Beta-lactamases with improved properties for therapy
AssigneeSynthetic Biologics, Inc.
InventorsMichael KALEKO, Sheila CONNELLY
Life of this patent
Estimating the term…
Abstract
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).
Claims73
A beta-lactamase comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO:
- 1 and one or more of the following mutations of Ambler classification:
- F33X, Q135X, G156X, A232X, A237X, A238X, S240X, T243X, R244X, S266X, and D276X, wherein X is any naturally-occurring amino acid and with the proviso that D276X is not present in the context of a single mutant.
The beta-lactamase of any one of the above claims, wherein the hydrophilic amino acid residue is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K) or an aromatic, polar and positively charged hydrophilic including histidine (H).
The beta-lactamase of any one of the above claims, wherein the hydrophilic amino acid residue is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).
The beta-lactamase of any one of the above claims, wherein the hydrophilic amino acid residue is a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E).
The beta-lactamase of any one of the above claims, wherein the hydrophobic amino acid is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
A beta-lactamase comprising an amino acid sequence having at least about 70% sequence identity with SEQ ID NO:
- 1 and one or more of the following mutations of Ambler classification:
- a hydrophobic residue other than phenylalanine (F) at position 33;
- a hydrophobic residue other than glutamine (Q) at position 135;
- a hydrophilic residue other than glycine (G) at position 156;
- a hydrophobic residue other than alanine (A) at position 232;
- a hydrophilic residue other than alanine (A) at position 237;
- a hydrophobic or hydrophilic residue other than alanine (A) at position 238;
- a hydrophilic residue other than serine (S) at position 240;
- a hydrophobic residue other than threonine (T) at position 243;
- a hydrophobic residue other than arginine (R) at position 244;
- a hydrophilic residue other than serine (S) at position 266;
- and
- a hydrophilic residue other than aspartate (D) at position 276,
- with the proviso that hydrophilic amino acid residue other than aspartic acid (D) at a position corresponding to position 276 is not present in the context of a single mutant.
The beta-lactamase of any one of the above claims, wherein the hydrophilic amino acid residue is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K) or an aromatic, polar and positively charged hydrophilic residue including histidine (H).
The beta-lactamase of any one of the above claims, wherein the hydrophilic amino acid residue is a polar and neutral of charge hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).
The beta-lactamase of any one of the above claims, wherein the hydrophilic amino acid residue is a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E).
The beta-lactamase of any one of the above claims, wherein the hydrophobic amino acid is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
The beta-lactamase of any one of the above claims, comprising one or more of the following mutations at positions of Ambler classification:
- F33Y, Q135M, G156R, A232G, A237S, A238G or T, S240P or D, T2431, R244T, S266N, D276N or R or K, provided that D276N or R or K is not in the context of a single mutant.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises Q135M.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises G156R and A238T.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises F33Y and D276N.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises F33Y, S240P, and D276N
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises F33Y, A238T, and D276N.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises A232G, A237S, A238G, and S240D.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises A232G, A237S, A238G, S240D, AND R244T.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises A232G, A237S, A238G, S240D, and D276R.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises A232G, A237S, A238G, S240D, and D276K.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises A232G, A237S, A238G, S240D, and Q135M.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises A238T.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises T2431, S266N, and D276N.
The beta-lactamase of any one of the above claims, wherein the beta-lactamase comprises A232G, A237S, A238G, S240D, and D276N.
A beta-lactamase comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO:
- 1 and the following of Ambler classification:
- a hydrophobic residue other than alanine (A) at position 232;
- a hydrophilic residue other than alanine (A) at position 237;
- a hydrophobic residue other than alanine (A) at position 238;
- a hydrophilic residue other than serine (S) at position 240;
- and
- a hydrophilic residue other than aspartate (D) at position 276.
A polynucleotide comprising a polynucleotide sequence encoding the beta-lactamase of any one of the above claims.
The beta-lactamase of any of the above claims, wherein the beta-lactamase hydrolyzes one or more of penicillins and cephalosporins.
The beta-lactamase of any of the above claims, characterized in that the beta-lactamase has low KM for cephalosporins.
The beta-lactamase of any of the above claims, characterized in that the beta-lactamase has a high Vmax.
The beta-lactamase of claim 344, wherein the high Vmax is greater than about 100 s−1, or about 1000 s−1, or about 10000s−1, or about 100000s−1, or about 1000000s−1.
The beta-lactamase of any of the above claims, characterized in that the beta-lactamase has improved catalytic efficiency against cephalosporins as compared to SEQ ID NO:
- 1.
The beta-lactamase of any of the above claims, characterized in that the beta-lactamase is active in the GI tract.
The beta-lactamase of any of the above claims, characterized in that the beta-lactamase is resistant to proteases in small intestine.
The beta-lactamase of any of the above claims, characterized in that the beta-lactamase is substantially active at a pH of about 6.0 to about 7.5.
The beta-lactamase of any of the above claims, characterized in that the beta-lactamase is resistant to one or more beta-lactamase inhibitors, optionally selected from tazobactam, sulbactam, and clavulanic acid.
A pharmaceutical composition, comprising a beta-lactamase of any of the above claims and one or more of and a pharmaceutically acceptable carrier or excipient.
The pharmaceutical composition of any of the above claims, further comprising an additional antibiotic degradation enzyme.
Dependencies are inferred from the claim text.