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 on March 1, 2022, U.S. Provisional Application No. 63 / 213,652 filed on June 22, 2021, and U.S. Provisional Application No. 63 / 181,118 filed on April 28, 2021, which are hereby incorporated by reference in their entirety for all purposes.

[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 face of an ABC transporter protein. For example, in some embodiments, the screening method comprises providing a chimeric ABC transporter in which one or more regions of the periplasmic, extracellular, and / or luminal face of an ABC transporter are replaced with one or more equivalent regions of the periplasmic, extracellular, and / or luminal face of a different ABC transporter, and screening for molecules that bind to the ABC transporter but do not bind to the chimeric ABC transporter. The present disclosure also relates to molecules that bind to the periplasmic, extracellular, and / or luminal face of an ABC transporter, identified, for example, by such screening. [Background Art]

[0003] ATP-binding cassette ("ABC") transporters constitute a superfamily of endogenous membrane proteins found in prokaryotes and eukaryotes, as well as in eukaryotic organelles involved in the ATP-powered transmembrane transport of many types of substrates across the membrane. ABC transporters have transmembrane pores that are either accessible from the inner side of the membrane (inward-facing conformation) or accessible from the outer side of the membrane (outward-facing conformation). Hydrolysis of ATP drives conformational changes in proteins, resulting in "flipping" of the transmembrane pore from the inside to the outside or vice versa to transport substrates across the membrane. See, for example, Figure 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 invert 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 membranes (cytoplasmic membrane). In contrast, eukaryotic cells have a single membrane that separates the outside of the cell from the cytosol. Eukaryotic organelles generally have a single membrane that separates the cytosol from the organelle lumen.

[0005] For various reasons, inhibiting the activity of ABC transporters may be desirable. For example, a molecule that can inhibit a specific ABC transporter in bacteria could be an effective antibiotic. 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 integration into the outer membrane. MsbA is a major component of the outer membrane and essential for the growth of Gram-negative bacteria. MsbA depletion disrupts LPS trafficking and causes cytolysis, and blocking MsbA ATPase activity inhibits bacterial growth. MsbA can be an antibiotic target because it has low identity with human ABC transporters; for example, 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 have utilized mutant cell types with more permeable outer membranes, e.g., the imp E. coli strain. Figure 3E. These screenings tended to be biased towards 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, molecules must first pass through both the outer and inner / cytoplasmic membranes, and most of the identified molecules are unable to do so, and therefore were ineffective in inhibiting ABC transporter activity in wild-type cells lacking an outer membrane with atypical permeability. Figure 6. Similarly, molecules that bind to the inward conformation in Gram-positive bacterial cells or to eukaryotic cells must pass through the cell membrane before binding to their target. When ABC transporters are in an outward conformation, there is a need for drug discovery methods that can be adapted to high-throughput screening techniques to select molecules that bind to the solvent-contactable region on the ABC transporter: the periplasm, extracellular and / or luminal surface, for example, the periplasm, extracellular and / or luminal cleft found within the periplasm, extracellular and / or luminal surface of the protein. Molecules that bind to this part of the ABC transporter do not need to cross many membranes to bind to their targets and, in some cases, can still act as inhibitors of the ABC transporter, for example, by competing for binding with the transporter's normal substrate or by blocking access to normal substrate-binding sites on the protein's outer surface. [Overview of the project]

[0007] To screen molecules that bind to the periplasm, extracellular, and / or luminal surface of ABC transporters, the inventors have developed a screening strategy using chimeric ABC transporter proteins in which one or more regions of the periplasm, extracellular, and / or luminal surface of an ABC transporter are replaced by one or more equivalent regions of the periplasm, extracellular, and / or luminal surface of different ABC transporters. Such molecules can be used, for example, in counter-selection screening where molecules that bind to ABC transporters but not to chimeric ABC transporters under screening conditions are identified as molecules that bind to the periplasm, extracellular, and / or luminal surface of ABC transporters. Chimeric ABC transporters and methods for identifying test molecules that bind to the periplasm, extracellular, and / or luminal surface regions of ABC transporters using them, as well as related binding molecules, molecular complexes, kits, and methods for using the identified molecules are described herein.

[0008] This disclosure includes, for example, one or a combination of the following embodiments:

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

[0010] Embodiment 2. A method for determining whether a test molecule binds to the periplasm, 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 the outward conformation; c) providing a chimeric ABC transporter in which one or more regions of the periplasm, extracellular, and / or luminal surface of the parent ABC transporter are replaced by one or more equivalent regions of the periplasm, extracellular, and / or luminal surface of different ABC transporters; and d) contacting the chimeric ABC transporter with the 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 periplasm, extracellular, and / or luminal surface of the parent ABC transporter.

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

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

[0013] Embodiment 5. The method according to 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 according to any one of Embodiments 1 to 5, wherein the parent ABC transporter is a type IV ABC transporter.

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

[0016] Embodiment 8. The method according to Embodiment 7, wherein the Gram-negative bacteria are selected from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas psychrotolerans, Candidatus accumulibacter, Janthinobacterium agaricidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

[0017] Embodiment 9. The method according to Embodiment 8, wherein the parent ABC transporter is derived from Escherichia coli.

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

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

[0020] Embodiment 12. The method according to Embodiment 11, wherein the Gram-negative bacteria are selected from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerance, Candidatus accumulibacter, Jansinobacterium agalisidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

[0021] Embodiment 13. The method according to any one of Embodiments 1 to 12, wherein the chimeric ABC transporter differs from the parent ABC transporter in that up to 50% of at least one periplasm, extracellular, or luminal loop and transmembrane segments on both sides of the loop are replaced with equivalent regions of a different ABC transporter.

[0022] Embodiment 14. The method according to Embodiment 13, wherein the chimeric ABC transporter differs from the parent ABC transporter in that at least one loop facing the periplasm, extracellular, or lumen and 50% of the transmembrane segments on both sides of the loop are replaced with equivalent regions 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 up to 25% of at least one peripheral, extracellular, or lumen-facing loop and the transmembrane segments on both sides of the loop are replaced with equivalent regions 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 up to 10% of at least one loop facing the periplasm, extracellular, or lumen and the transmembrane segments on both sides of the loop are replaced with equivalent regions 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 loops facing the periplasm, extracellular, or lumen, and up to 50% of the transmembrane segments on both sides of each loop, are replaced with equivalent regions of a different ABC transporter.

[0026] Embodiment 18. The method according to any one of Embodiments 1 to 17, wherein the parental ABC transporter and the different ABC transporters are each encoded by homologous genes originating from two different species.

[0027] Embodiment 19. The method according to 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 the EcMsbA residues Leu47-Pro68 in the periplasmic loop 1 (L1), EcMsbA residues Met159-Leu171 in the periplasmic loop 3 (L3), and EcMsbA residues Ala262-Ile292 in the periplasmic loop 5 (L5) are replaced with the equivalent region of Pseudomonas cyclotolerance (PpMsbA).

[0028] Embodiment 20. The method according to 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 the EcMsbA residues Leu47-Pro68 in peripheral loop 1 (L1), EcMsbA residues Met159-Leu171 in peripheral loop 3 (L3), and EcMsbA residues Ala262-Ile292 in peripheral loop 5 (L5) are replaced with the equivalent region of Candidatus acumulibacter (CaMsbA).

[0029] Embodiment 21. The method according to 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 the EcMsbA residues Leu47~Pro68 in peripheral loop 1 (L1), EcMsbA residues Met159~Leu171 in peripheral loop 3 (L3), and EcMsbA residues Ala262~Ile292 in peripheral loop 5 (L5) are replaced with the equivalent region (JaMsbA) of Jansinobacterium agalisidamunosum.

[0030] Embodiment 22. The method according to 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 the EcMsbA residues Leu47~Pro68 in peripheral loop 1 (L1), EcMsbA residues Met159~Leu171 in peripheral loop 3 (L3), and EcMsbA residues Ala262~Ile292 in peripheral loop 5 (L5) are replaced with the equivalent region of Thiomicrospira cyclica (TcMsbA).

[0031] Embodiment 23. The method according to 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 the 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) are replaced with the equivalent region (MqMsbA) of magnetospira strain-OH-2.

[0032] Embodiment 24. The method according to any one of Embodiments 1 to 23, wherein the molecule binds to the periplasm, extracellular, or luminal fissure of the parent ABC transporter.

[0033] Embodiment 25. The method according to 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 according to any one of Embodiments 1 to 25, further comprising performing a cell survival or proliferation assay and / or an ABC transporter function assay of the parent ABC transporter in the presence of the molecule.

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

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

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

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

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

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

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

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

[0043] Embodiment 35. K of 1 nM to 500 nM D The molecule according to Embodiment 27, which binds to the parent ABC transporter.

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

[0045] Embodiment 37. A molecule identified by the method according to any one of Embodiments 1 to 26, wherein the molecule 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 peptide binding fragment, a small molecule, or an antibody.

[0048] Embodiment 40. The peptide described in Embodiment 36, which is a macrocyclic molecule.

[0049] Embodiment 41. The macrocyclic molecule according to Embodiment 40, which is a 6-14-mer, 6-10-mer, 6-8-mer, or 8-10-mer macrocyclic molecule.

[0050] Embodiment 42. A macrocyclic molecule according to 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 that binds to the periplasm, extracellular space, or luminal fissure of a parent ABC transporter.

[0052] Embodiment 44. Macrocyclic molecular peptide G1118, G1119, or G1122.

[0053] Embodiment 45. Macrocyclic molecular peptide G1365.

[0054] Embodiment 46. Mg 2+ Molecules that compete with macrocyclic molecules G1118, G1119, and / or G1122 for binding to the peripheral surface 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 according to Embodiment 46, which inhibits at least 50, 60, 70, 80, 90, or 100% of the binding of G1118, G1119, and / or G1122 to the parent ABC transporter in a competitive assay.

[0056] Embodiment 48. Mg 2+ A molecule that competes with the macrocyclic molecule G1365 for binding to the peripheral surface of the parent ABC transporter when the parent ABC transporter is trapped in an outward conformation by treatment with ATP and vanadate.

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

[0058] Embodiment 50. A method for producing a chimeric ABC transporter, comprising replacing up to 50% of at least one peripheral, extracellular, or lumen-facing loop and transmembrane segments on both sides of the loop of a parent ABC transporter with equivalent regions of different ABC transporters.

[0059] Embodiment 51. The method according to Embodiment 50, wherein 50% of at least one peripheral, extracellular, or lumen-facing loop and transmembrane segments on both sides of the loop of the parent ABC transporter are replaced with equivalent regions of a different ABC transporter.

[0060] Embodiment 52. The method according to Embodiment 50, wherein up to 25% of at least one peripheral, extracellular, or lumen-facing loop and transmembrane segments on both sides of the loop of the parent ABC transporter are replaced with equivalent regions of a different ABC transporter.

[0061] Embodiment 53. The method according to Embodiment 50, wherein up to 10% of at least one peripheral, extracellular, or lumen-facing loop and transmembrane segments on both sides of the loop of the parent ABC transporter are replaced with equivalent regions of a different ABC transporter.

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

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

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

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

[0066] Embodiment 58. The method according to Embodiment 57, wherein the Gram-negative bacteria are selected from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerance, Candidatus accumulibacter, Jansinobacterium agalisidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

[0067] Embodiment 59. The method according to Embodiment 58, wherein the Gram-negative bacterium is Escherichia coli.

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

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

[0070] Embodiment 62. The method according to Embodiment 61, wherein the different ABC transporters are derived from bacteria selected from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerance, Candidatus accumulibacter, Jansinobacterium agalisidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

[0071] Embodiment 63. The method according to any one of Embodiments 50 to 62, wherein the parental ABC transporter and the different ABC transporter are each encoded by homologous genes originating from two different species.

[0072] Embodiment 64. The method according to 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 up to 50% of at least one peripheral, extracellular, or lumen-facing loop and transmembrane segments on both sides of the loop of a parent ABC transporter are replaced with equivalent regions of a different ABC transporter.

[0074] Embodiment 66. A chimeric ABC transporter according to Embodiment 65, wherein 50% of at least one peripheral, extracellular, or lumen-facing loop and transmembrane segments on both sides of the loop of the parent ABC transporter are replaced with equivalent regions of different ABC transporters.

[0075] Embodiment 67. A chimeric ABC transporter according to Embodiment 65, wherein up to 25% of at least one peripheral, extracellular, or lumen-facing loop and transmembrane segments on both sides of the loop of the parent ABC transporter are replaced with equivalent regions of different ABC transporters.

[0076] Embodiment 68. A chimeric ABC transporter according to Embodiment 65, wherein up to 10% of at least one peripheral, extracellular, or lumen-facing loop and transmembrane segments on both sides of the loop of the parent ABC transporter are replaced with equivalent regions of different ABC transporters.

[0077] Embodiment 69. A chimeric ABC transporter according to Embodiment 65, wherein at least two, at least three, or all of the loops facing the periplasm, extracellular, or lumen of the parent ABC transporter, and up to 50% of the transmembrane segments on both sides of each loop, are replaced with equivalent regions of different ABC transporters.

[0078] Embodiment 70. A chimeric ABC transporter according to 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 according to Embodiment 70, wherein the Gram-negative bacteria are selected from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerance, Candidatus accumulibacter, Jansinobacterium agalisidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

[0080] Embodiment 72. The chimeric ABC transporter according to Embodiment 71, wherein the parent ABC transporter is derived from Escherichia coli.

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

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

[0083] Embodiment 75. The chimeric ABC transporter according to Embodiment 74, wherein the Gram-negative bacteria are selected from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerance, Candidatus accumulibacter, Jansinobacterium agalisidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2.

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

[0085] Embodiment 77. A chimeric ABC transporter according to any one of Embodiments 65 to 76, wherein the parent ABC transporter and the different ABC transporter are each encoded by homologous genes originating from two different species.

[0086] Embodiment 78. The chimeric ABC transporter according to 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 containing a parent ABC transporter Escherichia coli MsbA (EcMsbA) in which one or more of the EcMsbA residues Leu47~Pro68 in peripheral loop 1 (L1), EcMsbA residues Met159~Leu171 in peripheral loop 3 (L3), and EcMsbA residues Ala262~Ile292 in peripheral loop 5 (L5) are replaced with the equivalent region (PpMsbA) of Pseudomonas cyclotolerance.

[0088] Embodiment 80. A chimeric ABC transporter containing EcMsbA, in which peripheral loop 1 (L1, EcMsbA residues Leu47~Pro68), peripheral loop 3 (L3, EcMsbA residues Met159~Leu171), and peripheral loop 5 (L5, EcMsbA residues Ala262~Ile292) are replaced with the equivalent region (CaMsbA) of Candidatus acumulibacter.

[0089] Embodiment 81. A chimeric ABC transporter containing EcMsbA, in which one or more of the EcMsbA residues Leu47-Pro68 in peripheral loop 1 (L1), EcMsbA residues Met159-Leu171 in peripheral loop 3 (L3), and EcMsbA residues Ala262-Ile292 in peripheral loop 5 (L5) are replaced with the equivalent region (JaMsbA) of Jansinobacterium agalisidamnosum.

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

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

[0092] Embodiment 84. A molecular complex comprising a chimeric ABC transporter according to any one of Embodiments 65 to 83, conjugated to a peptide, a small molecule, an antibody, a peptide conjugation fragment, a small molecule conjugation fragment, or an antibody conjugation fragment.

[0093] Embodiment 85. The complex according to Embodiment 84, wherein the peptide is a macrocyclic molecule.

[0094] Embodiment 86. The complex according to Embodiment 85, wherein the macrocyclic molecule is a 6-14-mer macrocyclic molecule.

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

[0096] Embodiment 88. The complex according to Embodiment 87, wherein the peptide is a macrocyclic molecule.

[0097] Embodiment 89. The complex according to Embodiment 88, wherein the macrocyclic molecule is a 6-14-mer macrocyclic molecule.

[0098] Embodiment 90. The complex according to Embodiment 89, wherein the macrocyclic molecule is G1118, G1119, G1122, or G1365.

[0099] Embodiment 91. A kit comprising a chimeric ABC transporter as described in any one of Embodiments 65 to 83 and a reagent for performing the method described in any one of Embodiments 1 to 26, wherein optionally the chimeric ABC transporter is mounted on a matrix or beads, and optionally the kit is as follows: a. From there, the chimeric ABC transporter is manipulated, involving the parent ABC transporter and / or a different ABC transporter; b. A matrix or beads for attaching ABC transporters, optionally streptavidin-coated beads, avidin-coated beads, or deglycosylated avidin-coated beads, or magnetic beads; c. One or more detergents for solubilizing ABC transporters 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 A kit that further includes one or more of the following.

[0100] Embodiment 92. The kit according to Embodiment 91, further comprising instructions for use.

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

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

[0103] Embodiment 95. Array: A peptide comprising ClacF-X1-X2-L-X3-X4-D-X5-X6-X7-X8-MeF-VC, wherein in the formula, i.X1 is W, V, or Y, ii. X2 is either W or Y, iii. X3 is either W or Y, iv.X4 is S, D, V, or H, v.X5 is N, and N is selected from any natural amino acid other than C, or from a non-natural 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 either W or Y, A peptide wherein, optionally, the peptide further includes 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.

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

[0105] Embodiment 97. The peptide according to Embodiment 96, wherein X1 is W.

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

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

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

[0109] Embodiment 101. The peptide according to Embodiment 100, wherein X4 is D.

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

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

[0112] Embodiment 104. The peptide according to Embodiment 103, wherein X5 is V.

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

[0114] Embodiment 106. The peptide according to Embodiment 105, wherein X6 is S.

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

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

[0117] Embodiment 109. A macrocyclic molecule formed from a peptide according to any one of Embodiments 95 to 108, wherein the molecule 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. Amino acid sequence: A peptide comprising ClacF-X1-Y-Bph-MeF-X2-VC, wherein in the formula, 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 according to Embodiment 110, wherein X1 is S, Y, L, V, A, R, K, or D.

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

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

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

[0123] Embodiment 115. The peptide according to Embodiment 114, wherein X2 is R.

[0124] Embodiment 116. A macrocyclic molecule formed from a peptide according to any one of Embodiments 110 to 115, wherein the molecule 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 macrocyclic molecule according to any one of Embodiments 95 to 116, wherein the peptide or macrocyclic molecule is conjugated to another molecule such as an antibiotic or antibacterial agent, and optionally the conjugation is at a C-terminal amino acid residue of the sequence.

[0126] Embodiment 118. The peptide or macrocyclic molecule according to Embodiment 117, wherein the antibiotic or antimicrobial agent is a polymyxin, for example, polymyxin B or polymyxin E. [Brief explanation of the drawing]

[0127] [Figure 1A] Figures of ABC transporter protein structures and diagrams showing how the proteins are positioned within the membrane relative to the periplasm / extracellular space and cytoplasm / cytosol are shown. Each individual amino acid is represented by a circle surrounding the amino acid, identified by a single-letter code. Additionally, some amino acid positions on the chain are numbered, for example, position 50 and position 60. Figure 1A shows the MsbA ABC transporter from E. coli. Periplasmic or extracellular loop regions that can be substituted for equivalent regions of different ABC transporters are shown and labeled to create chimeric ABC transporters. Transmembrane protein regions ("transmembrane segments" or "TM segments") that can be substituted for equivalent regions of ABC transporters from different ABC transporters are also shown. Rectangular boxes indicate regions that can be substituted in each protein. [Figure 1B] Figures of the protein structures of different ABC transporters and diagrams showing how the proteins are positioned within the membrane relative to the peripheral / extracellular space and cytoplasm / cytosol are shown. Each individual amino acid is represented by a circle surrounding the amino acid, identified by a single-letter code. Additionally, 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 approximately 25-30% sequence identity with MsbA. Periplasmic or extracellular loop regions that can be substituted with equivalent regions of different ABC transporters are shown and labeled to create chimeric ABC transporters. Transmembrane protein regions ("transmembrane segments" or "TM segments") that can be substituted with equivalent regions of ABC transporters derived from different ABC transporters are also shown. Rectangular boxes indicate regions that can be substituted with each protein. [Figure 1C-1]Figures of the protein structures of different ABC transporters and diagrams showing how the proteins are positioned within the membrane relative to the peripheral / extracellular space and cytoplasm / cytosol are shown. Each individual amino acid is represented by a circle surrounding the amino acid, identified by a single-letter code. Additionally, 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 approximately 25-30% sequence identity with MsbA. Periplasmic or extracellular loop regions that can be substituted with equivalent regions of different ABC transporters are shown and labeled to create chimeric ABC transporters. Transmembrane protein regions ("transmembrane segments" or "TM segments") that can be substituted with equivalent regions of ABC transporters derived from different ABC transporters are also shown. Rectangular boxes indicate regions that can be substituted with each protein. [Figure 1C-2] Figures of the protein structures of different ABC transporters and diagrams showing how the proteins are positioned within the membrane relative to the peripheral / extracellular space and cytoplasm / cytosol are shown. Each individual amino acid is represented by a circle surrounding the amino acid, identified by a single-letter code. Additionally, 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 approximately 25-30% sequence identity with MsbA. Periplasmic or extracellular loop regions that can be substituted with equivalent regions of different ABC transporters are shown and labeled to create chimeric ABC transporters. Transmembrane protein regions ("transmembrane segments" or "TM segments") that can be substituted with equivalent regions of ABC transporters derived from different ABC transporters are also shown. Rectangular boxes indicate regions that can be substituted with each protein. [Figure 2A]Figure 2A shows three-dimensional schematic diagrams of ABC transporters and chimeric ABC transporters, as well as how they are positioned within the membrane relative to the peripheral / extracellular space and cytoplasm / cytosol. The left panel of Figure 2A shows a three-dimensional schematic diagram of the Gram-negative bacterium MsbA positioned within the cell membrane (dark band intersecting the structure). The center panel of Figure 2A shows a three-dimensional schematic diagram of the macrocyclic molecule-MsbA complex, in which the macrocyclic molecule is bound to the peripheral surface of the protein, specifically to a peripheral cleavage. The right panel of Figure 2A shows chimeric MsbA (MsbA-chimera 5), ​​where the darker, filled amino acids at the top of the ribbon diagram are taken from equivalent regions in different ABC transporters. Such chimeras can be used, for example, to help select molecules that bind to the peripheral surface, including the peripheral cleavage of MsbA, such as the macrocyclic molecule shown in the center panel. [Figure 2B] This diagram shows three-dimensional schematics of ABC transporters and chimeric ABC transporters, as well as how they are positioned within the membrane relative to the peripheral / extracellular space and cytoplasm / cytosol. The left panel of Figure 2A shows a three-dimensional schematic of the Gram-negative bacterium MsbA, positioned within the cell membrane (dark band intersecting the structure). The center panel of Figure 2B shows the chimeric human ABCD4 transporter (PDB: 6JBJ), where the embedded amino acids of the protein are taken from equivalent regions in different ABC transporters. The left panel of Figure 2B also shows the chimeric MsbA protein, illustrating the similarities in the architecture of the two proteins. The right panel of Figure 2B shows the chimeric human ABCC1 transporter (PDB: 6BHU), where the embedded amino acids at the top of the ribbon diagram are taken from equivalent regions in different ABC transporters. [Figure 2C]This diagram shows three-dimensional schematics of ABC transporters and chimeric ABC transporters, as well as how they are positioned within the membrane relative to the periplasm / extracellular space and cytoplasm / cytosol. The left panel of Figure 2A shows a three-dimensional schematic of the Gram-negative bacterium MsbA, positioned within the cell membrane (dark band intersecting the structure). Figure 2C shows schematic diagrams of each of the three chimeric proteins from a periplasmic or extracellular perspective. Each periplasmic or extracellular loop is labeled. The embedded amino acids in the ribbon diagrams originate from equivalent regions in different ABC transporters. Each extracellular loop is also labeled. [Figure 3A] This section outlines a strategy for characterizing macrocyclic MsbA inhibitors. Figure 3A shows a schematic diagram of the INSITE screening strategy for identifying macrocyclic molecules that target the peripheral surface of EcMsbA. [Figure 3B] Strategies for characterizing macrocyclic MsbA inhibitors are presented. Figure 3B shows exemplary macrocyclic molecular inhibitors G1118 and G1365. These inhibitors contain thioether bonds as shown (non-natural 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] Strategies for characterizing macrocyclic MsbA inhibitors are presented. 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 the data are mean ± sem from three independent experiments. [Figure 3E]Strategies for characterizing macrocyclic MsbA inhibitors are presented. 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). Intima-elassing is indicated by arrows. Images represent >10 isolated cells for each condition. Scale bar is 0.1 μm. [Figure 3F] We present a strategy for characterizing macrocyclic MsbA inhibitors. Figure 3F shows electron micrographs comparing an E. coli imp strain with G1118 (right panel; arrows indicate intima elastocia) with an E. coli imp strain without G1118 (left panel). The IC50 of G1118 was determined to be 5 nM. [Figure 3G-L] Strategies for characterizing macrocyclic MsbA inhibitors are presented. Figures 3G-L show the dose-response curves of G1325 (Figure 3G; IC50=1.2μM; EC50=10μM), G1330 (Figure 3H; IC50=13μM; EC50=30μM), G1365 (Figure 3I; IC50=300nM; EC50=20μM), G1349 (Figure 3J; IC50=50μM; EC50=30μM), G1323 (Figure 3K; IC50=2.8μM), and G1320 (Figure 3L; IC50=185μM) against E. coli strains imp MsbAWT, imp MsbAHigh, and UPECWT. [Figure 3M] We present strategies for characterizing macrocyclic MsbA inhibitors. Figure 3M shows potential binding sites for G092 quinoline. [Figure 3N] Strategies for characterizing macrocyclic MsbA inhibitors are presented. Figure 3N shows potential binding sites for G1118 (Figure 3N). [Figure 4A-I]Figure 4A shows the binding of G1118 and G1365 at the peripheral fissure of MsbA. G1118 and LPS are shown on the map. Figure 4B shows a diagram of the binding of G1118 at the peripheral fissure of MsbA. The GKK tail is omitted for clarity. Figures 4C-D show macroscopic diagrams of the selective interaction between G1118 and MsbA. Figure 4E shows frequency plots of observed macrocyclic cDNAs in the G1118 family during sequencing after the final round of enrichment (non-natural 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 shown. Figures 4G-H show macroscopic diagrams of the selection interaction between G1365 and MsbA. Figure 4I shows frequency plots of observed macrocyclic cDNAs in the G1365 family during sequencing after the final round of enrichment (non-natural 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 the binding of G1118 and G1365 in peripheral fissures of MsbA. Figure 4J shows that G1365 binds near the membrane-exposed region of outward fissures. [Figure 4K-N] Figures 4K-N show the binding of G1118 and G1365 in peripheral fissures of MsbA. Figures 4K-N show that G1365 binds near the membrane-exposed region of outward fissures. [Figure 5A-B] Figure 5A shows LPS bound to the periphery binding site essential in the outward conformation. Figure 5A shows the periphery LPS binding site of MsbA on the medial lobule 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''-laurateacyl (2''-C12) chains are labeled. The Kdo residue has been omitted for clarity. [Figure 5C]Figure 5C shows LPS bound to surrounding binding sites essential for the outward conformation. Figure 5C shows the growth curve of *E. coli* MG1655 msbA-cKO lptD(imp4213) expressing the indicated WT or mutant allele of *E. coli* MsbA from a low-copy plasmid (Table 1). Data are mean ± sem from three independent experiments. [Figure 5D] This shows LPS bound to surrounding binding sites essential for the outward conformation. Figure 5D shows representative thin-section electron micrographs comparing cells representing msbA+, msbA-, and msbA mutant constructs shown in *E. coli* CFT073 UPEC lptD(imp4213) strain. Inner membrane refinement is indicated by arrows. The image represents >10 isolated cells for each variant. Scale bar is 0.1 μm. [Figure 5E] Figure 5E shows LPS bound to surrounding binding sites essential for the outward conformation. Figure 5E shows amino acids that, when mutated, cause growth failure in E. coli. [Figure 6] This panel illustrates a model for the selective recognition and transport of LPS by MsbA. Panel 1 - In the outward conformation, LPS can bind to surrounding binding sites along the inner membrane. Mature Kdo2-lipid A selectively binds to the positively charged aromatic residue on the 4'-phosphate side of LPS. Panel 2 - MsbA returns to the inward conformation, and LPS is locally enriched upon release from the surrounding binding site (PDB:6BL6). Facilitation of LPS diffusion into the central cavity occurs along the raised positively charged residue of MsbA. Panel 3 - LPS becomes enclosed within the central space in the entrance (PDB:5TV4) as previously described. Panel 4 - ATP binding triggers a major conformational change resulting in an outward state that releases LPS to the outer lobules of the inner membrane. LPS diffuses from MsbA for subsequent processing and transport to the outer membrane. [Figure 7] Crystallographic data collection and refined statistics are presented. [Figure 8] This document outlines the cryo-EM data acquisition parameters and model refinement statistics. [Figure 9A]The multiple sequence alignment of selected MsbA homologs is shown. In Figure 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 Figure 9A, SEQ ID NO: 4 is the E. coli MsbA protein sequence (1st row), SEQ ID NO: 5 is the Pseudomonas cyclotolerance MsbA protein sequence (2nd row), SEQ ID NO: 6 is the Candidatus acumulibacter MsbA protein sequence (3rd row), SEQ ID NO: 7 is the Jansinobacterium agalisidamuth MsbA protein sequence (4th row), SEQ ID NO: 8 is the Thiomicrospira cyclica MsbA protein sequence (5th row), and SEQ ID NO: 9 is the Magnetospira OH-2 MsbA protein sequence (6th row). [Figure 9B] The multiple sequence alignment of the selected MsbA homolog is shown. Figure 9B shows the overall sequence identity of the MsbA homolog in Figure 9A. [Figure 10A] The construction of the chimeric MsbA transporter is shown. Figure 10A shows exemplary sequences of E. coli MsbA-based chimeric proteins. Regions marked “Loop 1”, “Loop 2”, and “Loop 3” indicate the peripheral regions of the transmembrane helices and loops that were substituted in the production of the manipulated chimeric transporter proteins. In Figure 10A, SEQ ID NO: 4 is the E. coli MsbA protein sequence (1st row), SEQ ID NO: 12 is the E. coli MsbA-chimera 1 protein sequence (2nd row), 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 (3rd row), SEQ ID NO: 15 is the E. coli MsbA-chimera 4 protein sequence (4th row), and SEQ ID NO: 16 is the E. coli MsbA-chimera 5 protein sequence (5th row). [Figure 10B] The structure of the chimeric MsbA transporter is shown. Figure 10B shows the outward-facing E. coli MsbA and MsbA-chimera. The side view is shown in the upper panel, and the 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] The purification and evaluation of EcMsbA chimeras are shown. Figure 11A shows the SDS-PAGE of the purified EcMsbA chimeras visualized with Coomassie brilliant blue staining. Figure 11B shows the superposition of the size exclusion chromatography profile with the shown UV (280 nm) curve. [Figure 11C] The purification and evaluation of EcMsbA chimeras are shown. Figure 11C shows a comparison of ATPase activity of EcMsbA chimeras at decreasing concentrations compared to the WT protein. Data are mean ± sem from independent experiments. [Figure 11D] The purification and evaluation of EcMsbA chimeras are shown. Figure 11D shows exemplary chemical structures of quinoline and benzophenone inhibitors (Alexander et al., 2018; Ho et al., 2018). Data are mean ± sem from independent experiments. [Figure 11E] The purification and evaluation of EcMsbA chimeras are shown. Figure 11E shows the dose-response curves of the compound against purified EcMsbA and EcMsbA-chim5. The data are mean ± sem from three independent experiments. The IC50 value (in parentheses in Figure 11E) was determined by fitting the inhibitory dose-response curve to a nonlinear four-parameter inhibition model (Example 3). [Figure 12A] The crystal structure of MsbA-chimera 5, revealing the putative benzophenone receptor site, is shown. Figure 12A shows the overall structure of MsbA-chim5 in the inward conformation. LPS is shown as a sphere. The assigned benzophenone G758 is shown as a rod. 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, revealing the putative benzophenone receptor site, is shown. 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 within a shallow hydrophobic pocket around the residue, identified through resistance mapping studies (data not shown). [Figure 13A-B] This report presents the evaluation of the biochemical and phenotypic activity of macrocyclic MsbA inhibitors. Figure 13A shows the dose-response curves of G1118 against purified and Amphipole-reconstituted MsbA homologs derived from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Figure 13B shows the dose-response curves of G1118 against E. coli CFT073(WT) and CFT073 lptD(imp4213)(imp) strains. Data are mean ± sem from independent experiments. IC50 values ​​(in parentheses) were determined by fitting the dose-response curves to a nonlinear four-parameter inhibition model (Example 3). [Figure 13C-D] This report presents the evaluation of the biochemical and phenotypic activity of macrocyclic MsbA inhibitors. Figure 13C shows the dose-response curves of G1365 against purified and Amphipole-reconstituted MsbA homologs derived from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Figure 13D shows the dose-response curves of G1118 against E. coli CFT073(WT) and CFT073 lptD(imp4213)(imp) strains. Data are mean ± sem from three independent experiments. IC50 values ​​(in parentheses) were determined by fitting the dose-response curves to a nonlinear four-parameter inhibition model (Example 3). [Figure 14A] The biochemical evaluation of the complex used in the structural study is shown. Figure 14A shows the chemical structures of the G1118 and G1365 derivatives used in the structural study to isolate the PD-1365-resistant mutant (G1365·I2,Dopa3). Regions of the derivatives that are different from the G1118 or G1365 parent macrocyclic molecule, namely KK, R2, I2,Dopa3, and L2G4S2GEE, are indicated by circles. [Figure 14B-G] The biochemical evaluation of the complex used in the structural study is shown. Figures 14B-F show the dose-response curves of the macrocyclic parent and derivative molecules of G1118 and G1365 against purified EcMsbA. Figure 14G shows a comparison of the ATPase activity of EcMsbA at increasing concentrations in the presence or absence of Fab 12G7. Data are mean ± sem from three independent experiments (Figures 14B-G). IC50 values ​​(in parentheses in Figures 14B-F) were determined by fitting the inhibition dose-response curves to a nonlinear four-parameter inhibition model (Example 3). [Figure 15A] This shows cryo-EM data processing for MsbA in a complex with G1118. Figure 15A shows the processing pipeline. [Figure 15B-C] This shows cryo-EM data processing for MsbA in a complex with G1118. Figure 15B shows the local resolution plot calculated with Relion. Figure 15C shows the orientation distribution of all particles from the final round of 3D refinement. [Figure 15D] This shows the cryo-EM data processing for MsbA in a complex with G1118. Figure 15D shows the FSC plot from refinement in cisTEM. The maximum resolution used for alignment was 4.5 Å. [Figure 15E] This shows the cryo-EM data processing for MsbA in a complex with G1118. Figure 15E shows the density selected from the cryo-EM map. [Figure 16A-B] This shows macrocyclic molecular bonding at peripheral fissures in MsbA. In Figures 16A-B, the electrostatic surface of MsbA bonded to G1118 highlights the complex chemical environment of the macrocyclic molecular bonding site. Approximate film boundaries and non-enforced C2 symmetry axes are labeled. [Figure 16C-D] This shows macrocyclic molecular bonding at peripheral fissures of MsbA. Figures 16C-D show the electrostatic surface of MsbA bonded to G1365. [Figure 17A]This shows cryo-EM data processing for MsbA in a complex with G1365. Figure 17A shows the processing pipeline. [Figure 17B-C] The cryo-EM data processing for MsbA in a 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 the refinement in cisTEM. The maximum resolution used for alignment was 4.5 Å. [Figure 17D] This shows the cryo-EM data processing for MsbA in a complex with G1365. Figure 17D shows the local resolution plot calculated by RELION. [Figure 17E] This shows the cryo-EM data processing for MsbA in a complex with G1365. Figure 17E shows the density selected from the cryo-EM map. [Figure 18A-B] This shows the conserved surrounding LPS binding sites. Figure 18A shows the MsbA-G1118-LPS complex with symmetrically related LPS sites. Figure 18B shows the sequence conservation analysis of EcMsbA. [Figure 18C-D] The conserved surrounding LPS binding site is shown. Figure 18C shows the electrostatic potential on the surface of MsbA in the inward-facing LPS-binding conformation (PDB: 5TV4). The positive charge line is located from the LPS-binding site toward the central cavity. Missing side chain atoms were added using Maestro's Protein Preparation Wizard. The LPS binding position in the outward-facing G1118-binding structure is shown. In Figure 18D, the MsbA-G1118-LPS structure (blue) is aligned with the inward-facing MsbA (white, PDB: 5TV4). LPS residues within 15 Å were used for alignment. [Figure 19A]This shows the evaluation of the cell proliferation phenotype of MsbA-peripheral 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 either WT or the shown mutant of E. coli MsbA from the pLMG18 vector in the presence of 2% arabinose (under these conditions, untagged WT EcMsbA is also expressed). The blots are representative of n=2 independent experiments. [Figure 19B-E] This report evaluates the cell proliferation phenotype of MsbA-peripheral LPS binding site mutants. Figures 19B-E show the growth curves of E. coli MG1655 msbA-cKO lptD(imp4213) expressing the indicated WT or mutant allele of E. coli MsbA, or a plasmid-free control, in the presence or absence of arabinose. Expression of WT E. coli MsbA from chromosomes was induced using 2% arabinose, whereas in the absence of arabinose, the sole source of MsbA was constitutive expression from a pLMG18 vector expressing WT or the indicated mutant of E. coli MsbA. Data are mean ± sem from three independent experiments. [Figure 19F-I] This section describes the evaluation of the cell proliferation phenotype of MsbA-peripheral LPS binding site mutants. Figures 19F-19I show the growth curves of E. coli MG1655 msbA-cKO lptD(imp4213) expressing the indicated WT or mutant allele of E. coli MsbA, or a plasmid-free control, in the presence or absence of arabinose. Expression of WT E. coli MsbA from the chromosome was induced using 2% arabinose, whereas in the absence of arabinose, the sole source of MsbA was constitutive expression from a pLMG18 vector expressing WT or the indicated mutant of E. coli MsbA. [Figure 19J] This shows the evaluation of the cell proliferation phenotype of MsbA-peripheral LPS binding site mutants. Figure 19J shows a comparison of ATPase activity of EcMsbA mutants at reduced concentrations compared to the WT protein. Data are mean ± sem from three independent experiments. [Modes for carrying out the invention]

[0128] I. Definition Unless otherwise specified, scientific and technical terms used in connection with this invention shall have the meanings generally understood by those skilled in the art. To be used in accordance with this 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 specified. In the context of multiple dependent claims, the use of “or” refers only to the preceding independent or dependent claim as an alternative. Also, terms such as “element” or “component” include both elements and components consisting of one unit and elements and components consisting of multiple subunits, unless otherwise specified.

[0130] As used herein, the transitional term "essentially from" means, when referring to a step of a claimed method, that the method does not include additional steps beyond the specified step that would materially affect the basic and novel properties of the method. As used herein, the transitional term "essentially from" means, when referring to a composition or product such as a kit, that it does not include additional components beyond the specified one that would materially affect its basic and novel properties.

[0131] As used herein, the singular forms "a," "an," and "the" include multiple references unless otherwise clearly indicated by the context. For example, a reference to "(an) ABC transporter protein" includes multiple such transporter proteins, and a reference to "(the) cell" includes one or more cells and their equivalents known to those skilled in the art.

[0132] Where used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integers within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0133] Units, prefixes, and symbols are given in an International System of Units (SI) approved format. Numerical ranges include the numerical values ​​that define the range. The titles provided herein are not intended to limit the various aspects of this disclosure, which can be achieved by referring to the entire specification. Thus, the terms defined immediately below are more fully defined by referring to the entire specification.

[0134] As used herein, “ABC transporters” refers to ATP-binding cassette (“ABC”) transporters. ABC transporters constitute a superfamily of endogenous membrane proteins found in prokaryotes and eukaryotes that are involved in the ATP-powered transmembrane transport of many substrates, ranging from small inorganic and organic 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 generally have a characteristic architecture consisting of at least four domains: two transmembrane domains (TMDs) embedded within the membrane and two nucleotide-binding domains (NBDs). Hydrolysis of ATP on the NBDs drives conformational changes in the TMDs, resulting in alternating access from the inside and outside of the membrane to transport substrates in one direction across the membrane. Each TMD is composed of transmembrane alpha helices, typically having 6 to 10 transmembrane alpha helices, with most exporters having 6 transmembrane alpha helices per TMD. NBDs are highly conserved. In contrast, the TMDs that create the transport pathway are more variable, for example, depending on the substrate to which the ABC transporter is transporting.

[0136] One classification system described by Thomas et al., "Structural and functional diversity calls for a new classification of ABC transporters," 594 FEBS Letters 3767-3775 (2020) (the entire description of the ABC transporter classification system is incorporated by reference), groups ABC transporters into seven distinct types, I-VII, based on their TMD folds. This terminology is universally applicable and, although other information exists, is based in particular on ABC transporter structural information determined by sequence analysis, homology modeling, X-ray crystallography, and single-particle cryo-electron microscopy. 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 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 LptB2FG type based on distinct structural features. Type VII transporters have the following transmembrane helix configuration: 4+4. Therefore, as used herein, "Type IV" ABC transporters refer, for example, to ABC transporters classified as Type IV under this classification system, and "Type V" refers to Type V transporters classified under this system, and so on.

[0137] As used herein, the terms “outward conformation” and “inward conformation” refer to the alpha-helix conformations of two transmembrane domains or regions (TMDs) of an ABC transporter, packed in such a way that they form a transmembrane pore that is either accessible from the inner region of the membrane (inward) or accessible from the outer region of the membrane (outward). For example, in Gram-negative bacterial cells, ABC transporters incorporated into the inner membrane have an inward conformation in which the pore is open toward the cytoplasm and an outward conformation in which the pore is open toward the periplasm (the space between the inner and outer membranes). In Gram-positive bacterial cells or eukaryotic cells, ABC transporters incorporated into the cell membrane have an inward conformation in which the pore is open toward the cytoplasm and an outward conformation in which the pore is open toward the outside of the cell. In ABC transporters incorporated into the membranes of intracellular organelles such as mitochondria, ABC transporters have an outward conformation in which the pore is open toward the lumen of the organelle and an inward conformation in which the pore is open toward the cytoplasm / cytosol. Therefore, the outward conformation is one in which the pores are open to the lumen of the organelle.

[0138] The terms “periplasmic, extracellular, and / or luminal surfaces” of the ABC transporter refer to the surfaces or regions of the ABC transporter that are accessible when the transporter is in its outward conformation, depending on the membrane in which the transporter is located. “Periplasmic, extracellular, and / or luminal cleavage” refers to the pocket or pore formed by the packing of the alpha-helix that is accessible in the outward conformation of the protein. In some cases, for example, in the ABC transporter of Gram-negative bacteria, the outer portion of the ABC transporter faces the periplasmic surface, while in other cases, it faces the outside of the cell (e.g., extracellular) or it faces the lumen, where the ABC transporter is located within an intracellular organelle (e.g., lumen). Therefore, as used herein, “periplasmic cleavage” refers to the pocket or pore in the ABC transporter formed by the packing of the alpha-helix of the transmembrane domain, which is accessible to the periplasmic surface. As used herein, “extracellular slit” refers to a pocket or pore in an ABC transporter formed by the packing of alpha-helices of the transmembrane domain, which allows access to the extracellular environment. As used herein, “luminal slit” refers to a pocket or pore in an ABC transporter formed by the packing of alpha-helices of the transmembrane domain, which allows access to the lumen of intracellular organelles such as mitochondria, endoplasmic reticulum, and Golgi apparatus.

[0139] As used herein, a “chimeric ABC transporter” refers to an ABC transporter in which one or more loops facing the periplasm, extracellular, or lumen, and up to 50% of the transmembrane segments on either side of such loop(s), are replaced by equivalent regions from different ABC transporters. For example, the diagrams in Figures 1A–B show three periplasm (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”) inserted from a different ABC transporter is a region in the protein that would be at the same position if folded when residues are removed from the parent ABC transporter. In some cases, the region in the ABC transporter that is removed and replaced by a region derived from the chimeric ABC transporter can be determined using sequence alignment and structural information about the two proteins.

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

[0141] As used herein, the term “Gram-negative bacteria” refers to bacteria that do not retain crystal violet dye when Gram staining is performed. Gram staining is a common method for identifying common bacteria. In Gram staining, 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 characteristics 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 the solvent to remove the dye. In the final step, safranin stains Gram-negative cells red.

[0142] As used herein, the term “Gram-positive bacteria” refers to bacteria that retain crystal violet dye when Gram staining is performed. Gram staining is a common method for identifying common bacteria. In Gram staining, 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 characteristics 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 the solvent to remove the dye. In the final step, safranin stains Gram-negative cells red.

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

[0144] As used herein, the term "small molecule" refers to organic molecules having a molecular weight between 50 daltons and 2500 daltons.

[0145] As used herein, "macrocycle" or "macrocyclic molecule" refers to a cyclic polymer or the polymeric ring portion of a polymer. Macrocyclic molecules are in the size range of 500 to 2000 daltons. In some cases herein, macrocyclic molecules 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 come into direct contact with an ABC transporter. Binding fragments may be used in high-throughput screening.

[0147] In this disclosure, when referring to molecules that "bind" to an ABC transporter protein or a chimeric ABC transporter protein, the binding affinity is sufficiently strong, for example, that the interaction between 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) of 100 μM or less. D ) (that is, equivalent to affinity of 100 μM or more) is required, and in many cases K is 20 μM or less, 10 μM or less, 1 μM or less, or 500 nM or less. D It may be accompanied by.

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

[0149] The term "ATPase assay" refers to an assay used to measure the degree to which proteins, such as ABC transporter proteins, convert ATP to ADP.

[0150] The term “to treat,” and any words derived therefrom, as used herein, does not necessarily mean 100% or complete cure. Rather, treatment can mean, for example, reducing at least one symptom or condition, and in some cases, treatment can be of varying degrees, including, for example, about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. Furthermore, treatment can also include preventing, improving, or inhibiting one or more conditions or symptoms of a disorder, as well as delaying the onset of the disorder or its symptoms.

[0151] The terms “effective dose” or “therapeutic dose,” as used herein, refer to a sufficient amount of any of the molecules disclosed herein administered to alleviate, to some extent, one or more of the symptoms of a disease or condition being treated, such as an infection.

[0152] Further definitions, as needed, are included in the following sections.

[0153] II. Chimera ABC Transporter In some embodiments, the present invention includes a chimeric ABC transporter.

[0154] In some embodiments, the starting material for creating a chimeric ABC transporter is a specific ABC transporter ("parent" ABC transporter). In some embodiments, the parent ABC transporter is derived from eukaryotic cells. In some embodiments, the parent ABC transporter is a human ABC transporter. In some embodiments, the parent ABC transporter is derived from Gram-positive bacteria. In some embodiments, the parent ABC transporter is derived from Gram-negative bacteria. In some embodiments, the parent ABC transporter is one that can be embedded in the intracellular organelle membrane. In some embodiments, the parent ABC transporter is a transporter found in a specific bacterial species selected from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas cyclotolerance, Candidatus accumulibacter, Jansinobacterium agalisidamunosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2. In some embodiments, the parent ABC transporter is MsbA, for example, Escherichia coli MsbA.

[0155] In some embodiments, after a parent ABC transporter is selected, equivalent but different ABC transporters from different organisms or species are identified, and amino acid sequences or regions are taken from them to form a chimera using the parent transporter. In some cases, the objective is to identify different ABC transporters whose sequences are sufficiently different that the test molecule that binds to the parent ABC transporter does not bind to the different ABC transporter at a region that is equivalent but sufficiently similar except that the chimera folds properly into its exact architecture. In some embodiments, different ABC transporters are identified by searching for different ABC transporters that share 20–99% sequence identity with the parent ABC transporter. In some embodiments, the different ABC transporters originate from different, optionally related species or organisms, but from a homologous gene to the one being tested. For example, in some cases, the chimera may be constructed from homologous ABC transporters from two different bacterial species or genera, or from species such as humans and mice, or humans and primates, e.g., MsbA proteins from two different bacterial species. In some embodiments, the three-dimensional structure of a parent ABC transporter, if known, is compared to the three-dimensional structure of a different ABC transporter.

[0156] In some embodiments, if 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, if the parent ABC transporter is derived from a Gram-positive bacterium, the different ABC transporters are derived from another Gram-positive bacterium and, optionally, from homologous genes of other species. In some embodiments, if the parent ABC transporter is derived from a Gram-negative bacterium, the different ABC transporters are derived from a Gram-negative bacterium and, optionally, from homologous genes of other species. In some embodiments, the parent ABC transporter and the different ABC transporters are selected from two different species, selected from Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas cyclotolerance, Candidatus accumulibacter, Jansinobacterium agalisidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2. In some embodiments, the chimeric ABC transporter comprises a region of a parent ABC transporter selected from eukaryotic cells, Gram-positive bacteria, Gram-negative bacteria, Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerance, Candidatus accumulibacter, Jansinobacterium agallisidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2 and / or MsbA, and a region of a different ABC transporter selected from eukaryotic cells, Gram-positive bacteria, Gram-negative bacteria, Escherichia coli, Enterobacter cloaca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cyclotolerance, Candidatus accumulibacter, Jansinobacterium agallisidamnosum, Thiomicrospira cyclica, or Magnetospira strain-OH-2 and / or MsbA.

[0157] In some embodiments, ABC transporters are grouped into seven distinct types, I to VII, based on their transmembrane domain (TMD) folds, 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) (the entirety of which is incorporated by reference with respect to its description of the ABC transporter classification system). It may be advantageous to select different ABC transporters that are of the same type as the parent ABC transporter. 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 transporters are type V ABC transporters. In some embodiments, the parent ABC transporter is a type VI ABC transporter, and the different ABC transporters are type VI ABC transporters. In some embodiments, the parent ABC transporter is a type VII ABC transporter, and the different ABC transporters are type VII ABC transporters.

[0158] ABC transporters generally have a characteristic architecture comprising at least four domains: two transmembrane domains (TMDs) embedded within the membrane (the transmembrane segments may be part of the TMDs) and two nucleotide-binding domains (NBDs). Each TMD consists of transmembrane alpha helices, and each TMD typically has 6 to 10 transmembrane alpha helices, with most exporters having 6 transmembrane alpha helices per TMD. In some embodiments, ABC transporters have a loop facing the periplasm, extracellular space, or luminal space when the molecule is in an outward conformation, and transmembrane segments connected to both sides of such a loop. (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. TMDs are segments of amino acid chains that traverse membrane regions, and these are typically alpha-helices. The six TMDs in the schematic diagram are labeled 1-6. The schematic diagram also shows the peripheral and cytoplasmic loops of the membrane. Each loop has a first end that connects to a TMD, a second end that connects to a TMD, and a portion that contacts the peripheral or cytoplasm. The ABC transporter shown has three loops facing the peripheral plasma (e.g., loops 1, 2, and 3 corresponding to loops 1, 3, and 5 of MsbA (loops 2, 4, and 6 of MsbA face the cytoplasm)). Rectangular boxes indicate regions that can be replaced by equivalent regions from different ABC transporters, i.e., at least one of the peripheral plasma-facing loops, or all three of the loops, and up to 50% of the TMD segment.

[0160] Figure 1B shows a schematic diagram of the ABC transporter derived from the human ABC transporter ABCD4. The six TMDs in the schematic diagram are labeled 1 through 6. The schematic diagram also shows the extracellular loop and the cytosolic loop of the membrane. In the schematic diagram, the extracellular loop is labeled 1 through 3. Rectangular boxes indicate regions that can be replaced by equivalent regions derived from different ABC transporters.

[0161] Figure 1C shows a schematic diagram of the ABC transporter derived from the human ABC transporter ABCC1. While structurally different from the ABC transporters derived from E. coli and human ABCD4, as shown in Figures 1A and 1B, this transporter shares many of the same distinguishing characteristics. For example, it has extracellular loops, but instead of the three extracellular loops found in ABCD4, it has six extracellular loops labeled 1 through 6 in the schematic diagram. Similarly, it has 12 TMDs, which are labeled 1 through 12 in the schematic diagram. Rectangular boxes indicate regions that can be replaced by equivalent regions derived from different ABC transporters.

[0162] In each of the three schematic diagrams in Figures 1A–C, rectangular boxes indicate regions that may be replaced with equivalent regions derived from different ABC transporters. In some embodiments, up to 50%, 0–50%, 10–50%, 25–50%, 0–10%, 0–25%, or 10–25% of the TMD on both sides of the loop that is replaced in the chimera is also replaced. In some embodiments, the disclosure includes a chimeric ABC transporter in which up to 50% (i.e., 50%, 0–50%, 10–50%, 25–50%, 0–10%, 0–25%, or 10–25%) of the loop and transmembrane segments on both sides of the loop facing at least one peripheral, extracellular, or lumen is replaced with equivalent regions from different ABC transporters. In some embodiments, the present invention includes a chimeric ABC transporter in which at least one loop facing the periplasm, extracellular, or lumen and 50% of the transmembrane segments on both sides of the loop are replaced with equivalent regions of different ABC transporters. In some embodiments, the present invention includes a chimeric ABC transporter in which at least one loop facing the periplasm, extracellular, or lumen and up to 25% of the transmembrane segments on both sides of the loop are replaced with equivalent regions of different ABC transporters. In some embodiments, the present invention includes a chimeric ABC transporter in which at least one loop facing the periplasm, extracellular, or lumen and up to 10% of the transmembrane segments on both sides of the loop are replaced with equivalent regions of different ABC transporters. In some embodiments, the present invention includes chimeric ABC transporters in which up to 50% (i.e., 50%, 0-50%, 10-50%, 25-50%, 0-10%, 0-25%, or 10-25%) of the perimetrial, extracellular, or lumen-facing loops and the transmembrane segments on both sides of each loop are replaced with equivalent regions of different ABC transporters.

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

[0164] In some embodiments, once the locations of the ABC transporter and different ABC transporters, as well as at least one loop and / or transmembrane segment in each, are identified, a chimeric ABC transporter can be created by replacing the region of the ABC transporter with a region derived from the different ABC transporter. In some embodiments, for example, if the region derived from the ABC transporter is loop 1, then the equivalent region derived from the different ABC transporter is also loop 1. In some embodiments, for example, if the region derived from the ABC transporter is 25% of the transmembrane segment / TMD1, then the equivalent region derived from the different ABC transporter is also 25% of the transmembrane segment / TMD1.

[0165] In some embodiments, when the ABC transporter is MsbA, the chimeric ABC transporter contains an EcMsbA (amino acid sequence available in Uniprot P60752) in which one or more of the EcMsbA residues Leu47-Pro68 in peripheral loop 1 (L1), EcMsbA residues Met159-Leu171 in peripheral loop 3 (L3), and EcMsbA residues Ala262-Ile292 in peripheral loop 5 (L5) are replaced with the equivalent region of Pseudomonas cyclotolerance (PpMsbA; Uniprot A0A1G5PEL0). (In this specification, the term "loop" refers to both peripheral and cytoplasmic loops, with L1, L3, and L5 facing the peripheral plasm 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 Figure 1A.) The sequence is based on the amino acid sequence of the E. coli protein, which has deposit 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 contains EcMsbA in which peripheral loop 1 (L1, EcMsbA residues Leu47-Pro68), peripheral loop 3 (L3, EcMsbA residues Met159-Leu171), and peripheral loop 5 (L5, EcMsbA residues Ala262-Ile292) are replaced with the equivalent region of Candidatus acumulibacter species SK-12 (CaMsbA; Uniprot A0A011NLL4).

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

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

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

[0170] III. Screening methods using chimeric ABC transporters This disclosure includes, in particular, a method for identifying molecules that bind to the peripheral, extracellular, and / or luminal surfaces of specific ABC transporter proteins, using chimeric versions of proteins as described above. In some cases, the method identifies molecules that bind to cleavage in the peripheral, extracellular, and / or luminal surfaces of proteins.

[0171] In some embodiments, the method comprises determining whether a test molecule binds to the periplasm, 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 periplasm, extracellular, and / or luminal surface of a parent ABC transporter are replaced by one or more equivalent regions of the periplasm, extracellular, and / or luminal surface of a different ABC transporter; and (b) contacting the chimeric ABC transporter with a test molecule that binds to a 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 periplasm, extracellular, and / or luminal surface of the parent ABC transporter.

[0172] In some cases, for example, the molecule is first tested to determine whether it binds to the parent ABC transporter in the outward conformation by (a) trapping the parent ABC transporter in the 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 periplasm, extracellular, and / or luminal surface of the parent ABC transporter are replaced by one or more equivalent regions of the periplasm, extracellular, and / or luminal surface of a different ABC transporter, and (d) contacting the chimeric ABC transporter with the 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 periplasm, extracellular, and / or luminal surface of the ABC transporter. In any of these methods, in some embodiments, the method further includes trapping the chimeric ABC transporter in an outward conformation before contacting the chimeric ABC transporter with the test molecule. In the above method, the ABC transporter and / or chimeric ABC transporter is Mg 2+ It can be trapped in an outward conformation by treatment with ATP and vanadates (e.g., vanadate or ortho-vanadate anions).

[0173] These basic 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 to the inward conformation, as shown in the second panel on the left in Figure 6. As described herein, the transporter is Mg 2+Treatment with drugs such as ADP and vanadate combinations can trap the protein in an outward conformation, thus exposing the periplasmic, extracellular, or luminal surface of the protein and any associated periplasmic, extracellular, or luminal fissures. Figure 6. To selectively identify molecules that bind to the periplasmic, extracellular, and / or luminal surface or their associated fissures, counterselection 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 do not bind to the chimeric form under appropriate assay conditions, such as chimeric null binders, can be selected.

[0174] In some embodiments, the method is carried out using either a test molecule or an ABC transporter immobilized on beads or a matrix platform. In some cases, the parent ABC transporter and / or chimeric ABC transporter are immobilized on beads or a matrix platform using, for example, a set of biotin / streptavidin or a similar reagent. When beads are used for immobilization, they can have any shape, such as flakes or chips, spheres, or pellets. The matrix may consist of beads or smaller particles, and may be, for example, a slurry or a 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 to facilitate collection of the beads during the assay, for example, by the 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, thereby allowing the protein to attach to the matrix or beads.

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

[0176] In some embodiments, parental ABC transporters or chimeric ABC transporters are solubilized in surfactants or similar molecules that mimic biological membranes so that they maintain the appropriate folds and ability to form the correct outward conformation. Exemplary surfactants or related molecules or systems for solubilizing ABC transporters and / or chimeric ABC transporters include lauryl maltose neopentyl glycol (LMNG), and in some embodiments, 0.02% LMNG, as well as dodecyl-β-D-maltoside (DDM), brij-35, glycol-diosgenin, digitonin, amphiphore, e.g., amphiphore A8-35, and lipid nanodiscs. For example, surfactants can solubilize and stabilize transporters, while amphiphore can effectively encapsulate and stabilize hydrophobic portions of proteins, and proteins can also be stabilized within a kind of lipid bilayer created by lipid nanodiscs.

[0177] In some embodiments, a library of test molecules is screened. The library is brought into contact with chimeric ABC transporters on a matrix or beads 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 ABC transporters in the outward conformation compared to those in the outward conformation of the relevant chimeric ABC transporter may be identified as those that bind to the periplasm, 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 performed on the molecules selected in the above screening, for example, to determine their binding affinity to the parental ABC transporter and the chimeric ABC transporter, respectively, and to determine how they affect the function of the ABC transporter. Thus, for example, in some embodiments, an ATPase assay is performed to determine the ATP vs. ADP activity of the ABC transporter in the presence of the identified molecules. In some embodiments, the identified molecules that bind to the peripheral, extracellular, and / or luminal surface of the ABC transporter may act as inhibitors of the ATPase activity of the ABC transporter. ATPase assays are well known in the art, and various commercially available kits are available, for example, the Tanscreener ADP2 Assay (BellBrook Labs, Cat.#3010-1K) and the Molecular Probes ATP Determination Kit (Thermofisher, Cat.#A22066).

[0179] In some embodiments, the binding affinity of the identified molecule to the parent ABC transporter and / or chimeric ABC transporter can be determined. In some cases, this can be done with an ELISA assay similar to the one used for initial screening, for example, IC 50 A value is obtained. In some cases, a competitive ELISA assay may be performed, for example, using a parental ABC transporter bound to a matrix or beads and a chimeric ABC transporter released into solution (and vice versa). A molecule that preferentially binds to the parental ABC transporter over the corresponding chimeric ABC transporter should bind to the parental ABC transporter to approximately the same degree in the presence and absence of the chimeric ABC transporter. Such assays may be performed, for example, to confirm that a particular test molecule or molecule identified in a previous screening is selective for the peripheral, extracellular, and / or luminal surfaces of the transporter. In some embodiments, the binding assay may be performed in cell cultures to test binding to the parental ABC transporter in the cell membrane of the cell, or separately in its 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 molecules identified by screening on the function of the parent ABC transporter. For example, in some cases, molecules that bind to the peripheral, extracellular, and / or luminal surfaces of the ABC transporter act as transporter inhibitors. For example, if inhibition of ABC transporter activity is expected to result in, for example, loss of cell viability or reduced cell proliferation, a cell viability assay or proliferation assay can 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 molecules on the function of the transporter. In some cases, substrate transport by the transporter can be tested in the presence and absence of the molecule. For example, in the case of MsbA tested in the following examples, 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 the layering of the inner and outer membranes on the surface of bacterial cells, which is clearly visible by EM. See, for example, Figure 3E. Appropriate functional assays for the ABC transporter in question are known or can be readily developed based on knowledge of the art.

[0181] IV. Test molecules for screening methods In some embodiments, the disclosure includes screening methods for testing specific types of molecules, as well as molecules identified by any of the screening methods described herein as binding to the peripheral, extracellular, and / or luminal surface or clefts of specific ABC transporters. In some cases, the identified molecules do not bind to the chimeric ABC transporter used in the screening method, but they do bind to the parental ABC transporter on which the chimera is based. In some cases, the identified molecules bind to the parental ABC transporter with at least 10 times tighter affinity than the chimeric ABC transporter used for screening. In some cases, the identified molecules bind to the parental ABC transporter with at least 100 times tighter affinity than the chimeric ABC transporter used for screening. In some cases, the identified molecules bind to the parental 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- to 14-mer peptide, e.g., 6- to 12-mer, 6- to 10-mer, 6- to 8-mer, 8- to 12-mer, 8- to 10-mer, etc. In some embodiments, the peptide is 14-mer. See Table 3 and Figure 3F. In some embodiments, the peptide is 6- to 10-mer. In some embodiments, the peptide is 8- to 10-mer. In some embodiments, the peptide is 6- to 8-mer. In some embodiments, the peptide is 8-mer. See Figures 3E and 3G-L, and Table 5. In some embodiments, the peptide is 3-40-mer, 3-20-mer, 4-16-mer, 4-14-mer, or 6-14-mer, for example, 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 macrocyclic molecule. In some embodiments, the macrocyclic molecule is a 6- to 14-mer macrocyclic molecule, e.g., 6- to 12-mer, 6- to 10-mer, 6- to 8-mer, 8- to 12-mer, 8- to 10-mer, etc. In some embodiments, the macrocyclic molecule is a 14-mer macrocyclic molecule. See Table 3 and Figure 3F. In some embodiments, the macrocyclic molecule is a 6- to 10-mer macrocyclic molecule. In some embodiments, the macrocyclic molecule is an 8- to 10-mer macrocyclic molecule. In some embodiments, the macrocyclic molecule is a 6- to 8-mer macrocyclic molecule. In some embodiments, the macrocyclic molecule is an 8-mer macrocyclic molecule. See Figures 3E and 3G-L, and Table 5. In some embodiments, the macrocyclic molecule is a 3-40-mer, 3-20-mer, 4-16-mer, 4-14-mer, or 6-14-mer, for example, 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 macrocyclic molecule. In some embodiments, the macrocyclic molecule 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 described herein are small molecules. In some embodiments, the molecules tested are antibodies, which may include not only full-length antibodies such as IgG, IgM, IgA, IgD, and IgE, but also antigen-binding fragments of antibodies such as Fv, Fab', (Fab')2, scFv, nanobodies, single-chain antibodies, and bispecific or multispecific antibodies.

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

[0186] Certain macrocyclic molecules identified by screening for binders of E. coli MsbA are also included herein. In some embodiments, the macrocyclic molecule is G1118, a 14-mer macrocyclic molecule. See Figures 3F, 4A-D, and 6-. G1118 introduces 5 nM IC into E. coli MsbA, for example, in the imp E. coli strain (with a permeable outer membrane). 50 It was identified as a binding molecule. See Figure 3F. G1118 has the sequence ClAc-FWWLWDDVSWWMeFVCNH2 or ClAc-FWWLWDDVSWWMeFVCGKKNH2. In some embodiments, the macrocyclic molecule is G1365, which is an 8-mer macrocyclic molecule. See Figures 3E, 3G-L, and 4J-N. G1365 has the sequence ClAc-FVYBphMeFRVCNH2. G1365 is used to bind to imp E. coli MsbA with, for example, a 300 nM biochemical IC2. 50 It was identified as a binding agent, and when tested with imp strains having a permeable outer membrane, it has an affinity for Escherichia coli MsbA that is at least 10 times tighter than the affinity for MsbA from Acinetobacter baumannii (A. bau.). See Figures 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 by the screening described herein are ABC transporters with a K content of 20 μM or less. DIt binds to the ABC transporter with a K10 μM or less. In some embodiments, this molecule binds to the ABC transporter. D It binds to the ABC transporter with a K2M or less. In some embodiments, this molecule binds to the ABC transporter with a K2M or less. D It binds to the ABC transporter with a K of 500 nM or less. In some embodiments, this molecule binds to the ABC transporter. D It binds to the ABC transporter. In some embodiments, this molecule has a K of 1 nM or less. D It binds to the ABC transporter with 1-20 μM K. In some embodiments, this molecule is attached to the ABC transporter with 1-20 μM K. D It binds to the ABC transporter with 10-20 μM K. In some embodiments, this molecule is attached to the ABC transporter with 10-20 μM K. D It binds to the ABC transporter with 1 nM to 20 μM of K. D It binds to the ABC transporter with 1 nM to 500 nM of K. D Binding affinity can be determined by methods known in the art.

[0189] In some embodiments, molecules identified as binding to a specific ABC transporter may 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 periplasm, extracellular, or luminal surface or fissure of an ABC transporter by determining that it competes with already known molecules to bind to the periplasm, extracellular, or luminal surface or fissure of the same ABC transporter. For example, in the case of *E. coli* MsbA, macrocyclic molecules G1118 and G1365, as well as G1119 and G1122, may be used as competitors or positive controls in an assay to look for additional binders. In some such embodiments, screening is performed to determine if *E. coli* MsbA is Mg 2+ When trapped in an outward conformation, such as by treatment with ATP and vanadate, molecules that compete with G1118, G1119, and / or G1122 for binding to the peripheral surface of E. coli MsbA can be identified. In some embodiments, the molecules inhibit the binding of G1118, G1119, and / or G1122 to the ABC transporter by at least 50, 60, 70, 80, 90, or 100% in a competitive assay. In some such embodiments, screening can identify E. coli MsbA that is Mg 2+ When trapped in an outward conformation, such as by treatment with ATP and vanadate, molecules that compete with G1365 for binding to the peripheral surface of E. coli MsbA can be identified. In some embodiments, the molecules inhibit the binding of G1365 to the ABC transporter by at least 50, 60, 70, 80, 90, or 100% in the competitive assay.

[0190] In some embodiments, the 14-mer macrocyclic peptide may include 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; X5 is N, where N is any natural amino acid other than C, or Bph((S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid), Dopa(L-3,4-dihydroxyphenylalanine), MeF(N-methyl-L-phenylalanine), and MeG( Selected from non-natural amino acids selected from N-methyl-L-glycine; X6 is Y, K, A, S, D, R, or V; X7 is W, Y, or Bph; X8 is W or Y; optionally, the peptide further includes 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. 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 embodiments described above, the macrocyclic molecule is cyclized due to a thioether bond between the N-terminal chloroacetyl group of ClacF and the sulfhydryl group of the C residue.

[0191] In some embodiments, the 8-mer macrocyclic peptide may contain 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 macrocyclic molecule is cyclized due to a thioether bond between the N-terminal chloroacetyl group of ClacF and the sulfhydryl group of the C residue.

[0192] In other cases, the macrocyclic molecule has the sequence G1118, G1119, or G1122 (sequence number 1 or 2 (in the case of G1118), sequence number 10 (in the case of G1119), or sequence number 11 (in the case of G1122)). In yet other cases, the macrocyclic molecule has the sequence G1365 (sequence number 3).

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

[0194] V. Molecular complexes In some embodiments, the Disclosure includes molecular complexes comprising the ABC transporter described herein, bound to a molecule such as a peptide, small molecule, or antibody, peptide, small molecule, or antibody binding fragment. In some embodiments, the Invention includes molecular complexes comprising the ABC transporter and a macrocyclic molecule, which in some embodiments is a 6-14-mer, 6-10-mer, 6-8-mer, or 8-10-mer macrocyclic molecule. In some embodiments, this molecule is bound to the ABC transporter at KDs 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 molecular complexes comprising the chimeric ABC transporter described herein, bound to a molecule such as a peptide, small molecule, antibody, or peptide, small molecule, or antibody binding fragment. In some embodiments, the present invention comprises a molecular complex comprising a chimeric ABC transporter and a macrocyclic molecule, the macrocyclic molecule being an 8-10-mer macrocyclic molecule in some embodiments.

[0195] In some embodiments, the molecule is a peptide. In some embodiments, the peptide is a 6- to 14-mer peptide, e.g., 6- to 12-mer, 6- to 10-mer, 6- to 8-mer, 8- to 12-mer, 8- to 10-mer, etc. In some embodiments, the peptide is 14-mer. See Table 3 and Figure 3F. In some embodiments, the peptide is 6- to 10-mer. In some embodiments, the peptide is 8- to 10-mer. In some embodiments, the peptide is 6- to 8-mer. In some embodiments, the peptide is 8-mer. See Figures 3E and 3G-L, and Table 5. In some embodiments, the peptide is 3-40-mer, 3-20-mer, 4-16-mer, 4-14-mer, or 6-14-mer, for example, 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 macrocyclic molecule. In some embodiments, the macrocyclic molecule is a 6- to 14-mer macrocyclic molecule, e.g., 6- to 12-mer, 6- to 10-mer, 6- to 8-mer, 8- to 12-mer, 8- to 10-mer, etc. In some embodiments, the macrocyclic molecule is a 14-mer macrocyclic molecule. See Table 3 and Figure 3F. In some embodiments, the macrocyclic molecule is a 6- to 10-mer macrocyclic molecule. In some embodiments, the macrocyclic molecule is an 8- to 10-mer macrocyclic molecule. In some embodiments, the macrocyclic molecule is a 6- to 8-mer macrocyclic molecule. In some embodiments, the macrocyclic molecule is an 8-mer macrocyclic molecule. See Figures 3E and 3G-L, and Table 5. In some embodiments, the macrocyclic molecule is a 3-40-mer, 3-20-mer, 4-16-mer, 4-14-mer, or 6-14-mer, for example, 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 macrocyclic molecule. In some embodiments, the macrocyclic molecule has at least one lipophilic side chain and at least one positively charged side chain.

[0197] In some embodiments, the molecules in the complex are small molecules. In some embodiments, these molecules are antibodies, and these may include not only full-length antibodies such as IgG, IgM, IgA, IgD, and IgE, but also antigen-binding fragments of antibodies such as Fv, Fab', (Fab')2, scFv, nanobodies, single-chain antibodies, bispecific or multispecific antibodies.

[0198] In some embodiments, this molecule is a peptide binding fragment, a small molecule binding fragment, or an antibody binding fragment (e.g., an antigen binding fragment).

[0199] In some embodiments, the molecules in the complex are G1118, G1365, G1119, or G1122. In some embodiments, the molecules in the complex are, for example, the ABC transporter is Mg 2+ Upon treatment with ATP and vanadate, the molecules are trapped in an outward conformation and compete with macrocyclic molecules G1118, G1119, and / or G1122 for binding to the peripheral surface of ABC transporters such as E. coli MsbA. In some embodiments, the molecules inhibit the binding of G1118, G1119, and / or G1122 to ABC transporters by at least 50, 60, 70, 80, 90, or 100% in competitive assays. In some embodiments, the molecules are, for example, Mg 2+ When ABC transporters are trapped in an outward conformation by treatment with ATP and vanadate, the macrocyclic molecule G1365 competes with the periplasmic surface binding of ABC transporters such as E. coli MsbA. In some embodiments, the molecule inhibits the binding of G1365 to ABC transporters by at least 50, 60, 70, 80, 90, or 100% in competitive assays.

[0200] VI.Use In some embodiments, the ABC transporter is a bacterial ABC transporter. For example, in some cases, bacterial ABC transporters are candidate antibiotic targets due to relatively low sequence homology to mammalian ABC transporters, for example. In some cases, the bacterial ABC transporter is an MsbA transporter derived from a bacterial species or pathogen. In some cases, the screening methods described herein may be used to identify molecules that inhibit bacterial ABC transporters. Such molecules may have antibiotic activity. For example, the macrocyclic molecules G1118 and G1365 described herein have been found to inhibit the LPS transport activity of Escherichia coli MsbA and also inhibit cell proliferation, respectively. Thus, this disclosure also encompasses the use of molecules identified by the screening of bacterial ABC transporters described herein in the treatment of infections in subjects such as bacterial infections.

[0201] VII. Kit This disclosure also includes kits containing reagents related to the screening methods described herein. In some cases, the kits contain chimeric ABC transporters. In some cases, the kits contain reagents used in the screening methods described herein, with or without a specific chimeric ABC transporter. In some cases, the kits contain parental (non-chimeric) ABC transporters.

[0202] In some embodiments, the kits herein may include parental or chimeric ABC transporters attached to a matrix. In some embodiments, the kits herein may include ABC transporters attached to matrix particles such as beads. Such beads may have any shape, such as flakes or chips, spheres, or pellets. 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 transporters may or may not be pre-attached to the matrix. In some embodiments, reagents are included to facilitate the attachment of ABC transporters to beads or the matrix, such as through a biotin-streptavidin or similar system.

[0203] In some embodiments, the kit may include reagents related to the screening method herein. In some embodiments, the kit may include some or all of the reagents necessary to determine whether the test molecule binds to the peripheral, extracellular, and / or luminal surface of the ABC transporter. The kit may include, for example, one or more surfactants for solubilizing the ABC transporter and / or chimeric ABC transporter. Exemplary surfactants or related molecules or systems for solubilizing the ABC transporter and / or chimeric ABC transporter include lauryl maltose neopentyl glycol (LMNG), and in some embodiments, 0.02% LMNG, as well as dodecyl-β-D-maltoside (DDM), brij-35, glycol-diosgenin, digitonin, amphiphore, e.g., amphiphore A8-35, and lipid nanodiscs. The kit may include, for example, ATP, Mg2+, vanadate, and / or sodium orthovanadate, or other reagents for trapping the ABC transporter in an outward conformation. The kit may include, for example, one or more wash buffers. In some embodiments, the wash buffer may include Tris, MgCl2, 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 against an ABC transporter in or out of the presence of the test molecule.

[0204] In some embodiments, the kit may include a test molecule or a library of test molecules, e.g., peptides, small molecules, and / or antibodies. In some embodiments, the peptides in the kit may be macrocyclic molecules. In some embodiments, the kit may include test molecules that are conjugated fragments of peptides, small molecules, or antibodies. The kit may also include control molecules such as a positive control known to bind to the periplasm, extracellular, and / or luminal surface of a particular ABC transporter, or a negative control that does not bind at that site or binds to a chimeric ABC transporter but not to its parent ABC transporter.

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

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

[0207] The following are examples of the methods and compositions of the present disclosure. These examples are not intended to limit the present disclosure, but are for illustrative purposes only, and it should 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 the inversion of lipopolysaccharide (LPS) across the inner membrane (IM) and subsequently to the cell surface. Structural studies have revealed the alternating access mechanism for LPS transport by MsbA, but how substrate selectivity is achieved remained unclear. Previous structures of apoMsbA revealed an inward conformation in which LPS is bound to a central space within the entrance, shielded from a large phospholipid bilayer. In this encapsulated location, LPS is coordinated by a conserved ring of basic residues in which all acyl chains are confined to hydrophobic cavities.

[0209] Unbiased, high-throughput screening was performed on purified WT EcMsbA. By monitoring MsbA ATPase activity, it was found that quinoline and benzophenone class inhibitors trap the inward conformation (confirmation) by targeting the membrane-exposed binding site. While these efforts validated MsbA as a potential antimicrobial target, the small molecule series could not proceed due to its high lipophilicity and poor physiochemical properties. In particular, all small molecule modulators of ABC transporters that have been structurally characterized so far also bind to the inward state, increasing the likelihood that traditional drug discovery methods are inherently biased towards these inward states. Therefore, whether any state of the MsbA transport cycle is acceptable for drug discovery remains a fundamental unresolved question.

[0210] To overcome the challenges associated with targeting the hydrophobic membrane-embedded binding site in MsbA, a novel inhibitor discovery strategy was devised. To discover inhibitors with improved biochemical properties, the solvent-contactable region on the peripheral surface of MsbA was targeted. In particular, a key aspect of the MsbA transport cycle is the significant shift from an inward-facing to an outward-facing state, which exposes a large, solvent-contactable rift to the peripheral surface (Panel 1 in Figure 6). Under biochemical conditions, MsbA can be trapped in an outward-facing conformation that stabilizes this peripheral rift for potential inhibitor discovery (Figures 3A and 9A–12B, as well as Example 7). To bias selection towards the direction of the solvent-exposed peripheral rift, a counter-selection strategy was utilized using MsbA chimeras in which the peripheral surface of the transmembrane segment is collectively replaced with sequences derived from a distantly related ABC transporter (Figures 3A and 9A–12B, as well as Example 7). Following multiple screenings involving enrichment, G1118 and G1365 were identified as two macrocyclic compounds that bind to MsbA. Both G1118 and G1365 demonstrated selective and state-dependent binding to MsbA and exhibited potent biochemical and phenotypic activity. High-resolution structures of these macrocyclic molecules in complex with MsbA reveal the molecular basis for their state-dependent inhibition and represent the first known antagonists targeting outward cleavage in the ABC transporter. Thus, our strategy presents a template for the development of selective modulators of MsbA, and by extension, for the development of other challenging drug targets.

[0211] Example 1: Materials and methods for the preparation and expression of a chimeric MsbA transporter protein a. Design of chimeric proteins The wild-type MsbA transporter sequence derived 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 repeating the BLAST search, candidate MsbA transporters with approximately 40% overall sequence identity to EcMsbA, including within the peripheral loop region, were selected for further investigation. Subsequently, multiple sequence alignments and structural homology models (SWISSPROT) of the candidate MsbA homologs were manually analyzed to select candidate materials for subsequent chimeric construction. Based on these analyses, candidate MsbA transporters derived from the following Gram-negative bacterial strains were selected for chimeration with EcMsbA: Pseudomonas cyclotolerance (PpMsbA), Candidatus accumulibacter (CaMsbA), Jansinobacterium agallisidamnosum (JaMsbA), Thiomicrospira cyclica (TcMsbA), and Magnetospira strain-OH-2 (MqMsbA). To generate chimeras, the corresponding sequences in peripheral loop 1 (L1, EcMsbA residues Leu47-Pro68), peripheral loop 3 (L3, EcMsbA residues Met159-Leu171), and peripheral loop 5 (L5, EcMsbA residues Ala262-Ile292) were simultaneously replaced with equivalent regions of candidate MsbA homologs to generate chimeric constructs (Figure 10A).

[0212] Five chimeric constructs were synthesized (Genscript) and cloned into a modified pET-52b expression vector (EMD Millipore) for restriction-independent cloning. The chimeras were then overexpressed using the E. coli host Rosetta2 (DE3; EMD Millipore) by auto-induced fermentation at 17°C for 64 hours. Cells were collected, and the 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 peripheral sequences derived from ) were readily expressed and could be purified from E. coli with recovery yields similar to those of WT MsbA. The final buffer for the purified MsbA chimeric protein was 20 mM Tris (pH 8.0), 100 mM NaCl, and 0.005% lauryl maltose neopentyl glycol (LMNG; wt / v). Protein aliquots were rapidly frozen and stored at -80°C.

[0213] b. Protein expression and purification Wild-type (WT) MsbA transporters (E. coli; EcMsbA) derived from Escherichia coli, as well as peripheral 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 certain biochemical assays as shown; Anatrace), lauryl maltose neopentyl glycol (LMNG, for biochemical assays; Anatrace), or 3α-hydroxy-7α,12α-di-((O-β-D-maltosyl)-2-hydroxyethoxy)-colan (FA3, for crystallographic studies) as solubilizing surfactants. WT MsbA proteins derived from Enterobacter cloacae (EnMsbA), Klebsiella pneumoniae (KpMsbA), and Pseudomonas aeruginosa (PaMsbA) were similarly expressed and purified using LMNG as a solubilizing surfactant. The final buffer for the purified wild-type MsbA chimeric protein 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 rapidly frozen and stored at -80°C.

[0214] In some cases, the recognition motif for E. coli BirA biotin ligase (GLNDIFEAQKIEWHE) is incorporated between the amino-terminal FLAG affinity tag sequences, enabling efficient site-directed biotinylation (avidity) of purified MsbA. Evaluation of the ATPase activity and pharmacology of the chimeric MsbA has demonstrated that these large sequence changes can be tolerated without significant loss of biochemical activity or apparent structural changes at known antagonist receptor sites. In particular, MsbA in which the peripheral surface of EcMsbA is replaced with a sequence from a candidate MsbA homolog derived from Magnetospira spirillum strain-OH-2. Chim5 It remained sensitive to both the quinoline and benzophenone classes of previously identified small molecule MsbA inhibitors.

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

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

[0217] Cells expressing MsbA were collected, 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, LMNG was added to 1% (wt / v), and protein solubilization was performed by gentle stirring at 4°C overnight. After centrifugation at 185,000 ×g for 1 hour, 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)) at 4°C for 1 to 2 hours. 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 FLAG peptide. Each wild-type or mutant MsbA protein was passed through 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 to 10 mg / mL using a Vivaspin® centrifugal concentrator (50K molecular weight cut-off). Protein aliquots were flash-frozen and stored at -80°C.

[0218] c. Reconstitution into amphipol When use of amphipol-incorporated MsbA is indicated, purified MsbA in detergent was adjusted to 1 mg / mL and reconstituted into A8-35 amphipol (Anatrace) (Zoonens and Popot, 2014) as previously described (Ho et al., 2018). The final buffer for the purified amphipol-incorporated 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 MsbA sequences derived from the Enterobacteriaceae family of Gram-negative bacteria (taxid: 543) were identified using a BLAST search against the refseq_select database with 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 implemented in AL2CO (Pei and Grishin, 2001).

[0220] Example 2: Screening Materials and Methods for Identifying MsbA-binding Macrocycles a. Macrocyclic peptide library design Using counter-selection with a chimeric MsbA protein, peptide macrocycles were tested to 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 the initiator in a genetically reprogrammed in vitro translation system (Kashiwagi et al., 2013). The genetic code was designed 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 a downstream cysteine residue, yielding the macrocyclic peptide.

[0221] b. Selection of periplasmic MsbA-binding molecules Affinity selection of macrocyclic peptides binding to MsbA was performed using site-directed biotinylated EcMsbA solubilized in 0.02% LMNG, and the peptides were trapped in an outward conformation by including 50 μM ATP, 10 mM Mg2+, and 200 μM sodium ortho-vanadate throughout the selection process. Briefly, a 10 μM mRNA library was hybridized with a peptide-linker (11 μM) at room temperature (RT) for 3 minutes. The mRNA library was translated in a genetically reprogrammed in vitro translation system at 37°C for 30 minutes to produce a peptide-mRNA fusion library (Goto et al., 2011; Kawakami et al., 2013). Each reaction product contained 2 μM mRNA-peptide-linker conjugate, 12.5 μM initiator tRNA (ClAc-L-Phe aminoacylated tRNAfMet), and 25 μM of each elongator tRNA aminoacylated with specified non-regular / regular amino acids. Translation was performed on a 20 μL scale during the first selection. Post-translation, the reaction product was quenched with 17 mM EDTA. The product was then reverse transcribed using RNase H-inactive reverse transcriptase (Promega) at 42°C for 30 minutes, and the buffer was replaced with vanadate buffer: 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.02% LMNG (wt / v), 50 μM ATP, 1 mM DTT, and 200 μM 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 at 4°C for 60 minutes. Streptavidin-coated beads (Dynabeads® M-280 streptavidin, ThermoFisher Scientific) were added, and the mixture was incubated for 10 minutes to isolate macrocyclic molecules bound to the peripheral surface of EcMsbA. The beads were washed three times with cold vanadate buffer, and the cDNA was eluted from the beads by heating at 95°C for 5 minutes. The fractional recovery from the affinity selection step was evaluated by quantitative PCR using Sybr Green I on a LightCycler® thermal cycler (Roche). After six affinity maturation cycles, off-rate selection was further repeated two times by increasing the pre-elution washing stringency to identify high-affinity binders. The final enriched cDNA was sequenced using a MiSeq next-generation sequencer (Illumina).

[0223] c. Mutation scan of macrocyclic molecules G1118 and G1365 For mutation scans of the macrocyclic molecules G1118 (Table 4 below) and G1365 (Table 6 below), site-saturated DNA libraries were constructed based on these parent sequences by pooling multiple DNA templates, each containing a single "NNU" degenerate codon at a different position, so that any single amino acid change relative to the parent sequence would be sampled post-translated. The gene 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 peptide. After in vitro translation, a single affinity selection was independently performed using the two macrocyclic molecule libraries 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: the NGS frequency in the output pool was divided by the NGS frequency in the input pool and normalized to the parent value.

[0224] Example 3: Materials and methods for characterizing MsbA enzyme variants and strains a. plasmid The following plasmids (Table 1) were used in E. coli phenotypic studies. TIFF0007914136000001.tif75170

[0225] b. MsbA ATPase assay The ATPase activity of MsbA was measured using the Transcreener ADP2 FP Assay (BellBrook Labs). IC50 of MsbA inhibitors 50To determine the reaction, the compound was incubated with a 2× MsbA enzyme solution for 10 minutes, and then a 2× ATP solution was added to initiate the ATPase reaction. The final reaction conditions were MsbA enzyme 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), and 50 μM ATP. The following concentrations were used for each MsbA variant: 5 nM EcMsbA, EnMsbA, KpMsbA, and 80 nM PaMsbA reconstituted in Amphipole; 15 nM EcMsbA and EcMsbA-chim5 in LMNG surfactant; and 22 nM EcMsbA and 27 nM EcMsbA-12G7 complex in DDM surfactant. The enzymatic reaction products were incubated at room temperature for 1 hour and then quenched by adding Transcreener Detection buffer. IC 50 This was determined by fitting the inhibitory dose-response curve to a nonlinear four-parameter inhibitory model (GraphPad Prism).

[0226] c. Intracellular IC 50 decision Intracellular IC of various bacterial strains 50 (Table 2) was determined. TIFF0007914136000002.tif72170

[0227] Eight 2x dilutions of each compound, each containing 200 nL aliquots, were dispensed in double rows into 384-well plates using an Echo Liquid Handler (Labcyte). The strains were grown overnight for 16 hours in cationically modified Mueller-Hinton liquid medium containing 0.002% Tween, washed with PBS, and then osmotically treated in cationically modified Mueller-Hinton liquid medium containing 0.002% Tween and 10% glycerol. 600The mixture was resuspended in 1:1 solution and frozen in 100 ml aliquots. For each experiment, the aliquot was thawed on ice and diluted 1:1000 in cation-modified Mueller-Hinton liquid medium containing 0.002% Tween. 10 μL of cells / well was added to a 384-well plate containing the compound. The plate was incubated at 37°C for 6 hours, then 10 μL of BacTiter-Glo reagent (Promega) was added to each well, and luminescence was measured. IC50 was measured using Genedata Screener software. 50 I calculated it.

[0228] d. Time course of growth of E. coli supplemented with the MsbA allele Cultures of *E. coli* MG1655 msbA-cKO lptD(imp4213) carrying a pLMG18 vector expressing a wild-type (WT) or mutant allele 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 chromosomes. The overnight cultures were pelletized, washed with 5 mL of phosphate-buffered saline, and then oxidized in LB. 600 The cultures were resuspended in 1 oz solution. Then, each culture was diluted 1:100 in 5 mL of LB and 5 mL of LB + 2% arabinose. All cultures were grown on a roller drum at 37°C, and the OD600 was measured every hour. Growth curves were created by repeating the process three times on different days.

[0229] e. Western blot Cultures of *E. coli* MG1655 msbA-cKO lptD(imp4213) carrying a pLMG18 vector expressing a WT or mutant allele 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 chromosomes. A control strain without the plasmid was grown under the same conditions, but without chloramphenicol. The overnight cultures were diluted 1:100 in 15 mL of the same medium in a 50 mL conical tube and grown at 37°C for 3 hours with shaking. These cultures were pelletized, washed with 15 mL of PBS, and OD in LB. 600 The cultures were then resuspended in 1 oz solution. Each culture was then diluted 1:100 in 20 mL of LB for strains expressing the E. coli MsbA allele or strains without a plasmid control, and in 20 mL of LB + 1 mM IPTG for strains expressing the Acinetobacter baumannii MSbA allele. 7.5 mL of each cell with OD=1 was pelleted and lysed as a 2% arabinose sample. The diluted cultures were grown at 37°C for 3 hours, then pelleted and lysed.

[0230] To prepare the 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) with 15 mg / mL of lysozyme added, and incubated at 37°C for 30 minutes. The lysates were pelletized in a microcentrifuge at maximum speed at 4°C for 10 minutes, and the supernatant was transferred to a clean tube. The samples were dissolved in MES buffer on 20-well 4-12% Bis-Tris midi gels (BioRad), and the loading was controlled using GroEL as a loading control. 600The samples were normalized. Western blots were transferred to nitrocellulose using the iBlot system (Thermo Fisher Scientific). The membrane was blocked with Odyssey TBS blocking buffer (Li-Cor, #927-50000), and detected with 1:1000 anti-FLAG M2 (Sigma, #F3165) + 1:10000 anti-GroEL (Enzo, #ADI-SPS-875), followed by 1:10000 anti-IRDye 800CW mouse and anti-IRDye 680RD rabbit, respectively (Li-Cor, #925-32210 and #926-68071).

[0231] f. MIC assay The MIC of the culture medium was determined in cation-modified Mueller-Hinton liquid medium containing 0.002% Tween-80, as previously described (Ho et al., 2018).

[0232] Isolation of g.PD-1365-resistant mutants MG1655 lptD (imp4213) was placed 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), with a 0.005 OD (Oxygen Diffusion) concentration. 600 The cells were inoculated and grown at 37°C for 2 days. G1365·I2,Dopa3 is a close analogue of G1365 with two amino acid substitutions (Val2Ile and Tyr3Dopa; Figure 14A). When cultures from two wells representing growth were streaked onto LB agar plates containing 50 μM G1365·I2,Dopa3, only one culture formed colonies on the selection plate. PCR amplification and sequencing of the MsbA ORF from this resistant strain revealed a point mutation at residue 252, resulting in an amino acid change from aspartic acid to asparagine. 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: Materials and methods for electron microscopy Electron microscopy was performed on Escherichia coli CFT073 lptD(imp4213) or CFT073 lptD(imp4213)msbA-cond-ko cells rescued with the pLMG18 plasmid expressing the indicated E. coli MsbA variant, as described previously (HO et al., 2018). Where the use of a macrocyclic or small molecule inhibitor of MsbA is indicated, an overnight culture of CFT073 lptD(imp4213) grown in LB was diluted 1:100 in LB, grown to log phase at 37°C for 2.5 hours, then pelleted, washed twice with PBS, and adjusted to an OD in LB 600 = 0.1 by resuspension. Then 50 µl of a 10 mM stock of the indicated inhibitor was added to 5 mL of resuspended CFT073 lptD(imp4213), and the culture was incubated at 37°C for 3 hours. Cells were pelleted, washed, fixed and processed for transmission electron microscopy as described previously (HO et al., 2018).

[0234] Example 5: Synthesis of macrocyclic compounds a. Synthesis of benzophenone (G758) TIFF0007914136000003.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 hours 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. TIFF0007914136000004.tif14170

[0237] Step 2: 2-(4-methylpiperidine-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 hours and concentrated under reduced pressure to obtain a crude product. This crude 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). TIFF0007914136000005.tif14170

[0239] Step 3: 2-(4-methylpiperidine-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), oxalyl chloride (1.4 mL, 15.9 mmol) and two drops of NN-dimethylformamide were added. The resulting mixture was stirred at 20°C for 1.5 hours and concentrated under reduced pressure to obtain crude 2-(4-methyl-1-piperidyl)acetyl chloride (1.0 g, 5.7 mmol, yield 89.5%). The crude was used directly in the next step without further purification. TIFF0007914136000006.tif22170

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

[0242] A solution of 2-bromo-6-chloropyridine-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) was stirred in dichloromethane (20 mL) for 12 hours and diluted with dichloromethane (50 mL). The solution was washed with water (2 × 15 mL) and 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 obtain tert-butyl N-(2-bromo-6-chloro-3-pyridyl)carbamate (1.3 g, 4.2 mmol, yield 35.1%) as a white solid. TIFF0007914136000007.tif35170

[0243] Step 5: tert-butyl(6-chloro-2-((3-fluoropyridine-2-yl)(hydroxy)methyl)pyridine-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), butyllithium (2.5 M in hexane, 1.6 mL, 3.90 mmol) was added at -78°C. The mixture was stirred at -78°C for 1 hour, and then 3-fluoro-2-formylpyridine (610.1 mg, 4.88 mmol) was added. The resulting mixture was stirred at -78°C for 4 hours and quenched by adding saturated ammonium chloride (10 mL). The solution was diluted with ethyl acetate (50 mL), washed with water (2 × 10 mL) and 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, and tert-butyl N-[6-chloro-2- [(3-fluoro-2-pyridyl)-hydroxymethyl]-3-pyridyl]carbamate (550.0 mg, 1.55 mmol, yield 47.8%) was obtained as a yellow oily substance. TIFF0007914136000008.tif36170

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

[0246] tert-butyl N-[6-chloro-2-[(3-fluoro-2-pyridyl)-hydroxymethyl]-3-pyridyl] in dichloromethane (15 mL) A mixture of carbamate (460.0 mg, 1.30 mmol) and desmartin periodinane (2.2 g, 5.20 mmol) was stirred at 20°C for 3 hours. The reaction mixture was diluted with dichloromethane (20 mL), washed with water (2 × 10 mL) and 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 obtain tert-butyl N-[6-chloro-2-(3-fluoropyridine-2-carbonyl)-3-pyridyl)carbamate (300.0 mg, 0.85 mmol, yield 65.6%) as a white solid. TIFF0007914136000009.tif33170

[0247] Step 7: tert-butyl(6-cyano-2-(3-fluoropicolinoyl)pyridine-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)phosphine iron (72.5 mg, 0.13 mmol) in N,N-dimethylacetamide (10 mL) was heated at 90°C for 2 hours under microwave conditions and concentrated under reduced pressure. The residue was purified by column chromatography using elution with 0% to 50% ethyl acetate in petroleum ether to obtain tert-butyl N-[6-cyano-2-(3-fluoropyridine-2-carbonyl)-3-pyridyl]carbamate (150.0 mg, 0.44 mmol, yield 67.0%) as a white solid. TIFF0007914136000010.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 hour and concentrated under reduced pressure to obtain crude 5-amino-6-(3-fluoropyridine-2-carbonyl)pyridine-2-carbonitrile trifluoroacetate (84.0 mg, 0.25 mmol, yield 71.4%) as a yellow oily substance. TIFF0007914136000011.tif34170

[0251] Step 9: N-(6-cyano-2-(3-fluoropicolinoyl)pyridine-3-yl)-2-(4-methylpiperidine-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 hours and concentrated under reduced pressure. The residue was purified by reverse-phase chromatography (28-58 mg of acetonitrile in water / 0.05% NH4OH) to obtain N-[6-cyano-2-(3-fluoropyridine-2-carbonyl)-3-pyridyl]-2-(4-methyl-1-piperidyl)acetamide (9.40 mg, 0.02 mmol, yield 6.8%) 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; Measured value 382.1.

[0253] b. Synthesis of G1118, G1365, and their derivatives A macrocyclic thioether peptide was synthesized using standard Fmoc solid-phase peptide synthesis (SPPS). After coupling all amino acids, the deprotected N-terminus was chloroacetylated on resin, followed by global deprotection using a trifluoroacetic acid (TFA) deprotection cocktail. The peptide was then precipitated from the deprotection solution by adding more than 10 times excess diethyl ether. The crude peptide pellet was then dissolved and re-pelleted three times using diethyl ether. After the final wash, the pellet was allowed to dry, then resuspended in DMSO, and triethylamine was added to induce 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 peptide was purified using standard reversed-phase HPLC. Molecular mass was confirmed by single quadrupole LC / MS (LCMS-2020 system, Shimadzu).

[0254] c. Synthesis of other 14-mer peptides Further 14-mer peptides were designed according to the following formula (see Figure 4E for a graphical representation of a particular variant): ClacF-X1-X2-L-X3-X4-D-X5-X6-X7-X8-MeF-VC[wherein, i.X1 is W, V, or Y, ii. X2 is either W or Y, iii. X3 is either 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 from a non-natural 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 either W or Y] Optionally, the peptide further includes 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 following this formula were also tested for their inhibitory effect on MsbA ATPase activity in the assay described in Example 3 above, yielding 0.2-0.8 μM IC2. 50 We found that it possesses a value. The minimum inhibitory concentration (MIC) for inhibiting E. coli MsbA (EcMsbA) is greater than 100 μM, and for inhibiting cell proliferation of UPEC imp strains, EC 50 The concentration was approximately 2–8 μM for all but one of the molecules tested (exceeding 100 μM for the last molecule tested). Overall, these data indicate that macrocyclic peptide G1118 and related peptides following the above formula can generally inhibit MsbA and MsbA-related cellular activity.

[0255] Example 6: Materials and methods for determining protein structure a. Protein crystallization The inhibitor G758 was added to MsbA-chim5 at a concentration of 10 mg / ml / L, purified to a final concentration of 1 mM in FA3, and incubated on ice for 1 hour. Exogenous LPS was not added at any point. The G758-LPS-MsbA-chim5 complex was crystallized at 19°C using sitting-drop vapor diffusion by mixing the complex with the 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 in one week, to improve diffraction, the crystals were dehydrated by first immersing them in 10% ethylene glycol, 15% PEG 3350, and 100 mM HEPES (pH 7.0) for 30 minutes, and then in 20% ethylene glycol, 20% PEG 3350, and 100 mM HEPES (pH 7.0) for another 30 minutes, before rapid freezing in liquid nitrogen.

[0256] b. Structural determination, refinement, and analysis Diffraction data were collected at 100K using beamline 17ID of the Advanced Light Source. X-ray diffraction data were incorporated and scaled using autoPROC (Vonrhein et al., 2011), including 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 substitution using PHENIX (Adams et al., 2010), using a single 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 construction in Coot were guided by omit map scrutiny. In combination with the use of omit maps in the early stages of refinement, five chimeric sequences were replaced in the model with WT E. coli MsbA sequences. Strict shape and secondary structure constraints were applied throughout the refinement process to maintain strict stereochemistry, but non-crystallographic symmetry constraints were not applied until the end. Strong omission densities were evident very early on for G758 and LPS (central space within the inlet), resembling the densities of unassigned surfactants, but these were only modeled very late in the refinement process. The shape of the final G758-LPS-MsbA-chim5 model was evaluated 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. Sample preparation and data acquisition for cryo-electron microscopy (cryo-EM). Purified MsbA was mixed with Fab 12G7 in a 1:1 (w / w) ratio. 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 incubated with 20 mM MgCl2, 15 mM ATP, and 3.3 mM sodium vanadate on ice for 1 hour. The vanadate-trapped sample was further purified by size exclusion chromatography using a running buffer consisting of 20 mM Tris (pH 8), 100 mM NaCl, 0.03% αDDM, 20 mM MgCl2, 15 mM ATP, and 1 mM sodium vanadate. The peak fraction was concentrated to 2 mg / mL and incubated with 37 μM G1118 or 67 μM G1365 derivative (Figure 14A) and 0.007% Brij-35. After incubation with ligand on ice for 30 minutes, 3.5 μL of each sample was frozen on a glow discharge Ultrafoil 2 / 2 200 mesh grid using a Vitrobot at 4°C and 100% humidity. Cryo-EM data was collected on a Titan Krios (ThermoFisher Scientific, Waltham, MA) operating at 300 kV, equipped with a BioQuantum energy filter with a 20 eV slit width and a K2 Summit electron direct detection camera (Gatan, Inc., Pleasanton, CA). Images were recorded at a nominal magnification of 165,000x using a physical pixel size of 0.849 Å. Each image stack had a frame time of 0.25 seconds and approximately 40 e - / Å 2 It contains 40 frames with a total exposure of [value missing]. Data was collected within a fixed defocus range of 1.0 to 2.0 μm.

[0258] Cryo-EM data processing for d.G1118 Cryo-EM data for G1118 were processed using cisTEM (Grant et al., 2018) (Figure 15A-E). A total of 9,999 movies were corrected to full-frame motion using cisTEM and binned to a pixel size of 1.3 Å. The contrast transfer function parameters were fitted to CTFFIND-4 using the 30–4.0 Å bands of the spectrum, and 5,984 micrographs with better CTE resolution than 8 Å were selected for further processing. A total of 1,206,819 particles were picked up using cisTEM. An Ab initio model was generated using cisTEM with well-aligned particles in the 2D class average. The complete dataset was refined for an Ab initio model with C1 symmetry. Angular assignments from the consensus refinement were used for 3D classification without alignment. 410,386 particles from the best class were further manually refined by repeatedly increasing the resolution limit used for refinement. Densities for surfactant micelles and Fab were filtered to 20 Å by using a mask around MsbA. The refinement was converged to a resolution of 3.0 Å (Fourier shell correlation (FSC) = 0.143 determined by cisTEM) using a maximum frequency of 4 Å used for alignment. For model construction and figure generation, the map was sharpened in cisTEM with the following parameters: flattening from a resolution of 10 Å, application of a pre-cutoff B-factor of -90 Ų from the reciprocal space origin, and application of an exponential 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) (Figures 17A-E). A total of 10,273 micrographs were collected for G6671 and 17,575 for G3081. Each dataset was corrected for full-frame motion 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 bands of the spectrum. A total of 937,971 particles were picked up for G6671 and 993,151 particles for G3081 using cisTEM. Each dataset was individually subjected to a series of 2D and 3D classifications in RELION. Precise placement of macrocyclic molecules was not possible due to the limited resolution when processing each dataset individually. To increase the average output, 218,877 particles from the G3081 dataset and 222,975 particles from the G6671 dataset were merged, and refinement was used for a series of automated refinements in cisTEM. The density outside the MsbA mask was filtered to 20 Å. For MsbA model construction, an additional round of local refinement was performed with a high resolution limit of 4 Å to obtain 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 macrocyclic molecular model construction, obtaining a 3.2 Å map with a good density of G1365. For model construction and figure generation, maps were sharpened in cisTEM using the following parameters: flattening from a resolution of 8 Å, application of a pre-cutoff B-factor of either -90 Ų for MsbA maps or -120 Ų for TMD-focused refinement, and application of an index of merit filter. Electrostatic maps were calculated in PyMol using APBS.

[0260] f. Model refinement A homology model of *Escherichia coli* MsbA was generated using SWISS-MODEL (Waterhouse et al., 2018) based on the AMPPNP binding structure (PDB: 3B60) of *S. typhimurium* MsbA. The resulting model was fitted as a rigid body to a G1118 cryo-EM map. The model was refined through manual adjustments using ISOLDE (Croll, 2018) in Coot (Emsley and Cowtan, 2004) and ChimeraX (Goddard et al., 2018; Pettersen et al., 2021), followed by real-space refinement using phenix.real_space_refinement (Afonine et al., 2018). Macrocyclic molecules were parameterized using Corina (Molecular Networks GmbH) and manually fitted to density. The MsbA coordinates from the G1118 structure were then used as the initial model for G1365 before iterative model construction. The model was 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 counter-selection and outward conformational trapping (preliminary screening). To evaluate the ability of different selection schemes to identify 14-mer macrocyclic molecules that selectively bind to the peripheral surface of MsbA, the results of several selection schemes were compared by ELISA assay (Table 3). Many peptides demonstrated specific binding to MsbA. The peptides showed both vanadate-dependent binding and no reduction in recovery in the presence of chimeras. TIFF0007914136000012.tif255166

[0262] As shown in the upper left of Table 3, (a) selection performed in solution, in the presence of vanadate to trap EcMsbA in its outward conformation, and using counter-screening with chimeric MsbA, resulted in the identification of groups of macrocyclic molecules at frequencies of approximately 26% to 2% (Table 3; first 6 rows of the %freq column). Similar sets of specific macrocyclic molecules were identified when the screening was performed (b) in solution, with vanadate but without counter-selection, (c) on beads, without vanadate but with counter-selection, and (d) on beads, with vanadate but without counter-selection. (See the leftmost column and the "%freq" column in Table 3.) Subsequently, each of the identified macrocyclic molecules was subjected to ELISA assays in the presence and absence of vanadate to trap the EcMsbA protein in its outward conformation, and its binding to EcMsbA, the unrelated membrane protein OprF, and streptavidin beads (SA) was tested. The results were as follows: (a) In solution, molecules selected with vanadate using counter-screening bound well to EcMsbA in the presence of vanadate, but poorly in the absence of vanadate ("+ / - ratio" column, first 6 rows), and did not bind well to either OprF alone or streptavidin beads alone under either condition. Most of the molecules identified in the screening under conditions (b), (c), and (d) bound to EcMsbA to a similar degree whether or not they were trapped in the outward conformation with vanadate, indicating that both trapping the protein in the outward conformation and using chimeric protein-versus-screening are more effective in identifying molecules that bind to the peripheral surface 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 the chimeric EcMsbA protein as a binding competitor. Similar degrees of binding in the presence and absence of EcMsbA, and therefore ratios greater than 1.0, indicate that the molecules are selective for EcMsbA and that the chimeric protein does not act as a competitor for binding to EcMsbA.(See the right-hand column of Table 3.) The molecules identified under the screening conditions of (a) had similar levels of binding in the presence and absence of the chimera, as indicated by ratios of 1.0 or greater in the assay. This is consistent with the binding of these molecules to the peripheral surface of EcMsbA.

[0263] b. Discovery of site-specific properties of the MsbA inhibitor G1118 To enrich molecules that specifically target the aqueous peripheral fissure of E. coli MsbA (EcMsbA), we first used a structure-based homology model to evaluate the peripheral surface of the transmembrane segment using repeated BLAST searches (Figures 9A-12B). We then induced the manipulation of five unique chimeric constructs, each collectively replacing the sequence of a putative MsbA transporter protein derived from a distantly related Gram-negative bacterium. Of the five constructed chimeras, 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 can be tolerated while maintaining biochemical activity (Figure 11C). In particular, MsbA-chim5, in which the entire peripheral surface of EcMsbA was replaced with a sequence derived from the MsbA homolog from Magnetospira spirirum, still maintained comparable ATPase activity and showed sensitivity to both the quinoline (G247 and G907) and benzophenone (G758) classes of previously identified small molecule inhibitors of wild-type (WT) EcMsbA (Figures 11D-E and 12A-B).

[0264] Next, macrocyclic molecules were selected against WT EcMsbA transporters trapped in a purified outward conformation, followed by counter-selection using similarly trapped MsbA chimeras in which the peripheral surface of the transmembrane segment was collectively replaced with the sequence of a putative MsbA transporter protein derived from a distantly related Gram-negative bacterium (Figure 3A and 9A-12B). Selection was performed using an in vitro translated peptide macrocyclic molecule mRNA display 12-14mer library containing 10 molecules (Karow and Georgopoulos, 1993) incorporating native and non-native amino acids (Example 2). After eight selections and counter-selections, the 14-mer macrocyclic molecule, G1118, showed significant enrichment in WT EcMsbA (Figure 3B). G1118 was found to have a 50% inhibitory concentration (IC) of approximately 110 nM. 50 It was found that this affects the ATPase activity of EcMsbA, and is consistent with targeting of peripheral fissures (Figure 3C), with a titer of less than 1 / 30th of that of purified MsbA-chim5 (IC1). 50 G1118 also showed IC50 (>5 μM) for the related Enterobacter cloaca and Klebsiella pneumoniae MsbA transporters, respectively. 50 Although inhibition was achieved (Figure 13A-D), it showed only weak activity against Pseudomonas aeruginosa MsbA, which has lower sequence identity with EcMsbA (approximately 39%) (IC 50 (=3.1 μM). E. coli cells treated with G1118 induced a distinctive intermembryonic phenotype (Doerrler et al., 2001) that reflects MsbA-depleted bacteria, consistent with pharmacological inhibition of MsbA (Figure 3E). Due to the relatively large size of G1118 (2,085 Da), cellular activity was limited to cells containing the lptD(imp4213) allele, which confers a defect in outer membrane (OM) permeability, and the minimum inhibitory concentration (MIC) measured was approximately 210 μg / mL (Figure 13B). Further characterization of G1118 is discussed in Example 8 below.

[0265] c. Discovery of site-specific properties of MsbA inhibitor G1365 To discover macrocyclic molecules with OM permeability and growth inhibitory activity against WT E. coli strains, smaller macrocyclic molecular scaffolds were identified. These smaller macrocyclic molecular scaffolds also target MsbA at periplasmic fissures by performing additional selection for EcMsbA and MsbA-chim5 using an 8-10mer macrocyclic molecular library. Furthermore, since small amphiphilic molecules containing primary amines are known to be most likely to accumulate in cells (Richter et al., 2017), a codon table biased towards lipophilic and amine-containing side chains was utilized.

[0266] We discovered an 8-mer macrocyclic molecule, G1365. G1365 is an MsbA-chim5 transporter (IC2). 50 Compared to (>5 μM) (Figures 3B and 3D), the concentration and ATPase inhibitory activity (IC) against WT EcMsbA were significantly improved. 50 It showed an IC50 (1.3 μM). In biochemical assays, G1365 inhibited the MsbA transporters of the related Enterobacter cloaca and Klebsiella pneumoniae at approximately 700 nM and 660 nM IC50, respectively. 50 Inhibition was achieved with (Figure 13C), but it exhibited milder inhibitory activity against MsbA of a distantly related Pseudomonas aeruginosa (IC). 50 Treatment of E. coli imp4213 with G1365 reflected the MsbA-depleted bacterial inner membrane refinement phenotype, consistent with on-target MsbA activity (Figure 3E). In contrast to the larger 14-mer macrocyclic molecule G1118, G1365 promoted the growth of WT E. coli cells with intact outer membranes at approximately 20 μM IC50. 50 The inhibitory effect was found to last for 6 hours (Figures 13B and 13D), but the overnight MIC could not be determined. Whole-genome sequencing analysis was subsequently performed on isolated E. coli imp4213 mutants resistant to G1365 derivatives (see Methods), revealing that a single nucleotide mutation in msbA resulted in an amino acid change, Asp252Asn, within the peripheral fissure of MsbA. Further characterization of G1365 is discussed in Example 9 below.

[0267] The data comprehensively identify MsbA as a cellular target of G1365 and highlight the potential for discovering synthetic macrocyclic molecules capable of traversing the intact bacterial outer membrane.

[0268] d. Additional 14-mer macrocyclic peptide activators The 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 induce extracellular membrane stacking to a greater extent than G1118 (data for G1119 and G1122 are not shown; see Figure 3F for data for G1118). IC on inhibition of MsbA ATPase activity by G1118, G1119, and G1122 50 Perform the following (see Example 8 for details on the method): 5nM IC at G1118 and G1119 50 Value and 2nM IC in G1122 50 The 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. For ATPase activity in assays such as those described in Example 3 above, 0.2–0.8 μM IC50 was used. 50 We found that it possesses a value. The minimum inhibitory concentration (MIC) for inhibiting Escherichia coli MsbA (EcMsbA) (see Example 3 for the method) was greater than 100 μM, and the EC for inhibiting cell proliferation of UPEC imp strains 50The concentration was approximately 2–8 μM for all but one of the molecules tested (exceeding 100 μM for the last molecule tested). Overall, these data indicate that macrocyclic peptide G1118 and related peptides following the above formula can generally inhibit MsbA and MsbA-related cellular activity.

[0271] Example 8: G1118 binds to MsbA. a. Biochemical characterization of G1118 Using the materials and methods described in Examples 1-5 above, G1118 was identified as a macrocyclic molecule that binds to MsbA. The 14-residue peptide macrocyclic molecule G1118 was identified after eight enrichments and screenings. G1118 exhibited potent inhibitory activity against EcMsbA (50% inhibitory concentration (IC) of approximately 110 nM). 50 )) showed still weak activity against counter-selective chimeras (IC 50 >5 μM; Figures 3B-C and 13A). Treatment of outer membrane-permeable E. coli cells carrying the lpt(imp4213) allele with G1118 (Sampson et al., 1989) reflected a distinctive inner membrane refinement phenotype 2 (Figure 3E), characteristic of MsbA depletion, but no activity was observed in WT E. coli with an intact outer membrane. G1118 is Mg 2+G1118 is a 14-mer macrocyclic compound identified by macrocyclic selection of purified WT transporters trapped in an outward conformation by treatment with ATP and vanadate, followed by counter-selection using similarly trapped periplasmic chimeras, which removes macrocyclic molecules that bind to regions other than the periplasmic fissure. G1118 exhibits potent inhibitory activity against EcMsbA. After counter-selection with chimeric MsbA, G1118 continued to show significant enrichment against WT EcMsbA, and its inhibitory activity is consistent with targeting the periplasmic fissure, as G1118 showed a titer of less than 1 / 30th of that of the purified chimera. Escherichia coli treated with G1118 also reflected a unique membrane-refining phenotype of MsbA-depleted bacteria, consistent with on-target activity. However, the cellular activity of G1118 was limited to cells also containing 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. Since G1118 corresponds to the first potent and selective inhibitor of MsbA that targets peripheral rifts, the molecular basis for its antagonism was studied using the structural biology method described in Example 6 above.

[0272] b.G1118 binds to MsbA at its peripheral binding site and traps MsbA in an outward-facing position. To clarify the molecular mechanism by which G1118 antagonizes MsbA, the 3.0 Å cryo-EM structure of the MsbA-G1118 complex was determined in the presence of ADP-vanadate, using antibody antigen-binding fragments (Fab) to aid in particle alignment (Figures 4A-D, 14A-C, 15A-E, and 8). The materials and methods were as described in Example 6 above. In the complex with G1118, the peripheral pocket exposed to the solvent, where the MsbA homodimer, adopts an outwardly open conformation formed between the transmembrane helices TM1, TM3, and TM6 of each MsbA subunit (Figures 4A-D). Strong features in the cryo-EM map indicating the presence of a single G1118 macrocyclic molecule in the peripheral fissure validate the design of the chimeric selection strategy. G1118 is an amphiphilic peptide that complements the complex physicochemical properties of peripheral fissures in MsbA, capturing polar, van der Waals, and hydrophobic contacts, and the solvent can contact a surface area of ​​approximately 880A. 2 Fill in the blanks (Figure 16A-B). The binding of G1118 is incompatible with the rearrangement required for MsbA to return to its inward conformation (Ho et al., 2018; Mi et al., 2017; Ward et al., 2007), clearly defining this macrocyclic molecule as a state-dependent inhibitor that locks the transporter in the outward conformation and interferes with LPS transport. Since G1118 was observed to cause cell toxicity by inhibiting MsbA (Figure 3E), the co-complex structure is likely to correspond to a physiologically relevant inhibitor complex.

[0273] The peripheral receptor site for G1118 on MsbA is found near the central axis of the transporter, which extends above and below the membrane-aqueous contact surface (Figure 4B, and 16A-B). The MsbA dimer is highly symmetric (CαRMSD of approximately 0.4 Å), and G1118 is Trp10 G1118 and Trp11 G1118 By using the indole ring to perform a pseudosymmetric interaction and packing between Leu45, Leu52, and Ile292', this inherent symmetry is utilized on both sides of the transporter between TM1 and TM6' (Figure 4C). Ser9 G1118 and Trp3G1118 These form a pair of hydrogen bonds attached to the carbonyl skeletons of Lys49 and Ser289' of MsbA, respectively (Figure 4D). On the polar plane of G1118, Asp7 G1118 A salt bridge was directly observed between G1118 and Arg296, which is a highly conserved basic residue deep within the central cavity of MsbA that has been previously shown to coordinate LPS to an inward conformation (Figure 4C) (Ho et al., 2018; Mi et al., 2017). Analysis of enriched relevant sequences and mutation scans of G1118 confirmed the crucial interaction and amphiphilicity of the macrocyclic molecule required to interact with MsbA (Figure 4E and Table 4). TIFF0007914136000013.tif116170

[0274] Table 4 above shows a mutation scan of the macrocyclic molecule G1118. The enrichment factors are shown for each macrocyclic molecule containing the substitutions shown at positions 2-13 of the macrocyclic core (where enrichment factor = (% frequency [target] / % frequency [input]), normalized by the parent value). Amino acids identical to the parent are shown with a black border. Natural amino acids are shown with a standard single letter. Non-natural 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 pharmacological and receptor sites within the ABC transporter family.

[0275] In summary, the structure of the complex guided the selection of residues for mutation analysis. 50In vitro curves showed that these residues weaken macrocyclic molecule binding. Conversely, analysis of enriched sequences from RNA display selection pointed to the role of residues in affinity to macrocyclic molecules. Overall, the structure and cellular activity validated the macrocyclic molecule selection strategy and peripheral fissures as drug-potential locations that definitively validate our protein engineering and selection strategies. Due to the large size of G1118, the wild-type activity hindered the optimization of derivatives, likely due to difficulties in permeating the LPS-rich outer membrane.

[0276] Example 9: Identification of G1365 a. Biochemical characterization of G1365 In an attempt to select smaller macrocyclic molecules that can overcome the OM permeability barrier, another series of selections and chimeric counterselections were carried out using 8-10mer macrocyclic molecules. The materials and methods were as described above in Examples 1-5. Codon tables biased towards lipophilic and positively charged side chains were also used.

[0277] Using these methods, G1365 was identified (Example 7 and Figure 3B). G1365 showed biochemical activity against MsbA (Figure 3D; IC). 50 (=1.3 μM), and E. coli lptD(imp4213) cells treated with G1365 also exhibited the MsbA depleted membrane phenotype (Figure 3E). In particular, G1365 also inhibited the growth of WT E. coli cells with intact outer membranes for 6 hours (approximately 20 μM IC50). 50 (Figures 13B and 13D) highlight the potential for discovering synthetic macrocyclic molecules that can traverse the intact bacterial outer membrane. Specifically, the effects of G1365 and other macrocyclic molecules 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., those with normal OM) were determined at different concentrations of the macrocyclic molecules (Figures 3E and 3G-L, and Table 5). TIFF0007914136000014.tif89170

[0278] Biochemical IC2 for G1365 in this experiment 50 It is 300 nM, and the EC2 is 300 nM for wild-type E. coli cells. 50 The concentration was 20 μM. See Figures 3G-L and Table 5 below. To further confirm that G1365 is selective for MsbA derived from E. coli, the minimum inhibitory concentrations (MICs) of G1365 and other macrocyclic molecules for inhibiting growth in 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, while in Acinetobacter baumannii it exceeded 100 μM, a difference of approximately 10 times in concentration, indicating that G1365 preferentially interacts with MsbA from E. coli compared to MsbA from Acinetobacter baumannii. The MICs for other macrocyclic molecules tested did not differ between the two bacterial strains, indicating that they are not selective between the two MsbA proteins. The molecular basis for their antagonism was studied using the structural biology techniques described in Example 6 above.

[0279] b.G1365 binds deep within the peripheral fissure of MsbA. To understand the molecular basis of the inhibition, the cryo-EM structure of the MsbA-12G7-G1365 complex was determined. The materials and methods were as described in Example 6 above. The 3.1 Å cryo-EM structure of MsbA in complex with a potent derivative of G1365 is shown (Figures 14A-B, 17A-E, and 8). In particular, G1365 is an amphiphilic molecule, and its smaller size allows it to bind to peripheral fissures of MsbA nearly 10 Å deeper 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 peripheral rift (Figures 4A, 4B, 4F-K, and 16A-D), suggesting that this amphiphilic macrocyclic molecule can directly access the receptor site from the lipid bilayer.

[0280] G1365 is identified as a state-dependent inhibitor because it traps MsbA in an outward conformation that is incompatible with the transition to the inward state. The binding site to G1365 is adjacent to the membrane-exposed region of the transporter having binding sites formed by TM1, TM3, and TM6, and Arg6 G1365 The guanidinium side chains are oriented towards the lipid bilayer (Figure 4F~H). Each is MeF5. G1365 Between Arg148 and Bhp4 G1365 The cation-π interaction between and Arg296 stabilizes the binding of G1365 within the receptor site. The extended biphenyl residue Bhp4 G1365 This likely extends beyond the axis of symmetry of MsbA, interfering with the binding of the second macrocyclic molecule. Enriched relevant sequence and mutation scans of G1365 confirm the critical interaction of the macrocyclic molecule required to interact with MsbA (Figure 4I and Table 5). TIFF0007914136000015.tif115170

[0281] Table 6 above shows a mutation scan of the macrocyclic molecule G1365. Exactly as in Table 4 above, these are the enrichment factors (where enrichment factor = (% frequency [target] / % frequency [input])) for macrocyclic molecules containing substitutions shown at positions 2-7 of the macrocyclic core. Amino acids identical to the parent are indicated by a black border. Natural amino acids are indicated by a standard single letter. Non-natural 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 can also logically explain the Asp252Asn resistance mutation (Example 7 above), because this should interfere with the interaction between Arg296 and G1365 (Figure 4H).

[0282] Unexpectedly, the guanidinium side chain of G1365 Arg6 faces toward the lipid bilayer. The bulky biphenyl residue extends across the pseudo-symmetry axis toward Arg296 from other protomers. This structure also logically explains the mechanism for the D252N resistance mutation by disrupting the salt bridge between Lys299 on TM6 and presumably interfering with the interaction between Arg296 and G1365. The structure of G1365 identifies a unique receptor site compared to G1118, even though the outward-open conformation of MsbA trapped by the two molecules is very similar. Therefore, like G1118, G1365 is a state-dependent inhibitor of MsbA, despite its small binding footprint.

[0283] Example 10: Identification of an unexpected LPS binding site on MsbA It is now unknown how MsbA initially binds to its rare substrate LPS rather than the abundant phospholipids in the membrane bilayer to achieve selective transport. In the cryo-EM map, two well-resolved LPS molecules bind around MsbA (Figures 4A and 18A-D). These prominent map features clearly place the characteristic bisphosphoglucosamine head group of lipid A at a previously unrecognized symmetry-related coordination site at the level of the inner leaflet of the MsbA dimer (Figure 18A). Importantly, the visualized LPS molecules are far away from the single binding site previously observed in the central cavity within the inward-facing entrance of MsbA (Ho et al., 2018; Mi et al., 2017). Since LPS was not added to the samples used for cryo-EM, the identification of these distinct peripheral LPS binding sites on outward-facing MsbA suggests that they are physiologically relevant.

[0284] The peripheral inner leaflet LPS binding site on MsbA is formed by TM2, TM4' and TM5', and upon LPS binding, 865Å 2The transporter surface area is filled. Three distinctive features of MsbA-bound LPS extend along the lipid A core of the molecule (Figure 5A). First, the highly conserved basic side chain from MsbA coordinates to the characteristic phosphate on the glucosamine head group of lipid A (Figure 18B). Arg188 and Lys243 form salt bridges with 1-phosphate (Figure 5A), while Lys95 and Arg238 form salt bridges with 4'-phosphate (Figure 5B). Second, the hexa-acylated tail of lipid A interacts with the extended hydrophobic surface on the transporter, where the 2'-hydroxymyristic acid and 2''-laurate ester bonds are embedded in the conserved aromatic side chains of Tyr87 and Trp91, respectively. Thirdly, refined 3-deoxy-D-manno-octa-2-urosonic acid (Kdo) core-saccharides derived from lipid A are coordinated in the extended polar intracellular stalk region on the transporter (Figure 18C). These strategic multi-point contacts with lipid A at the surrounding LPS binding site reveal the structural basis for MsbA's selective binding to LPS over bulk membrane phospholipids.

[0285] Since the outward conformation of MsbA has historically been associated with the release of substrates to peripheral parenchymal lobules (Ho et al., 2018; Mi et al., 2017; Ward et al., 2007), finding LPS bound to MsbA along the medial lobule in this state was initially surprising. We generated targeted double mutants of residues that interact with LPS. We evaluated the ability of these double mutants to aid the growth of E. coli cells lacking WT MsbA (Figure 19A-J). We found that basic residues coordinating to the 4'-phosphate of lipid A (Lys95 and Arg238) and aromatic side chains supporting the 2'-hydroxymyristic acid and 2''-laurate chain esters of lipid A (Tyr87 and Trp91) were essential. This is because the mutations to alanine inhibited cell proliferation and produced a unique inner membrane refinement phenotype that reflects WT MsbA-depleted bacteria (Figure 5C-E) (Doerrler et al., 2001). In contrast, the side chains coordinating to the 1-phosphate of lipid A (Arg188 and Lys243) or proximal non-LPS-interacting residues are not necessarily required. Unexpectedly, (1) essential peri-LPS binding sites were found on the medial lobule of MsbA in an outward conformation, and (2) previously unknown substrate selectivity determinants with important physiological significance were identified.

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

[0287] A site-directed ligand discovery strategy was devised to selectively inhibit ABC transporters by targeting cleavage exposed to the outward-facing solvent using engineered chimeric proteins. G1118 and G1365 are two macrocyclic compounds that target unprecedented receptor sites within outward-facing cleavage, defining novel pharmacology within the ABC transporter superfamily. These macrocyclic molecules may serve as initial lead compounds for the discovery of further antimicrobial agents. Similar site-directed ligand discovery efforts using engineered chimeras can identify novel receptor sites in other protein classes and are particularly useful for discovering new pharmacology.

[0288] Previous studies have shed light on the alternating access mechanism of transport employed by MsbA (Ho et al., 2018; Mi et al., 2017; Ward et al., 2007), but how MsbA achieves selectivity for LPS over large amounts of membrane phospholipids remained unclear. While the inward-facing structure of MsbA revealed LPS bound to the central cavity, these structures capture an intermediate state in the transport cycle and fail to provide insight into how LPS is selectively loaded from the bulk phospholipid bilayer into the central cavity. This disclosure discusses the discovery of an outward-facing state of MsbA bound to its substrate, unexpectedly revealing that MsbA first recognizes LPS through a conserved, essential side-chain network located along the inner membrane lobule of the transporter. Based on these observations, several principles underlying 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 termination of the catalytic cycle (Hollenstein et al., 2007). As discussed in the above examples, the data require a more holistic model, where the LPS binding site presented by the outward state helps to significantly increase the local concentration of LPS within the medial lobule around the transporter (Figure 6). In the outward conformation, MsbA strategically targets the unique chemical structure of core lipid A in the surrounding region through multipoint interactions with a characteristic biphosphoglucosamine head group, hexaacylated chain, and Kdo-core sugars to achieve selective binding to LPS over phospholipids (Figure 5A-B). Targeted mutagenesis and physiological studies have established the essential properties of conserved residues in this previously unrecognized LPS binding site. Interference from surrounding binding sites during the transition to the inward conformation is expected to release LPS, facilitating selective loading into the central cavity, possibly along a series of basic residues extending from the surrounding binding sites to the cavity within the entrance (Figure 18C-D). In summary, MsbA links the release of LPS to the outer lobule with the binding of the substrate to the inner lobule, revealing that the outward state, previously considered the final stage of the ABC exporter cycle, may be intrinsically and directly linked to the initial state.

[0290] Visualization of the surrounding LPS recognition complex provides initial insight into how MsbA can distinguish between mature and immature LPS species. The Lys95 and Arg238 side chains are essential, forming a direct salt bridge to the 4'-phosphate of lipid A (Figure 5B), providing a structural rationale for why the precursor 1-monophosphorylated lipid A species is not transported.23 The essential nature of the Lys95 and Arg238-mediated interaction also explains why suppressor mutations of 4'-kinase LpxK have not been identified and logically explains why MsbA and LpxK are chromosomally linked24 or why they exist as a single linked protein in some bacteria.25 Furthermore, the Tyr87 and Trp91 side chains are essential and located directly beneath the ester bonds of the 2'-hydroxymyristic acid and 2"-laurate acyl chains (Figure 5B), which are added by the slow-acting enzymes LpxL and LpxM in the lipid A biosynthesis pathway.26,27 This observation provides molecular evidence for why hexa-acylated LPS is a preferred substrate for EcMsbA.28,29 The picture emerging from the surrounding LPS recognition site, in addition to selectively enriching LPS over phospholipids, suggests The key is that MsbA employs unique quality control to ensure that mature LPS species are selected for transport across the inner membrane into the peripheral plasma. The degree of refinement of this molecule will help ensure that the integration of the outer membrane barrier is not impaired 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 outward conformations to selectively concentrate and examine potential substrates.

[0291] References TIFF0007914136000016.tif122170TIFF0007914136000017.tif214170TIFF00079141360 00018.tif204170TIFF0007914136000019.tif213170TIFF0007914136000020.tif103170

[0292] array The following table illustrates certain sequences referenced herein. Dashes are placed between residues or functional groups of certain peptides listed below. TIFF0007914136000021.tif255164TIFF0007914136000022.tif255164TIFF0007914136000023.tif255170TIFF0007914136000024.tif159170

[0293] The invention described herein is described in some detail with examples and embodiments for clarity of understanding, but the description and embodiments are not to be construed as limiting the scope of the invention. All patent and scientific literature disclosures cited herein are expressly incorporated in their entirety by reference.

Claims

1. A chimeric ABC transporter containing a parent ABC transporter Escherichia coli MsbA (EcMsbA) in which one or more of the EcMsbA residues Leu47-Pro68 in peripheral loop 1 (L1), EcMsbA residues Met159-Leu171 in peripheral loop 3 (L3), and EcMsbA residues Ala262-Ile292 in peripheral loop 5 (L5) are replaced with the equivalent region of Pseudomonas cyclotolerance (PpMsbA), the equivalent region of Candidatus acumulibacter (CaMsbA), or the equivalent region of Thiomicrospira cyclica (TcMsbA).

2. A method for determining whether a test molecule binds to the peripheral, extracellular, and / or luminal surface of a parent ABC transporter, a) To provide the chimeric ABC transporter described in claim 1, and b) Contacting a 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 peripheral, extracellular, and / or luminal surface of the parent ABC transporter. Methods that include...

3. A method for determining whether a test molecule binds to the peripheral, extracellular, and / or luminal surface of a parent ABC transporter, a) Trap the parent ABC transporter into an outward-facing conformation. b) Select a test molecule that binds to the parent ABC transporter in an outward conformation. c) To provide the chimeric ABC transporter described in claim 1, and d) Contacting the chimeric ABC transporter with the test molecule of b) that binds to the parent ABC transporter in its outward conformation, and determining that the test molecule does not bind to the chimeric ABC transporter, then it is determined that the test molecule binds to the peripheral, extracellular, and / or luminal surface of the parent ABC transporter. A method that includes this.

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

5. The complex according to claim 4, wherein the peptide is a macrocyclic molecule.

6. The composite according to claim 5, wherein the macrocyclic molecule is a 6-14-mer macrocyclic molecule.

7. A kit comprising the chimeric ABC transporter described in claim 1 and a reagent for carrying out the method according to claim 2 or 3, wherein optionally the chimeric ABC transporter is attached to a matrix or beads, and optionally the kit is: a. Parent ABC transporter and / or different ABC transporters; b. A matrix or beads for attaching ABC transporters, optionally streptavidin-coated beads, avidin-coated beads, or deglycosylated avidin-coated beads, or magnetic beads; c. One or more detergents for solubilizing ABC transporters 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 A kit that further includes one or more of the following.

8. The kit according to claim 7, further comprising an instruction manual.

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