Bifunctional cocaine esterase multi-site mutants and uses thereof

Abstract

The application discloses a bifunctional cocaine esterase mutant. The cocaine esterase mutant of the application has greatly improved catalytic efficiency for cocaine and benzoyl ecgonidine, a toxic metabolite of cocaine, and thus has very important clinical value for realizing complete detoxification of cocaine and lays a brand-new material foundation for development of a cocaine detoxification drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine. Specifically, this invention relates to bifunctional cocaine esterase multisite mutants and their applications. Background Technology

[0002] Cocaine abuse is a global health and social problem. As a potent central nervous system stimulant, cocaine stimulates the dopamine reward circuit, producing a strong sense of euphoria in users and is highly addictive. Cocaine use can cause severe cardiovascular effects, easily leading to acute poisoning and death from overdose.

[0003] Bacterial cocaine esterase (CocE) efficiently catalyzes the hydrolysis of cocaine. Subsequent development of thermostable mutants (such as T172R / G173Q) holds promise as effective treatments for cocaine abuse, but they exhibit low activity against the toxic metabolite benzoyl stigmine (BZE). After cocaine enters the body, approximately half is hydrolyzed into BZE in the liver under the catalysis of CES1. Compared to cocaine, BZE has a longer half-life and higher toxicity in vivo, which is the main reason for the long-term toxicity of cocaine.

[0004] To completely eliminate the toxic effects of cocaine, it is necessary to eliminate not only the toxicity of cocaine itself, but also the toxicity caused by its toxic metabolite BZE. Cocaine and BZE molecules have a charge difference, and CocE mutations often make it difficult to simultaneously catalyze both substrates.

[0005] Therefore, developing a bifunctional metabolic enzyme that can rapidly degrade both cocaine and BZE is quite challenging, but it has significant clinical value for achieving complete cocaine detoxification. Summary of the Invention

[0006] The purpose of this invention is to develop a bifunctional cocaine esterase capable of rapidly degrading both cocaine and BZE, and its application in achieving complete cocaine detoxification.

[0007] Another object of the present invention is to provide a method for efficiently decomposing cocaine and BZE using the aforementioned bifunctional cocaine esterase.

[0008] Another objective of this invention is to provide a method for treating patients with cocaine and BZE poisoning.

[0009] In a first aspect, the present invention provides a cocaine esterase mutant, wherein the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: 42, 43, 44, 48, 49, 50, 51, 52, 53, 54, 55, 56, 87, 116, 118, 119, 141, 150, 162, 165, 166, 169, 170, 172, 173, 196, 261, 288, 290, 301, 407, 408.

[0010] In a specific embodiment, the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at position 55 and / or position 407 corresponding to the amino acid sequence shown in SEQ ID NO:1.

[0011] In a specific embodiment, the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO:1: 51, 116, 172, 173, 196, 301.

[0012] In a specific embodiment, the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at the following sites corresponding to the amino acid sequence shown in SEQ ID NO:1: 51, 55, 116, 172, 173, 196, 301, 407.

[0013] In a specific embodiment, the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO:1: The 42nd position is mutated to E or D, or a conserved substitution residue of E or D; The 43rd position is mutated to E or D, or a conserved substitution residue of E or D; The 44th position is mutated to K, or a conserved substitution residue of K is made. The 48th position is mutated to E, or a conserved substitution residue of E is made. The 49th position is mutated to D or E, or a conserved substitution residue of D or E; The 50th position is mutated to E or D, or a conserved substitution residue of E or D; The 51st position is mutated to L, or a conserved substitution residue of L is made. The 52nd position is mutated to D, E, or L, or a conserved substitution residue of D, E, or L; The 53rd position is mutated to D or E, or a conserved substitution residue of D or E; The 54th position is mutated to D or E, or a conserved substitution residue of D or E; The 55th position is mutated to D, E, F, V or K, or a conserved substitution residue of D, E, F, V or K; The 56th position is mutated to E or D, or a conserved substitution residue of E or D; The 87th position is mutated to W, or a conserved substitution residue of W is made. The 116th position is mutated to K, or a conserved substitution residue of K is made. The 118th position is mutated to D, E, or K, or a conserved substitution residue of D, E, or K; The 119th position is mutated to K, D, or E, or a conserved substitution residue of K, D, or E; The 141st position is mutated to G or K, or a conserved substitution residue of G or K; The 150th position is mutated to C, or a conserved substitution residue of C is made. The 162nd position is mutated to G, or a conserved substitution residue of G is made. The 165th position is mutated to D or E, or a conserved substitution residue of D or E; The 166th position is mutated to D or E, or a conserved substitution residue of D or E; The 169th position is mutated to E or D, or a conserved substitution residue of E or D; The 170th position is mutated to E or D, or a conserved substitution residue of E or D; The 172nd position is mutated to R, or a conserved substitution residue of R is made. The mutation at position 173 is Q, or a conserved substitution residue for Q; The 196th position is mutated to C, E, R, K or D, or a conserved substitution residue of C, E, R, K or D; The 261st position is mutated to E or D, or a conserved substitution residue of E or D is made. The 288th position is mutated to D, A, F, L, E, R, or K, or a conserved substitution residue of D, A, F, L, E, R, or K; The 290th position is mutated to D or E, or a conserved substitution residue of D or E; The 301st position is mutated to C, or a conserved substitution residue of C is made. The 407 position is mutated to D, E, R, V or I, or a conserved substitution residue of D, E, R, V or I; The 408th position is mutated to D or E, or a conserved substitution residue of D or E.

[0014] In a specific embodiment, the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO:1: The 51st position is mutated to L, or a conserved substitution residue of L is made. The 55th position is mutated to D, E, F, V or K, or a conserved substitution residue of D, E, F, V or K; The 116th position is mutated to K, or a conserved substitution residue of K is made. The 172nd position is mutated to R, or a conserved substitution residue of R is made. The mutation at position 173 is Q, or a conserved substitution residue for Q; The 196th position is mutated to C, E, R, K or D, or a conserved substitution residue of C, E, R, K or D; The 301st position is mutated to C, or a conserved substitution residue of C is made. The 407 position is mutated to D, E, R, V or I, or a conserved substitution residue of D, E, R, V or I.

[0015] In a specific embodiment, the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at the following sites corresponding to the amino acid sequence shown in SEQ ID NO:1: The 51st position is mutated to L, or a conserved substitution residue of L is made. The 55th position is mutated to D, or a conserved substitution residue of D is made. The 116th position is mutated to K, or a conserved substitution residue of K is made. The 172nd position is mutated to R, or a conserved substitution residue of R is made. The mutation at position 173 is Q, or a conserved substitution residue for Q; The 196th position is mutated to C, or a conserved substitution residue of C is made. The 301st position is mutated to C, or a conserved substitution residue of C is made. The 407 position is mutated to I, or a conserved substitution residue of I is made.

[0016] In a preferred embodiment, the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at the following sites corresponding to the amino acid sequence shown in SEQ ID NO:1: Q55D / T172R / G173Q / L196C / I301C、Q55E / T172R / G173Q / L196C / I301C、T172R / G173Q / L196C / S288D / I301C、T172R / G173Q / L196C / S288E / I301C、Q55D / T172R / G173Q / L196C / S288D / I301C、Q55D / T172R / G173Q / L196C / S288E / I301C、A51L / Q55E / V116K / T172R / G173Q / L196C / I301C、A51L / Q55K / V116K / T172R / G173Q / L196C / I301C、A51L / Q55F / V116K / T172R / G173Q / L196C / I301C、A51L / Q55V / V116K / T172R / G173Q / L196C / I301C、A51L / V116K / T172R / G173Q / L196C / S288F / I301C、A51L / V116K / T172R / G173Q / L196C / S288E / I301C、A51L / V116K / T172R / G173Q / L196C / S288D / I301C、A51L / V116K / T172R / G173Q / L196C / S288L / I301C、A51L / V116K / M141G / T172R / G173Q / L196C / I301C、A51L / V116K / M141K / T172R / G173Q / L196C / I301C、A51L / W52L / V116K / T172R / G173Q / L196C / I301C、Y44K / A51L / V116K / T172R / G173Q / L196C / I301C、A51L / V116K / Y118K / T172R / G173Q / L196C / I301C、A51L / Q55E / V116K / T172R / G173Q / L196C / S288E / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、N42D / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、N42E / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、P43E / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、P43D / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、D48E / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、V49E / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、V49D / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、F50D / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、F50E / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C、A51L / W52E / Q55D / V116K / T172R / G173Q / L196C / I301C、A51L / W52D / Q55D / V116K / T172R / G173Q / L196C / I301C、A51L / S53D / Q55D / V116K / T172R / G173Q / L196C / I301C、A51L / S53E / Q55D / V116K / T172R / G173Q / L196C / I301C、A51L / T54D / Q55D / V116K / T172R / G173Q / L196C / I301C、A51L / T54E / Q55D / V116K / T172R / G173Q / L196C / I301C、A51L / Q55D / S56E / V116K / T172R / G173Q / L196C / I301C、A51L / Q55D / S56D / V116K / T172R / G173Q / L196C / I301C、A51L / Q55D / H87W / V116K / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / Y118D / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / Y118E / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / L119E / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / L119K / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / L119D / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / P150C / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / A162G / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / G165E / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / G165D / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / W166D / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / W166E / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / L169E / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / L169D / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / I170E / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / I170D / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / T172R / G173Q / L196E / I301C、A51L / Q55D / V116K / T172R / G173Q / L196D / I301C、A51L / Q55D / V116K / T172R / G173Q / L196K / I301C、A51L / Q55D / V116K / T172R / G173Q / L196R / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / F261E / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / F261D / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / S288A / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / S288F / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / S288L / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / S288E / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / S288K / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / S288R / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / S288D / I301C、A51L / Q55D / V116K / T172R / G173Q / L196C / L290D / I301C, A51L / Q55D / V116K / T172R / G173Q / L196C / L290E / I301C, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407V, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I、A51L / Q55D / V116K / T172R / G173Q / L196C / I 301C / L407R, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407D, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407E, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / F408D, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / F408E; Preferably, the following mutations are present: A51L / Q55D / V116K / T172R / G173Q / L196C / I301C, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I; more preferably, the mutation shown in A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I is present.

[0017] In a second aspect, the present invention provides an isolated polynucleotide molecule that encodes the cocaine esterase mutant described in the first aspect.

[0018] In a third aspect, the present invention provides an expression vector comprising the polynucleotide molecule described in the second aspect.

[0019] In a fourth aspect, the present invention provides a host cell comprising the expression vector described in the third aspect, or the host cell having the polynucleotide molecule described in the second aspect integrated into its genome.

[0020] In a fifth aspect, the present invention provides a composition comprising the cocaine esterase mutant described in the first aspect.

[0021] In a preferred embodiment, the composition is a pharmaceutical composition.

[0022] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0023] In a sixth aspect, the present invention provides an immobilized enzyme comprising the cocaine esterase mutant described in the first aspect.

[0024] In a seventh aspect, the present invention provides a method for the simultaneous hydrolysis of cocaine and its metabolites, the method comprising the step of contacting cocaine and its metabolites with the cocaine esterase mutant described in the first aspect, the composition described in the fifth aspect, or the immobilized enzyme described in the sixth aspect.

[0025] In a preferred embodiment, the metabolite is benzoyl saccharide (BZE).

[0026] In a preferred embodiment, the method for hydrolyzing cocaine and its metabolites is an in vitro method.

[0027] In an eighth aspect, the present invention provides a method for treating cocaine and its metabolite poisoning, the method comprising the step of administering a therapeutically effective amount of the cocaine esterase mutant of the first aspect or the composition of the fifth aspect to a subject in need of such treatment.

[0028] In a preferred embodiment, the metabolite is benzoyl saccharide (BZE).

[0029] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0030] Figure 1 The structural formulas of cocaine and benzoyl sphagine are shown; Figure 2 Enzyme kinetics of cocaine and BZE catalyzed in vitro by the highly active CocE mutant are shown, where E196-301 represents T172R / G173Q / L196C / I301C, E116-51 represents A51L / V116K / T172R / G173Q / L196C / I301C, and E55-407 represents A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I; Figure 3The in vivo clearance time-dose curves of cocaine and BZE by the CocE mutant, as well as the efficiency of EME and ECG in the formation of the products, are shown. Among them, E196-301 represents T172R / G173Q / L196C / I301C, E116-51 represents A51L / V116K / T172R / G173Q / L196C / I301C, and E55-407 represents A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I. Figure 4 Enzyme kinetics curves of CocE mutant catalyzing cocaine and BZE in vitro are shown. Detailed Implementation

[0031] Through extensive and in-depth research, the inventors unexpectedly discovered that further mutations of known cocaine esterases resulted in mutants that not only retained the catalytic efficiency against the toxic cocaine metabolite benzoyl sphagnum, but also exhibited significantly enhanced catalytic activity against cocaine, thus yielding a bifunctional metabolic enzyme capable of rapidly degrading both cocaine and benzoyl sphagnum. This invention was completed based on this discovery.

[0032] Bifunctional cocaine esterase Because cocaine is hydrolyzed into benzoyl zeolite (BZE), a metabolite with a longer half-life and greater toxicity, after entering the human body, it is necessary not only to eliminate the toxicity of cocaine itself, but also to eliminate the toxicity caused by the toxic metabolite BZE.

[0033] Existing bacterial cocaine esterases (CocE) can efficiently catalyze the hydrolysis of cocaine, but exhibit low activity towards BZE. Therefore, researchers have attempted to develop CocE mutants to improve the catalytic efficiency of BZE. However, due to the charge difference between cocaine and BZE molecules, CocE mutants struggle to catalyze both substrates simultaneously. For example, US 12180519 B2 discloses a cocaine esterase mutant, A51L / V116K / T172R / G173Q / L196C / I301C, which significantly improves the catalytic efficiency of BZE; however, this mutant exhibits a significant decrease in catalytic activity towards cocaine.

[0034] To develop a bifunctional metabolic enzyme that can rapidly degrade both cocaine and BZE, the inventors further mutated the existing cocaine esterase. The resulting cocaine esterase mutant not only retained the highly efficient activity of catalyzing the hydrolysis of cocaine, but also significantly improved the catalytic efficiency for the cocaine metabolite benzoyl sphagnum.

[0035] In a specific embodiment, the present invention provides a cocaine esterase mutant whose amino acid sequence contains an amino acid mutation at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: 42, 43, 44, 48, 49, 50, 51, 52, 53, 54, 55, 56, 87, 116, 118, 119, 141, 150, 162, 165, 166, 169, 170, 172, 173, 196, 261, 288, 290, 301, 407, 408. In particular, the inventors have found that amino acid mutations at positions 55 and / or 407 of the amino acid sequence corresponding to SEQ ID NO: 1 are crucial for the obtained cocaine esterase mutant to possess both catalytic activity for the hydrolysis of cocaine and catalytic activity for the hydrolysis of the cocaine metabolite benzoyl bud base.

[0036] In a preferred embodiment, the amino acid sequence of the cocaine esterase mutant of the present invention has amino acid mutations at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: 51, 116, 172, 173, 196, 301. In a further preferred embodiment, the amino acid sequence of the cocaine esterase mutant of the present invention has amino acid mutations at the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: 51, 55, 116, 172, 173, 196, 301, 407.

[0037] As is well known to those skilled in the art, finding suitable mutation sites is more important for the activity of the resulting mutants. Therefore, under the teachings of this invention, those skilled in the art can prepare various mutants and test the activity of the resulting mutants. In a preferred embodiment, the amino acid sequence of the cocaine esterase mutant of this invention contains the following mutations: A51L / Q55D / V116K / T172R / G173Q / L196C / I301C, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I; more preferably, the mutation shown in A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I is present.

[0038] Furthermore, it will be readily apparent to those skilled in the art that altering a few amino acid residues in certain regions of a polypeptide does not substantially change its biological activity; for example, appropriately substituting certain amino acids to obtain a sequence does not affect its activity (see Watson et al., Molecular Biology of The Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224). Therefore, those skilled in the art can perform such substitutions (including residue substitution, deletion, insertion, and addition) and ensure that the resulting polypeptide retains its initial function.

[0039] Therefore, it is obvious that further mutations can be made into the cocaine esterase mutant of the present invention to obtain a further mutant that still possesses both the activity of catalyzing the hydrolysis of cocaine and the activity of catalyzing the hydrolysis of the cocaine metabolite benzoyl spore base. For example, it is known to those skilled in the art that adding or removing several amino acid residues at either end of a polypeptide, such as preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue, will not affect the function of the resulting mutant. For example, for ease of purification, those skilled in the art often add a 6×His tag to either end of the obtained protein, and such a protein has the same function as a protein without a 6×His tag.

[0040] The present invention also provides polynucleotides encoding the polypeptides of the present invention. The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding this polypeptide, or may include polynucleotides that also include additional coding and / or non-coding sequences.

[0041] The cocaine esterase mutant gene can be a probe prepared under stringent conditions with the amino acid sequence shown in SEQ ID NO: 1, such as DNA that hybridizes to a sequence complementary to part or all of the amino acid sequence shown in SEQ ID NO: 1, as long as its initial function is maintained. The “stringent conditions” refer to conditions that allow so-called specific hybridization to occur without non-specific hybridization. For example, conditions where highly homologous DNAs, such as DNAs with more than 80% homology, hybridize with each other, and DNAs with less than 80% homology do not hybridize with each other, or typical Southern hybridization washing conditions, i.e., washing once, preferably 2-3 times, at a salt concentration and temperature equivalent to 60°C, 1*SSC, 0.1% SDS, preferably 60°C, 0.1*SSC, 0.1% SDS, more preferably 68°C, 0.1*SSC, 0.1% SDS.

[0042] Furthermore, because codon degeneracy varies from host to host, any codon in the cocaine esterase gene can be replaced with a corresponding equivalent codon. That is, the cocaine esterase gene can be any of the cocaine esterase genes mentioned above, for example, due to the degeneracy of the genetic code. For instance, the cocaine esterase gene can be a modified gene that has the optimal codon based on the frequency of the codons used in the host.

[0043] Therefore, the terms “containing,” “having,” or “including” used in this article include “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0044] As used herein, the term "active fragment" has the same or similar meaning as conventionally understood by those skilled in the art, referring to a fragment whose amino acid sequence is part of the complete protein or polypeptide sequence, but which possesses the same or similar function or activity as the complete protein or polypeptide. Specifically, in this invention, "active fragment" refers to any amino acid sequence having a cocaine esterase mutant.

[0045] Based on the teachings of this invention and the cocaine esterase mutant specifically obtained by this invention, those skilled in the art can easily obtain active fragments with the same or similar activities or functions, and such active fragments should naturally fall within the protection scope of this invention.

[0046] "corresponds to" As used herein, the term "corresponds to" has the meaning commonly understood by one of ordinary skill in the art. Specifically, "corresponds to" means that, after homology or sequence identity alignment, one sequence corresponds to a specified position in another sequence. Therefore, for example, regarding "corresponding to the 11th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1," if a 6×His tag is added to one end of the amino acid sequence shown in SEQ ID NO: 1, then the 11th position corresponding to the 17th position in the resulting mutant might be the 17th position.

[0047] In specific embodiments, the homology or sequence similarity can be 90% or more, preferably 95% or more, more preferably 96%, 97%, 98%, or 99% homology. Therefore, cocaine esterase mutants that have 90% or more sequence similarity or homology with the specific cocaine esterase of the present invention, and have the amino acid residue mutation at the above-mentioned sites, are also included within the scope of protection of the present invention.

[0048] The corresponding position of any amino acid sequence to the amino acid sequence shown in SEQ ID NO: 1 can be determined by comparing the amino acid sequences. Methods for determining sequence homology or identity known to those skilled in the art include, but are not limited to: Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, edited by Gribskov, M. and Devereux, J., M. Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0049] host cells As used herein, the term "host cell" has the meaning commonly understood by those skilled in the art, namely, a host cell capable of producing the cocaine esterase mutant of the present invention. In other words, the present invention can utilize any host cell, as long as the cocaine esterase mutant of the present invention can be expressed in that host cell.

[0050] For example, the host cells suitable for use in this invention are selected from prokaryotic cells. In specific embodiments, the host cells suitable for use in this invention include, but are not limited to, Escherichia coli cells, such as BL21(DE3), Shuffle, and Origami.

[0051] Immobilized enzymes As used herein, the term "immobilized enzyme" has the meaning conventionally understood by one of ordinary skill in the art. Specifically, the term refers to a water-soluble enzyme that has been treated by physical or chemical methods to bind to or encapsulate a water-insoluble macromolecular carrier, thereby forming a water-soluble gel or semi-permeable membrane microcapsule that reduces its fluidity.

[0052] Immobilized enzymes retain their enzymatic activity, acting on the substrate in a solid state during catalytic reactions. Immobilization generally increases enzyme stability, facilitates separation from the reaction system, and allows for easy control and repeated use. It also facilitates transportation and storage, which is beneficial for automated production. Immobilized enzymes are an enzyme application technology that has developed over the past decade and shows promising application prospects in industrial production, chemical analysis, and pharmaceuticals.

[0053] Those skilled in the art, based on the teachings herein, will readily recognize that the cocaine esterase mutant of the present invention can be processed into an immobilized enzyme or used in a whole-cell catalytic form for the hydrolysis of cocaine and its toxic metabolites.

[0054] The pharmaceutical composition of the present invention Based on the cocaine esterase mutant of the present invention, the present invention provides a pharmaceutical composition containing a therapeutically effective amount of the cocaine esterase mutant of the present invention, and a pharmaceutically acceptable carrier or excipient.

[0055] The pharmaceutical compositions of the present invention can be formulated into dosage forms suitable for various routes of administration, but are primarily suitable for intravenous administration. The dosage is the amount used to effectively improve or eliminate symptoms of cocaine and / or its toxic metabolites.

[0056] The cocaine esterase mutant or pharmaceutical composition of the present invention can be administered to any mammal, provided that they can obtain a therapeutic effect from the cocaine esterase mutant or pharmaceutical composition of the present invention. Humans are the most important of these mammals.

[0057] The pharmaceutical compositions of the present invention can be manufactured using known methods. The dosage of the pharmaceutical compositions of the present invention can be determined by the treating physician based on the actual situation.

[0058] Methods for simultaneous hydrolysis of cocaine and BZE Based on the bifunctional cocaine esterase of the present invention, the present invention also provides a method for the simultaneous hydrolysis of cocaine and its metabolites, comprising the step of contacting cocaine and its metabolites with a cocaine esterase mutant of the present invention, a pharmaceutical composition, or an immobilized enzyme. The method for the simultaneous hydrolysis of cocaine and its metabolites is preferably an in vitro method.

[0059] Furthermore, the present invention also provides a method for treating cocaine and its metabolite poisoning, comprising administering a therapeutically effective amount of the cocaine esterase mutant of the present invention, or a pharmaceutical composition thereof, to a subject in need, thereby effectively improving or eliminating symptoms of poisoning by one or more cocaine and / or its toxic metabolites. Administration methods include, but are not limited to, various administration methods well known in the art, but primarily intravenous administration.

[0060] In this document, the terms "synchronous" or "simultaneously" have the same meaning, referring to the fact that the cocaine esterase mutant of the present invention can catalyze both the hydrolysis of cocaine and the hydrolysis of the toxic metabolite BZE of cocaine. In specific embodiments, the simultaneous hydrolysis of cocaine and its metabolites by the cocaine esterase mutant of the present invention refers to effectively improving or eliminating the symptoms of poisoning from one or more cocaine and / or its toxic metabolites.

[0061] Based on the teachings of this invention, those skilled in the art will undoubtedly understand that the cocaine esterase mutant of this invention possesses pharmaceutical activity. Therefore, the cocaine esterase mutant of this invention can be used as a drug for the treatment of diseases. Based on the teachings of this invention and conventional techniques in the art, those skilled in the art can, without creative effort, detect various characteristics that the cocaine esterase mutant of this invention should possess as a drug, such as drugability, bioavailability, toxicity, etc.

[0062] Advantages of this invention: 1. Based on the prior art, the inventors further mutated the cocaine esterase, and the resulting cocaine esterase significantly improved the catalytic efficiency of both cocaine and its toxic metabolite benzoyl sphagine. 2. The cocaine esterase of the present invention is a bifunctional metabolic enzyme that can rapidly degrade both cocaine and BZE, and therefore has very important clinical value for achieving complete cocaine detoxification. 3. The cocaine esterase of the present invention lays a completely new material basis for the development of cocaine antidote drugs.

[0063] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0064] Example Example 1. Obtaining CocE mutants using site-directed mutagenesis. The wild-type CocE described in this invention comprises 574 amino acids, with the following sequence: MVDGNYSVASNVMVPMRDGVRLAVDLYRPDADGPVPVLLVRNPYDKFDVFAWSTQSTNWLEFVRDGYAVVIQDTRGLFASEGEFVPHVDDEADAEDTLSWILEQAWCDGNVGMFGVSYLGVTQWQAAVSGVGGLKAIAPSMASADLYRAPWYGPGGALSVEALLGWSALIGTGLITSRSDARPEDAADFVQLAAILNDVAGAASVTPLAEQPLLGRLIPWVIDQVVDHPDNDESWQSISLFERLGGLATPALITAGWYDGFVGESLRTFVAV KDNADARLVVGPWSHSNLTGRNADRKFGIAATYPIQEATTMHKAFFDRHLRGETDALAGVPKVRLFVMGIDEWRDETDWPLPDTAYTPFYLGGSGAANTSTGGGTLSTSISGTESADTYLYDPADPVPSLGGTLLFHNGDNGPADQRPIHD RDDVLCYSTEVLTDPVEVTGTVSARLFVSSSAVDTDFTAKLVDVFPDGRAIALCDGIVRMRYRETLVNPTLIEAGEIYEVAIDMLATSNVFLPGHRIMVQVSSSNFPKYDRNSNTGGVIAREQLEEMCTAVNRIHRGPEHPSHIVLPIIKR (SEQ ID NO: 1).

[0065] First, wild-type CocE cDNA (GenBank# AF173165.1, synthesized by Shanghai Jierui Biotechnology) was constructed into the E. coli expression vector pET-22b(+) (provided by Shanghai Jierui Biotechnology, containing the C-terminal -6×His target gene inserted into NdeI and XhoI restriction sites). Using the wild-type CocE plasmid as a template, primers for the mutant were designed, and the mutant PCR product was amplified by PCR (KOD One MaterMix, Shanghai Toyobo). After removing the DNA template from the product with DpnI (Thermo Scientific, FD1703), the product was transformed into competent cells (DH5α) to circularize the PCR product. The transformed competent cell culture was plated on LB solid medium containing 100 μg / mL ampicillin and cultured at 37°C for 15 hours. Single clones were selected, and the mutant plasmid was extracted using a plasmid extraction kit. The obtained mutant plasmid was confirmed by DNA sequencing. For cells with multiple mutations, one mutation was performed before proceeding to the next round of mutations. The primer designs for the mutants are shown in Table 1.

[0066] Table 1 Primer sequences used for site-directed mutagenesis Example 2: Protein Expression and Purification The successfully constructed mutant plasmid was transformed into *E. coli* BL21 competent cells for protein expression. The bacterial culture was inoculated into LB liquid medium (containing 100 μg / mL ampicillin) and cultured at 37°C and 180 rpm on a shaker until OD600 = 0.6–0.8. The culture was then cooled to 15°C. IPTG (Sigma, 367-93-1) was added to a final concentration of 1 mM, and protein expression was induced at 15°C and 180 rpm for 15 hours. The bacterial cells were collected and resuspended in 50 mM Tris-HCl (pH 7.4) buffer containing 150 mM NaCl. E. coli cells were lysed using a pre-cooled high-pressure cell disruptor (SCIENTZ JG-IA, Ningbo Xinzhi). After centrifugation at 9000 rpm for 45 min, the supernatant was collected. The supernatant and nickel medium (Chromsep, 11-0110-03) were mixed by rotation at 4°C for 2 hours to bind the 6×His protein to the medium. The binding buffer was added to a gravity column and allowed to flow out naturally under gravity. The target protein was purified by elution using a gradient of imidazole concentrations. The eluted fraction was collected into a 30K (Millipore) ultrafiltration tube, and the buffer was replaced by centrifugation. The protein was stored in Solution S solution (50 mM HEPES, 20% D-sorbitol, 1 M glycine, pH 7.4). Protein concentration was determined using a Bradford assay kit (Sangon Biotech, C503031-1000).

[0067] Example 3: In vitro activity analysis of hydrolytic enzymes First, an HPLC method was established for the detection of the substrate cocaine, BZE, and the product benzoic acid (BA). Cocaine, BZE, and BA all exhibit strong UV absorption at 230 nm. HPLC analysis was performed using acetonitrile and 0.1% formic acid as the mobile phase, separated by a C18 liquid chromatography column. The absorbance of cocaine, BZE, and BA at 230 nm was detected using a UV detector, and linear standard curves for cocaine, BZE, and BA were obtained. The activity of CocE in the reaction of cocaine and BZE was determined at 37℃, with three replicates per group. The enzymatic reaction was initiated by diluting 50 μL of substrate BZE / cocaine solution with 50 μL of enzyme solution (0.1 M phosphate buffer (pH 7.4)). Specific reaction conditions are shown in Tables 2 and 3.

[0068] Table 2. In vitro catalytic reaction conditions of mutant enzymes with substrate BZE Table 3. In vitro catalytic reaction conditions of the mutant enzyme with the substrate cocaine The reaction was then terminated by adding 50 μL of 10% perchloric acid and 50 μL of acetonitrile. The mixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was diluted to an appropriate ratio and injected in 50 µL. The peak times, peak areas, and standard curves of cocaine, BZE, and BA were compared to calculate the residual concentrations of cocaine and BZE and the concentration of BA generated in the reaction sample. By calculating the reaction rate of BA catalyzed by the enzyme at different substrate concentrations, enzyme kinetic curves were plotted using GraphPad Prism 8, and Michaelis-Menten kinetic analysis was performed to obtain the catalytic parameters of each mutant for cocaine and BZE. k cat and K M The results are shown in Table 4. For mutations with lower activity, only the activity at a single substrate concentration point was measured, and the results are shown in Table 5. A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I shows further improved activity compared to the currently known highly active mutants A51L / V116K / T172R / G173Q / L196C / I301C and T172R / G173Q / L196C / I301C (catalyzing cocaine). k cat = 2248 min -1 , K M = 39.3 µM, k cat / K M = 5.7 × 10 7 min -1 M -1 ; Catalyst BZE k cat = 2664min -1 , K M = 78.7 µM, k cat / K M = 3.4×10 7 min -1 M -1 It exhibits high catalytic activity for both cocaine and its toxic metabolite BZE.

[0069] Table 4. Catalytic kinetic parameters of the mutant enzyme for cocaine and BZE Remark: K eff The catalytic efficiency of the corresponding enzyme for the substrate BZE ( k cat / K M ).

[0070] RCE refers to the ratio of the catalytic efficiency of the mutant enzyme on the substrate to the catalytic efficiency of A51L / V116K / T172R / G173Q / L196C / I301C on the substrate.

[0071] RCE cocaine * RCE BZE The product of the mutant enzyme's catalytic efficiency for cocaine and BZE and the ratio of the catalytic efficiency of A51L / V116K / T172R / G173Q / L196C / I301C for cocaine and BZE reflects the overall catalytic effect of the mutant enzyme on the two substrates compared to A51L / V116K / T172R / G173Q / L196C / I301C.

[0072] Table 5. Single-point catalytic reaction rates of mutant enzymes for cocaine and BZE Additionally, mutants P43D / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C, P43E / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C, V49D / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C, V49E / A51L / Q55D / V116K / T172R / G173Q / L196C / I301C, and A51L / S53E / Q55D / V116K / T172R / G173Q / L196C / I301C, A51L / Q55D / V116K / Y118D / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / Y118E / T172R / G173Q / L196C / I301C、A 51L / Q55D / V116K / L119E / T172R / G173Q / L196C / I301C, A51L / Q55D / V116K / G165D / T172R / G173Q / L196C / I301C、A51L / Q55D / V116K / W166D / T172R / G173Q / L196C / I301C, A51L / Q55D / V116K / W166E / T172R / G173Q / L196C / I301C, A51L / Q55D / V116K / I170D / T172R / G173Q / L196C / I301C, A51L / Q55D / V116K / T172R / G173Q / L1 96C / F261D / I301C, A51L / Q55D / V116K / T172R / G173Q / L196C / F261E / I301C, A51L / Q55D / V116K / T172R / G173Q / L196C / L290E / I301C, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407D, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407E, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / F408D, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / F408E. Single-point activity testing conditions were the same as for A51L / Q55D / V116K / T172R / G173Q / L196C / I301C. No product formation was detected, indicating that the activity is relatively lower than that of A51L / Q55D / V116K / T172R / G173Q / L196C / I301C is lower.

[0073] Example 4. In vivo activity analysis of highly active hydrolytic enzymes To compare the efficiency of the A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I mutant obtained in this invention in eliminating cocaine and BZE in vivo compared to the currently available highly active mutants A51L / V116K / T172R / G173Q / L196C / I301C and T172R / G173Q / L196C / I301C, rats were intravenously injected with saline, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I, and A51L / V116K / T172R / G173Q / L196C / I301C / L407I at a dose of 0.6 mg / kg via tail vein injection. G173Q / L196C / I301C and T172R / G173Q / L196C / I301C were administered via tail vein injection of 5 mg / kg cocaine and 2 mg / kg BZE 20 seconds later. Blood samples were collected at 2, 5, 15, 30, 45, 60, 90, and 120 minutes after cocaine injection to measure the concentrations of cocaine, BZE, cocaine product EME, and BZE product ECG, comparing the ability of different proteins to metabolize cocaine and BZE in vivo. Figure 3 As shown in the figure, the A51L / V116K / T172R / G173Q / L196C / I301C group only effectively reduced BZE, with poor effects on cocaine degradation; the T172R / G173Q / L196C / I301C group only effectively reduced cocaine, with poor effects on BZE degradation; while the A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I group effectively reduced cocaine levels in rats and also had a good clearance effect on BZE. These results indicate that the designed mutant, A51L / Q55D / V116K / T172R / G173Q / L196C / I301C / L407I, as a bifunctional cocaine metabolic enzyme, can simultaneously and efficiently catalyze the degradation of cocaine and its main toxic metabolite, BZE.

[0074] Although embodiments of this application have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting this application. Various changes and modifications may be made to this application without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the claims of this application.

[0075] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A cocaine esterase mutant, wherein the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO:1: 42、43、44、48、49、50、51、52、53、54、55、56、87、116、118、119、141、150、162、165、166、169、170、172、173、196、261、288、290、301、407、408。 2. The cocaine esterase mutant of claim 1, wherein the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at position 55 and / or position 407 corresponding to the amino acid sequence shown in SEQ ID NO:

1.

3. The cocaine esterase mutant of claim 2, wherein the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: 51、116、172、173、196、301。 4. The cocaine esterase mutant of claim 2, wherein the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: 51、55、116、172、173、196、301、407。 5. The cocaine esterase mutant according to any one of claims 1-4, wherein the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: The 42nd position is mutated to E or D, or a conserved substitution residue of E or D; The 43rd position is mutated to E or D, or a conserved substitution residue of E or D; The 44th position is mutated to K, or a conserved substitution residue of K is made. The 48th position is mutated to E, or a conserved substitution residue of E is made. The 49th position is mutated to D or E, or a conserved substitution residue of D or E; The 50th position is mutated to E or D, or a conserved substitution residue of E or D; The 51st position is mutated to L, or a conserved substitution residue of L is made. The 52nd position is mutated to D, E, or L, or a conserved substitution residue of D, E, or L; The 53rd position is mutated to D or E, or a conserved substitution residue of D or E; The 54th position is mutated to D or E, or a conserved substitution residue of D or E; The 55th position is mutated to D, E, F, V or K, or a conserved substitution residue of D, E, F, V or K; The 56th position is mutated to E or D, or a conserved substitution residue of E or D; The 87th position is mutated to W, or a conserved substitution residue of W is made. The 116th position is mutated to K, or a conserved substitution residue of K is made. The 118th position is mutated to D, E, or K, or a conserved substitution residue of D, E, or K; The 119th position is mutated to K, D, or E, or a conserved substitution residue of K, D, or E; The 141st position is mutated to G or K, or a conserved substitution residue of G or K; The 150th position is mutated to C, or a conserved substitution residue of C is made. The 162nd position is mutated to G, or a conserved substitution residue of G is made. The 165th position is mutated to D or E, or a conserved substitution residue of D or E; The 166th position is mutated to D or E, or a conserved substitution residue of D or E; The 169th position is mutated to E or D, or a conserved substitution residue of E or D; The 170th position is mutated to E or D, or a conserved substitution residue of E or D; The 172nd position is mutated to R, or a conserved substitution residue of R is made. The mutation at position 173 is Q, or a conserved substitution residue for Q; The 196th position is mutated to C, E, R, K or D, or a conserved substitution residue of C, E, R, K or D; The 261st position is mutated to E or D, or a conserved substitution residue of E or D is made. The 288th position is mutated to D, A, F, L, E, R, or K, or a conserved substitution residue of D, A, F, L, E, R, or K; The 290th position is mutated to D or E, or a conserved substitution residue of D or E; The 301st position is mutated to C, or a conserved substitution residue of C is made. The 407 position is mutated to D, E, R, V or I, or a conserved substitution residue of D, E, R, V or I; The 408th position is mutated to D or E, or a conserved substitution residue of D or E.

6. The cocaine esterase mutant of claim 5, wherein the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at one or more of the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: The 51st position is mutated to L, or a conserved substitution residue of L is made. The 55th position is mutated to D, E, F, V or K, or a conserved substitution residue of D, E, F, V or K; The 116th position is mutated to K, or a conserved substitution residue of K is made. The 172nd position is mutated to R, or a conserved substitution residue of R is made. The mutation at position 173 is Q, or a conserved substitution residue for Q; The 196th position is mutated to C, E, R, K or D, or a conserved substitution residue of C, E, R, K or D; The 301st position is mutated to C, or a conserved substitution residue of C is made. The 407 position is mutated to D, E, R, V or I, or a conserved substitution residue of D, E, R, V or I.

7. The cocaine esterase mutant of claim 6, wherein the amino acid sequence of the cocaine esterase mutant has an amino acid mutation at the following sites corresponding to the amino acid sequence shown in SEQ ID NO: 1: The 51st position is mutated to L, or a conserved substitution residue of L is made. The 55th position is mutated to D, or a conserved substitution residue of D is made. The 116th position is mutated to K, or a conserved substitution residue of K is made. The 172nd position is mutated to R, or a conserved substitution residue of R is made. The mutation at position 173 is Q, or a conserved substitution residue for Q; The 196th position is mutated to C, or a conserved substitution residue of C is made. The 301st position is mutated to C, or a conserved substitution residue of C is made. The 407 position is mutated to I, or a conserved substitution residue of I is made.

8. An isolated polynucleotide molecule, said polynucleotide molecule encoding a cocaine esterase mutant according to any one of claims 1-7.

9. An expression vector comprising the polynucleotide molecule of claim 8.

10. A host cell comprising the expression vector of claim 9, or the host cell having the polynucleotide molecule of claim 8 integrated into its genome.

11. A composition comprising the cocaine esterase mutant according to any one of claims 1-7.

12. An immobilized enzyme comprising a cocaine esterase mutant according to any one of claims 1-7.

13. A method for simultaneous hydrolysis of cocaine and its metabolites, the method comprising the step of contacting cocaine and its metabolites with a cocaine esterase mutant according to any one of claims 1-7, the composition according to claim 11, or the immobilized enzyme according to claim 12.

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