Beta-lactamases with improved properties for therapy
This invention relates to, in part, compositions of beta-lactamases and methods of using these enzymes in, for example, gastrointestinal tract (GI tract) disorders such as C. difficile infection (CDI).
1. A beta-lactamase comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1 and the following mutations according to Ambler classification:
an aliphatic hydrophobic residue other than alanine (A) at position 232,
a polar and neutral hydrophilic residue other than alanine (A) at position 237,
an aliphatic hydrophobic residue other than alanine (A) at position 238, and
a polar and negative hydrophilic residue other than serine (S) at position 240.
2. The beta-lactamase of claim 1 , wherein the beta-lactamase has a further mutation of a polar and neutral hydrophilic or a polar and positive hydrophilic residue other than aspartate (D) at position 276 according to Ambler classification.
3. The beta-lactamase of claim 1 , wherein the aliphatic hydrophobic residue is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).
4. The beta-lactamase of any one of claims 1 - 2 , wherein the polar and neutral hydrophilic residue is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).
5. The beta-lactamase of claim 1 , wherein the polar and negative hydrophilic residue is selected from aspartate (D) and glutamate (E).
6. The beta-lactamase of claim 2 , wherein the polar and positive hydrophilic residue is selected from arginine (R) and lysine (K).
7. The beta-lactamase of any one of claims 1 - 2 , wherein the beta-lactamase hydrolyzes one or more of penicillins and cephalosporins.
8. The beta-lactamase of claim 7 , wherein the penicillin is ampicillin.
9. The beta-lactamase of claim 7 , wherein the cephalosporin is selected from ceftriaxone, cefotaxime, cefozolin, cefoperazone, cefepime, cefuroxime, and ceftazidime.
10. The beta-lactamase of any one of claims 1 - 2 , wherein the beta-lactamase has improved catalytic efficiency against cephalosporins as compared to SEQ ID NO: 1.
11. The beta-lactamase of claim 10 , wherein the beta-lactamase has about a 10 fold to about a 1000 fold improved catalytic efficiency against a cephalosporin as compared to SEQ ID NO: 1.
12. The beta-lactamase of claim 11 , wherein the cephalosporin is selected from ceftriaxone, cefotaxime, cefozolin, cefoperazone, cefepime, cefuroxime, and ceftazidime.
13. The beta-lactamase of any one of claims 1 - 2 , wherein the beta-lactamase has improved catalytic efficiency against a cephalosporin as compared to P3A.
14. The beta-lactamase of claim 13 , wherein the cephalosporin is selected from cefotaxime, cefepime, and ceftriaxone.
15. The beta-lactamase of any one of claims 1 - 2 , wherein the beta-lactamase is active in the GI tract.
16. The beta-lactamase of claim 15 , wherein the beta-lactamase is stable in the small intestine, optionally selected from one or more of the duodenum, jejunum, and ileum.
17. A polynucleotide comprising a polynucleotide sequence encoding the beta-lactamase of any one of claims 1 - 2 .
18. A host cell comprising the polynucleotide of claim 17 .
19. A pharmaceutical composition, comprising the beta-lactamase of any one of claims 1 - 2 and a pharmaceutically acceptable carrier or excipient.
20. The pharmaceutical composition of claim 19 , wherein the composition is formulated for oral administration, optionally selected from a tablet, multi-particulate sprinkle, and a multi-particulate capsule.
21. A method for preventing an antibiotic-induced adverse effect in the gastrointestinal (GI) tract, comprising administering an effective amount of a beta-lactamase to a patient in need thereof, wherein the beta-lactamase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1 and the following mutations according to Ambler classification:
an aliphatic hydrophobic residue other than alanine (A) at position 232,
a polar and neutral hydrophilic residue other than alanine (A) at position 237,
an aliphatic hydrophobic residue other than alanine (A) at position 238, and
a polar and negative hydrophilic residue other than serine (S) at position 240.
22. The method of claim 21 , wherein the beta-lactamase has a further mutation of a polar and neutral hydrophilic or a polar and positive hydrophilic residue other than aspartate (D) at position 276 according to Ambler classification.
23. The method of claim 21 , wherein the aliphatic hydrophobic residue is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).
24. The method of any one of claims 21 - 22 , wherein the polar and neutral hydrophilic residue is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).
25. The method of claim 21 , wherein the polar and negative hydrophilic residue is selected from aspartate (D) and glutamate (E).
26. The method of claim 22 , wherein the polar and positive hydrophilic residue is selected from aspartate (D) and glutamate (E).
27. The method of any one of claims 21 - 22 , wherein the subject is being administered or will be administered an antibiotic.
28. A method for treating or preventing an antibiotic induced C. difficile infection (CDI) and/or a C. difficile -associated disease, comprising administering an effective amount of a beta-lactamase to a patient in need thereof, wherein the beta-lactamase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1 and the following mutations according to Ambler classification:
an aliphatic hydrophobic residue other than alanine (A) at position 232,
a polar and neutral hydrophilic residue other than alanine (A) at position 237,
an aliphatic hydrophobic residue other than alanine (A) at position 238, and
a polar and negative hydrophilic residue other than serine (S) at position 240.
29. The method of claim 28 , wherein the beta-lactamase has a further mutation of a polar and neutral hydrophilic or a polar and positive hydrophilic residue other than aspartate (D) at position 276 according to Ambler classification.
30. The method of claim 28 , wherein the aliphatic hydrophobic residue is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).
31. The method of any one of claims 28 - 29 , wherein the polar and neutral hydrophilic residue is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).
32. The method of claim 28 , wherein the polar and negative hydrophilic residue is selected from aspartate (D) and glutamate (E).
33. The beta-lactamase of claim 29 , wherein the polar and positive hydrophilic residue is selected from arginine (R) and lysine (K).
34. The method of any one of claims 28 - 29 , wherein the subject is being administered or will be administered an antibiotic.
35. The method of any one of claims 28 - 29 , wherein the C. difficile -associated disease is antibiotic-associated diarrhea (AAD).