as the initial model for G1365 prior to iterative model building. Models were validated using phenix.validation_cryoEM with built-in MolProbity scoring. Figures were generated using UCSF ChimeraX (Goddard et al., 2018; Pettersen et al., 2021).

[0261] Example 7: Discovery and characterization of macrocyclic MsbA inhibitors a. Comparison of molecules selected with and without counterselection and outward conformation trapping (preliminary screening) To evaluate the ability of different selection schemes to identify 14-mer macrocycles that selectively bind to the periplasmic face of MsbA, the results of several selection schemes were compared by ELISA assay (Table 3). A number of peptides were used to demonstrate specific binding to MsbA. The peptides show both vanadate-dependent binding and no reduction in recovery in the presence of the chimera. TIFF2024519413000013.tif255166

[0262] As shown in the top, left side of Table 3, (a) selections performed in solution, in the presence of vanadate to trap EcMsbA in its outward conformation, and with a counterscreen using chimeric MsbA, led to the identification of a group of macrocycles at frequencies of about 26% to 2% (Table 3; first six lines of the %freq column). Similar sets of specific macrocycles were identified when the screens were performed (b) in solution with vanadate but without counterselection, (c) on beads without vanadate but including counterselection, and (d) on beads with vanadate but without counterselection. (See the left-most column and "%freq" column of Table 3.) Each of the identified macrocycles was then subjected to ELISA assays in the presence and absence of vanadate to trap the EcMsbA protein in its outward conformation and tested for binding to EcMsbA, to the unrelated membrane protein OprF, and to streptavidin beads (SA). The results showed that (a) in solution, molecules selected with vanadate using a counterscreen bound well to EcMsbA in the presence of vanadate but poorly without vanadate ("+ / - ratio" column, first six lines) and did not bind well to either OprF alone or to streptavidin beads alone under either condition; The majority of the molecules in the screens identified under conditions (b), (c), and (d) bound to a similar extent to EcMsbA whether or not they were trapped in an outward conformation with vanadate, indicating that trapping proteins in an outward conformation and using chimeric protein counterscreens are both more effective in identifying molecules that bind to the periplasmic face of EcMsbA. Competitive ELISA experiments were also performed to test the binding of the identified molecules to EcMsbA on beads in the presence and absence of chimeric EcMsbA protein as a binding competitor. A similar extent of binding in the presence and absence of EcMsbA, thus a ratio of 1.0 or greater, indicates that the molecule is selective for EcMsbA and that the chimera does not act as a competitor for binding to EcMsbA.(See the right-hand column of Table 3.) Molecules identified under the screening conditions of (a) had similar degrees of binding in the presence and absence of the chimera, as indicated by ratios of 1.0 or greater in the assay, consistent with their binding to the periplasmic face of EcMsbA.

[0263] b. Site-directed discovery of MsbA inhibitor G1118 To enrich for molecules that specifically target the aqueous periplasmic cleft of E. coli MsbA (EcMsbA), structure-based homology modeling was first used to evaluate the periplasmic face of the transmembrane segment using iterative BLAST searches (Figures 9A-12B), leading to the engineering of five unique chimeric constructs that collectively replace each with the sequence of a putative MsbA transporter protein from a distantly related Gram-negative bacterium. Of the five chimeras constructed, chimera 2, chimera 4, and chimera 5 (MsbA-chim5) were stably expressed and purified from E. coli (Figures 11A-B). Evaluation of the ATPase activity and pharmacology of the chimeras revealed that these large sequence changes could be tolerated while maintaining biochemical activity (Figure 11C). Notably, MsbA-chim5, in which the entire periplasmic face of EcMsbA was replaced with sequences derived from the MsbA homolog from Magnetospirillum, still maintained comparable ATPase activity and exhibited sensitivity to both the quinoline (G247 and G907) and benzophenone (G758) classes of small molecule inhibitors of previously identified wild-type (WT) EcMsbA (Figures S11D-E and S12A-B).

[0264] Next, selection of macrocycles against WT EcMsbA transporter trapped in a purified outward conformation was followed by counter-selection using a similarly trapped MsbA chimera in which the periplasmic face of the transmembrane segment had been replaced en bloc with the sequence of a putative MsbA transporter protein from a distantly related Gram-negative bacterium (Figures 3A and 9A-12B). Selections used an in vitro translated peptide macrocycle mRNA display 12-14mer library containing 10 (Karow and Georgopoulos, 1993) molecules incorporating natural and unnatural amino acids (Example 2). After eight rounds of selection and counter-selection, the 14-mer macrocycle, G1118, showed significant enrichment with WT EcMsbA (Figure 3B). G1118 exhibited a 50% inhibitory concentration (IC) of approximately 110 nM. 50 ) was found to affect the ATPase activity of EcMsbA with less than 30-fold lower potency (IC) against purified MsbA-chim5, consistent with targeting to the periplasmic cleft (Fig. 3C). 50 G1118 also inhibited the related Enterobacter cloacae and Klebsiella pneumoniae MsbA transporters with IC values ​​of ∼240 nM and ∼120 nM, respectively. 50 (Fig. 13A-D), but had only weak activity against P. aeruginosa MsbA, which has lower sequence identity (approximately 39%) with EcMsbA (IC 50 = 3.1 μM). E. coli cells treated with G1118 elicited a unique inner membrane elaboration phenotype reflecting bacteria depleted of MsbA (Doerrler et al., 2001), consistent with pharmacological inhibition of MsbA (Figure 3E). Due to the relatively large size of G1118 (2,085 Da), cellular activity was restricted to cells containing the lptD(imp4213) allele that confers an outer membrane (OM) permeability defect, with a measured minimum inhibitory concentration (MIC) of approximately 210 μg / mL (Figure 13B). Further characterization of G1118 is discussed in Example 8 below.

[0265] c. Site-directed discovery of MsbA inhibitor G1365 To discover macrocycles with OM-permeabilizing and growth-inhibitory activity against WT E. coli strains, we identified smaller macrocycle scaffolds. These smaller macrocycle scaffolds also target MsbA in the periplasmic cleft by performing additional selections against EcMsbA and MsbA-chim5 using an 8-10mer macrocycle library. We also utilized a codon table biased towards lipophilic and amine-containing side chains, as it is known that small amphipathic molecules containing primary amines are most likely to accumulate in cells (Richter et al., 2017).

[0266] We discovered an 8-mer macrocycle, G1365, which binds the MsbA-chim5 transporter (IC 50 >5 μM) (Figures 3B and 3D) compared with WT EcMsbA, which showed significantly higher concentration and ATPase inhibitory activity (IC 50 In biochemical assays, G1365 inhibited the related Enterobacter cloacae and Klebsiella pneumoniae MsbA transporters with IC values ​​of approximately 700 nM and 660 nM, respectively. 50 (Fig. 13C), but had a more moderate inhibitory activity against MsbA of the distantly related Pseudomonas aeruginosa (IC 50 = 7.6 μM). G1365 treatment of E. coli imp4213 mirrored the inner membrane elaboration phenotype of MsbA-depleted bacteria, consistent with on-target MsbA activity (Figure 3E). In contrast to the larger 14-mer macrocycle G1118, G1365 inhibited the growth of WT E. coli cells with intact outer membranes with an IC of approximately 20 μM. 50 was found to be inhibitory for 6 hours (Figures 13B and 13D), although an overnight MIC could not be determined. Whole genome sequencing analysis was then performed on the isolated E. coli imp4213 mutant resistant to derivatives of G1365 (see Methods), revealing that a single nucleotide mutation in msbA results in an amino acid change, Asp252Asn, within the periplasmic cleft of MsbA. Further characterization of G1365 is discussed below in Example 9.

[0267] The data collectively identify MsbA as the cellular target of G1365 and highlight the possibility of discovering synthetic macrocycles capable of crossing the intact bacterial outer membrane.

[0268] d. Additional 14-mer macrocyclic peptide activators Additional macrocyclic peptide activators tested were G1119 (ClAc-FWWLWSDMeGDWWMeFVC-NH2; SEQ ID NO:10) and G1122 (ClAc-FRYLWMeAWGLVWDNC-NH2; SEQ ID NO:11).

[0269] Cryo-EM analysis of G1119 and G1122 showed that they caused lamination of the outer cell membrane to a greater extent than G1118 (data for G1119 and G1122 not shown, see Fig. 3F for data for G1118). IC for inhibition of ATPase activity of MsbA by G1118, G1119, and G1122 50 was performed (see Example 8 for methods) and found an IC of 5 nM for G1118 and G1119. 50 value and IC of 2 nM for G1122 50 value was determined.

[0270] Eleven different molecules designed according to the formula ClacF-X1-X2-L-X3-X4-D-X5-X6-X7-X8-MeF-VC (see Example 5 above) were also tested for their inhibitory effect on MsbA. They showed IC values ​​of 0.2-0.8 μM for ATPase activity in the assay as described in Example 3 above. 50 The minimum inhibitory concentration (MIC) for inhibition of E. coli MsbA (EcMsbA) (see Example 3 for methods) was found to be greater than 100 μM, and the EC 50ranged from about 2 to about 8 μM for all but one of the molecules tested (exceeding 100 μM for the last molecule tested). Overall, these data indicate that the macrocyclic peptide G1118 and related peptides according to the formula above are generally capable of inhibiting MsbA and cellular activities associated with MsbA.

[0271] Example 8: G1118 binds to MsbA Biochemical characterization of G1118 Using the materials and methods described in Examples 1-5 above, G1118 was identified as a macrocycle that binds to MsbA. The 14-residue peptide macrocycle G1118 was identified after eight rounds of enrichment and screening. G1118 exhibited potent inhibitory activity against EcMsbA (50% inhibitory concentration (IC) of approximately 110 nM). 50 )) and still showed weak activity against the counterselected chimeras (IC 50 G1118 treatment of outer membrane permeabilized E. coli cells carrying the lpt(imp4213) allele (Sampson et al., 1989) reflected a distinct inner membrane elaboration phenotype 2 (Figure 3E) characteristic of MsbA depletion, whereas no activity was observed against WT E. coli with an intact outer membrane. G1118 inhibited the Mg 2+G1118 is a 14-mer macrocycle identified by macrocycle selection against purified WT transporter trapped in an outward conformation by treatment with ATP and vanadate, followed by counterselection with a similarly trapped periplasmic chimera to remove macrocycles that bind to regions other than the periplasmic cleft. G1118 has potent inhibitory activity against EcMsbA. After counterselection with chimeric MsbA, G1118 continued to show significant enrichment against WT EcMsbA, and its inhibitory activity is consistent with targeting to the periplasmic cleft, as G1118 was less potent than the purified chimera by 30-fold. E. coli treated with G1118 also reflected the unique membrane elaboration phenotype of bacteria depleted of MsbA, consistent with on-target activity. However, the cellular activity of G1118 was restricted to cells that also contained the imp4213 allele, which confers a defect in outer membrane (OM) permeability, suggesting that G1118 has difficulty crossing the OM to reach the MsbA protein. Because G1118 represents the first potent and selective inhibitor of MsbA that targets the periplasmic cleft, the molecular basis for its antagonistic action was investigated using structural biology methods described above in Example 6.

[0272] b. G1118 binds to MsbA at the periplasmic binding site and traps MsbA in the outward-facing state To clarify the molecular mechanism by which G1118 antagonizes MsbA, a 3.0 Å cryo-EM structure of the MsbA-G1118 complex in the presence of ADP-vanadate was determined using an antigen-binding fragment of an antibody (Fab) to aid in particle alignment (Figures 4A-D, 14A-C, 15A-E, and 8). Materials and methods were as described in Example 6 above. In complex with G1118, the MsbA homodimer adopts an outward-open conformation (Figure 4A-D), with a solvent-exposed peripheral pocket formed between the transmembrane helices TM1, TM3, and TM6 of each MsbA subunit. Strong features in the cryo-EM maps signaling the presence of a single G1118 macrocycle in the periplasmic cleft validate the design of the chimera selection strategy. G1118 is an amphipathic peptide that complements the complex physicochemical properties of the periplasmic cleft of MsbA, complementing polar, van der Waals and hydrophobic contacts and conserving approximately 880 A of solvent-accessible surface area. 2 (Figure S16A-B). Binding of G1118 is incompatible with the rearrangement required for MsbA to return to an inward-facing conformation (Ho et al., 2018; Mi et al., 2017; Ward et al., 2007), defining this macrocycle as a state-dependent inhibitor that locks the transporter in an outward conformation and prevents LPS transport. Since G1118 was observed to cause cell killing by inhibiting MsbA (Figure 3E), the cocomplex structure likely corresponds to a physiologically relevant inhibitor complex.

[0273] The periplasmic receptor site for G1118 on MsbA is found near the central axis of the transporter that extends above and below the membrane-aqueous interface (Figures 4B and 16A-B). The MsbA dimer is highly symmetric (Cα RMSD of approximately 0.4 Å) and G1118 is located at the Trp10 G1118 and Trp11 G1118 We exploit this inherent symmetry on both sides of the transporter between TM1 and TM6' by making pseudo-symmetric interactions with the indole ring of Ser9, packing between Leu45, Leu52, and Ile292' (Figure 4C). G1118 and Trp3G1118 form a pair of hydrogen bonds with the backbone carbonyls of Lys49 and Ser289' of MsbA, respectively (Fig. 4D). On the polar face of G1118, Asp7 G1118 A salt bridge was directly observed between G1118 and Arg296, a highly conserved basic residue deep within the central cavity of MsbA previously found to coordinate LPS in an inward-facing conformation (Figure 4C) (Ho et al., 2018; Mi et al., 2017). Analysis of enriched related sequences of G1118 and mutational scanning confirm the critical interactions and amphipathic nature of the macrocycle required to interact with MsbA (Figure 4E and Table 4). TIFF2024519413000014.tif116170

[0274] Table 4 above shows the mutation scan of macrocycle G1118. The enrichment factors for each macrocycle containing the indicated substitutions at positions 2-13 of the macrocycle core are shown (where enrichment factor = (% frequency [target] / % frequency [input]), normalized to the parent value). Amino acids that are identical to the parent are shown with a black border. Natural amino acids are shown with standard single letters. Unnatural amino acids are as follows: (1) MeF = F * , N-methyl-L-phenylalanine; (2) MeG=G * , N-methyl-L-glycine; (3) Dopa, L-3,4-dihydroxyphenylalanine; and (4) Bph=B * , (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid. Overall, our biochemical and structural studies identify novel pharmacology and receptor sites within the ABC transporter family.

[0275] In summary, the structure of the complex guided the selection of residues for mutational analysis. IC 50In vitro curves showed that these residues weakened macrocycle binding. Conversely, analysis of enriched sequences from RNA display selections pointed to a role of residues relative to the macrocycle for affinity. Overall, the structures and cellular activities validated the macrocycle selection strategy and the periplasmic cleft as a druggable site that conclusively validated our protein engineering and selection strategy. Due to the large size of G1118, and possibly due to its difficulty in penetrating the LPS-rich outer membrane, wild-type activity prevented optimization of derivatives.

[0276] Example 9: Identification of G1365 Biochemical characterization of G1365 In an attempt to select for smaller macrocycles capable of overcoming the OM permeability barrier, another round of selections and chimeric counterselections were performed using 8-10 mer macrocycles. Materials and methods were as described above in Examples 1-5. A codon table biased towards lipophilic and positively charged side chains was also used.

[0277] Using these approaches, G1365 was identified (Example 7 and FIG. 3B). G1365 exhibited biochemical activity against MsbA (FIG. 3D; IC 50 = 1.3 µM), E. coli lptD(imp4213) cells treated with G1365 also revealed an MsbA-depleted membrane phenotype (Figure 3E). Notably, G1365 also inhibited the growth of WT E. coli cells with intact outer membranes over a 6-h period (IC of approximately 20 µM). 50 13B and 13D), highlighting the possibility of discovering synthetic macrocycles that can cross the intact bacterial outer membrane. Specifically, the effect of G1365 and other macrocycles on the viability of imp E. coli cells expressing normal and high levels of MsbA (MsbA WT and MsbA High) as well as wild-type E. coli cells (UPEC WT; i.e., with a normal OM) was determined at different concentrations of macrocycles (Figures 3E and 3G-L, and Table 5). TIFF2024519413000015.tif89170

[0278] Biochemical IC for G1365 in this study 50 is 300 nM, and the EC 50 was 20 μM. See Figure 3G-L and Table 5 below. To further confirm that G1365 is selective for MsbA from E. coli, the minimum inhibitory concentrations (MICs) of G1365 and other macrocycles for growth inhibition of imp E. coli and a different bacterial species, Acinetobacter baumannii (A. bau.), were determined. The MIC of G1365 in imp E. coli was 12.5 μM, whereas it was over 100 μM in A. baumannii, an approximately 10-fold difference in concentration, indicating that G1365 preferentially interacts with E. coli MsbA compared to A. baumannii MsbA. The MICs for the other tested macrocycles did not differ between the two bacterial strains, indicating that they are not selective between the two MsbA proteins. The molecular basis for the antagonistic action was investigated using the structural biology approach described in Example 6 above.

[0279] b. G1365 binds deep within the periplasmic cleft of MsbA To understand the molecular basis of inhibition, the cryo-EM structure of the MsbA-12G7-G1365 complex was determined. Materials and methods were as described in Example 6 above. 3.1 Å cryo-EM structure of MsbA in complex with a potent derivative of G1365 (Figures 14A-B, 17A-E, and 8). Notably, G1365 is an amphipathic molecule, and its smaller size allows it to bind nearly 10 Å deeper into the periplasmic cleft of MsbA compared to G1118 (Figures 4A, 4B, 4F, and 16A-D). Unlike G1118, which binds along the central axis of MsbA, the G1365 receptor site is found at the edge of the periplasmic cleft (Figures 4A, 4B, 4F–K, and 16A–D), suggesting that this amphipathic macrocycle can directly access the receptor site from the lipid bilayer.

[0280] We identify G1365 as a state-dependent inhibitor because it traps MsbA in an outward conformation that is incompatible with a transition to the inward state. The binding site for G1365 is adjacent to the membrane-exposed region of the transporter with binding sites formed by TM1, TM3, and TM6 and is contiguous with Arg6. G1365 The guanidinium side chains of MeF5 are oriented toward the lipid bilayer (Figure 4F-H). G1365 between Arg148 and Bhp4 G1365 The cation-π interaction between G1365 and Arg296 stabilizes the binding of G1365 within the receptor site. G1365 likely reaches beyond the axis of symmetry of MsbA to interfere with the binding of a second macrocycle. Enriched related sequences and mutational scans of G1365 confirm the critical interactions of the macrocycle required to interact with MsbA (Fig. 4I and Table 5). TIFF2024519413000016.tif115170

[0281] Table 6 above shows the mutation scan of macrocycle G1365. Just like in Table 4 above, the enrichment factors for macrocycles containing the indicated substitutions at positions 2-7 of the macrocycle core (where enrichment factor = (% frequency [target] / % frequency [input]), normalized to the parent value). Amino acids that are identical to the parent are shown with a black border. Natural amino acids are shown with standard single letters. Unnatural amino acids are as follows: (1) MeF = F * , N-methyl-L-phenylalanine; (2) MeG=G * , N-methyl-L-glycine; (3) Dopa, L-3,4-dihydroxyphenylalanine; and / or (4) Bph=B * , (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid. The binding pose of G1365 could also rationalize the Asp252Asn-resistant mutation (Example 7 above), since this should disrupt the interaction between Arg296 and G1365 (Figure 4H).

[0282] Unexpectedly, the guanidinium side chain of G1365Arg6 points towards the lipid bilayer. The bulky biphenyl residue reaches beyond the pseudo-axis of symmetry towards the other protomarker Arg296. The structure also logically explains the mechanism for the D252N resistance mutation by disrupting the salt bridge between Lys299 on TM6, presumably disrupting the interaction between Arg296 and G1365. The structure of G1365 identifies a unique receptor site compared to G1118, even though the outward-open conformations of MsbA trapped by the two molecules are very similar. Thus, like G1118, G1365 is a state-dependent inhibitor of MsbA, despite its smaller binding footprint.

[0283] Example 10: Identification of an unexpected LPS-binding site on MsbA It is now known how MsbA initially binds and achieves selective transport of its rare substrate LPS rather than the abundant phospholipids in the membrane bilayer. In cryo-EM maps, two well-resolved LPS molecules are bound at the periphery of MsbA (Figs. 4A and 18A-D). A striking feature of these maps is the unequivocal placement of the characteristic biphosphoglucosamine head group of lipid A in a previously unrecognized symmetrically related coordination site at the level of the inner membrane leaflet of the MsbA dimer (Fig. 18A). Importantly, the visualized LPS molecules are far removed from the previously observed single binding site (Ho et al., 2018; Mi et al., 2017) within the central space within the entrance of the inward-facing state of MsbA. Since LPS was not added to the samples used for cryo-EM, the identification of these unique peripheral LPS binding sites on the outward-facing state of MsbA suggests that they are physiologically relevant.

[0284] The LPS-binding site around the inner membrane leaflet on MsbA is formed by TM2, TM4', and TM5' and expands by 865 Å upon LPS binding. 2of the transporter surface area is filled. Three distinctive features of MsbA-bound LPS are spread along the lipid A core of the molecule (Fig. 5A). First, a highly conserved basic side chain from MsbA coordinates to a characteristic phosphate on the glucosamine head group of lipid A (Fig. S18B). Arg188 and Lys243 form salt bridges with the 1-phosphate (Fig. 5A), while Lys95 and Arg238 form salt bridges with the 4'-phosphate (Fig. 5B). Second, the hexa-acylated tail of lipid A interacts with an extended hydrophobic surface on the transporter, where the 2'-hydroxymyristic acid and 2''-laurate ester bonds are telescoped against the conserved aromatic side chains of Tyr87 and Trp91, respectively. Third, an elaborated 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) core sugar derived from lipid A is coordinated at an extended polar intracellular stalk region on the transporter (Figure S18C). These strategic multipoint contacts made to lipid A at the peripheral LPS-binding site reveal the structural basis for MsbA's selective binding to LPS over bulk membrane phospholipids.

[0285] Because the outward conformation of MsbA has previously been associated with the release of substrates into the periplasmic membrane leaflet (Ho et al., 2018; Mi et al., 2017; Ward et al., 2007), it was initially surprising to find LPS bound to MsbA along the inner leaflet in this state. We generated targeted double mutations of residues that interact with LPS. The double mutants were assessed for their ability to support the growth of E. coli cells lacking WT MsbA (Figure S19A-J). Basic residues that coordinate the 4'-phosphate of lipid A (Lys95 and Arg238) and aromatic side chains that support the 2'-hydroxymyristic and 2''-lauric acid chain esters of lipid A (Tyr87 and Trp91) were found to be essential. This is because their mutation to alanine blocked cell growth and created a unique inner membrane elaboration phenotype that mirrored bacteria depleted of WT MsbA (Figure 5C-E) (Doerrler et al., 2001). In contrast, the side chains coordinating the 1-phosphate of lipid A (Arg188 and Lys243) or proximal non-LPS-interacting residues are not necessarily required. Unexpectedly, (1) an essential peripheral LPS-binding site was discovered on the inner leaflet of MsbA in an outward conformation, and (2) a previously unknown substrate selectivity determinant with important physiological significance was identified.

[0286] Example 11: Discussion This disclosure discusses the site-directed discovery of potent and state-dependent inhibitors of ABC transporters. This disclosure discusses tools and new insights related to the mechanism of action of MsbA. Using these tools, many of the challenges associated with the unbiased discovery of ligands that bind to integral membrane receptor sites have been overcome.

[0287] A site-directed ligand discovery strategy was devised to selectively inhibit ABC transporters by targeting the solvent-exposed cleft of the outward-facing state using engineered chimeric proteins. G1118 and G1365 are two macrocycles that target previously undescribed receptor sites within the outward-facing cleft, defining new pharmacology within the ABC transporter superfamily, and these macrocycles may represent initial lead compounds for the discovery of additional antibacterial agents. Similar site-directed ligand discovery efforts using engineered chimeras could identify new receptor sites in other protein classes and be particularly useful for discovering new pharmacology.

[0288] Although previous studies have shed light on the alternate access mechanism of transport employed by MsbA (Ho et al., 2018; Mi et al., 2017; Ward et al., 2007), it has remained unclear how MsbA achieves selectivity for LPS over bulk membrane phospholipids. Although inward-facing structures of MsbA have revealed LPS bound within the central cavity, these structures fail to capture intermediate states of the transport cycle and provide insight into how LPS is selectively loaded from the bulk phospholipid bilayer into the central cavity. The present disclosure discusses the discovery of MsbA in an outward-facing state bound to its substrate and unexpectedly reveals that MsbA first recognizes LPS through a conserved, integral network of side chains located along the inner membrane leaflet of the transporter. Based on these observations, several principles that may underlie the mechanism of LPS selectivity by MsbA are discussed below.

[0289] Until now, the outward conformation in ABC transporters has been strictly associated with substrate release and the termination of the catalytic cycle (Hollenstein et al., 2007). As discussed in the above examples, the data call for a more holistic model, whereby the LPS binding site presented by the outward state serves to greatly increase the local concentration of LPS within the inner leaflet around the transporter (Figure 6). In the outward conformation, MsbA strategically targets the unique chemical structure of the core lipid A at the peripheral site through multipoint interactions with the characteristic biphosphoglucosamine head group, hexaacylated chains, and Kdo-core sugars to achieve selective binding to LPS over phospholipids (Figure 5A-B). Targeted mutagenesis and physiological studies have established the essential nature of the conserved residues in this previously unrecognized LPS binding site. Disturbance of the peripheral binding sites upon transition to the inward conformation is predicted to release LPS and facilitate its selective loading into the central cavity, presumably along a stretch of basic residues that stretches from the peripheral binding sites to the intraenteric space (Figure S1C-D). Taken together, MsbA couples the release of LPS to the outer leaflet with the binding of substrate to the inner leaflet, revealing that the outward state, previously regarded as the final step in the ABC exporter cycle, can be uniquely and directly coupled to the first one.

[0290] Visualization of the periplasmic LPS recognition complex provides the first insight into how MsbA is able to discriminate between mature and immature LPS species. The Lys95 and Arg238 side chains are essential and form a direct salt bridge to the 4'-phosphate of lipid A (Figure 5B), providing a structural basis for why precursor 1-mono-phosphorylated lipid A species is not transported.23 The essential nature of the Lys95 and Arg238 mediated interaction also explains why suppressor mutations of the 4'-kinase LpxK have not been identified and logically explains why MsbA and LpxK are chromosomally linked24 or exist as a single linked protein in some bacteria.25 Furthermore, Tyr87 and Trp91 side chains are essential and lie immediately below the ester bonds of the 2'-hydroxymyristic and 2"-lauric acid acyl chains (Figure 5B), which are added by the slow-acting enzymes, LpxL and LpxM, in the lipid A biosynthetic pathway. This observation provides a molecular basis for why hexa-acylated LPS is the preferred substrate for EcMsbA. The picture that emerges from the peripheral LPS recognition site is that, in addition to selectively enriching LPS over phospholipids, Our findings suggest that MsbA employs intrinsic quality control to ensure that mature LPS species are selected for transport across the inner membrane into the periplasm. This degree of molecular sophistication may help ensure that the integrity of the outer membrane barrier is not compromised by the passage of immature LPS species. Overall, our findings shed light on the selective recognition and transport of lipids and introduce a paradigm in which ABC transporters can utilize an outward-facing conformation to selectively enrich and interrogate potential substrates.

[0291] References TIFF2024519413000017.tif122170TIFF2024519413000018.tif214170TIFF20245194130 00019.tif204170TIFF2024519413000020.tif213170TIFF2024519413000021.tif103170

[0292] array The following table describes certain sequences referenced herein. Dashes are added between the residues or functional groups of certain peptides listed below. TIFF2024519413000022.tif255164TIFF2024519413000023.tif255164TIFF2024519413000024.tif255170TIFF2024519413000025.tif159170

[0293] The foregoing invention has been described in some detail by way of illustrations and examples for clarity of understanding, but the descriptions and examples are not to be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

Claims

1. A chimeric ABC transporter comprising the parent ABC transporter Escherichia coli MsbA (EcMsbA) in which one or more of EcMsbA residues Leu47-Pro68 in periplasmic loop 1 (L1), EcMsbA residues Met159-Leu171 in periplasmic loop 3 (L3), and EcMsbA residues Ala262-Ile292 in periplasmic loop 5 (L5) have been replaced with the equivalent region from Pseudomonas psychotolerans (PpMsbA), Candidatus accumlibacter (CaMsbA), or Thiomicrospira cyclica (TcMsbA).

2. A method for determining whether a test molecule binds to the periplasmic, extracellular, and / or luminal surface of a parent ABC transporter, comprising: a) providing a chimeric ABC transporter according to claim 1; and b) contacting the chimeric ABC transporter with a test molecule that binds to the parent ABC transporter in an outward conformation, whereby if the test molecule does not bind to the chimeric ABC transporter, it is determined that the test molecule binds to the periplasmic, extracellular, and / or luminal surface of the parent ABC transporter; A method comprising:

3. A method for determining whether a test molecule binds to the periplasmic, extracellular, and / or luminal surface of a parent ABC transporter, comprising: a) trapping the parent ABC transporter in an outward conformation; b) selecting a test molecule that binds to the parent ABC transporter in an outward conformation; c) providing a chimeric ABC transporter according to claim 1; and d) contacting the chimeric ABC transporter with a test molecule of (b) that binds to the parent ABC transporter in an outward conformation, whereby if the test molecule does not bind to the chimeric ABC transporter, it is determined that the test molecule binds to the periplasmic, extracellular, and / or luminal surface of the parent ABC transporter; The method includes:

4. 13. A molecular complex comprising the chimeric ABC transporter of claim 1 bound to a peptide, a small molecule, an antibody, a binding fragment of a peptide, a binding fragment of a small molecule, or a binding fragment of an antibody.

5. The conjugate of claim 4 , wherein the peptide is a macrocycle.

6. The conjugate of claim 5, wherein the macrocycle is a 6-14-mer macrocycle.

7. 4. A kit comprising the chimeric ABC transporter of claim 1 and reagents for carrying out the method of claim 2 or 3, optionally wherein the chimeric ABC transporter is attached to a matrix or beads, and optionally wherein the kit comprises: a. a parent ABC transporter and / or a different ABC transporter from which the chimeric ABC transporter is engineered; b. A matrix or beads for attachment of the ABC transporter, optionally streptavidin-, avidin-, or deglycosylated avidin-coated beads, or magnetic beads; c. one or more detergents to solubilize the ABC transporter on the matrix or beads; d. at least one wash buffer; e. at least one elution buffer; f. at least one positive or negative control molecule The kit further comprises one or more of:

8. 8. The kit of claim 7, further comprising instructions for use.

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