High-activity Mutants Research Articles

Direct Screening of PET Hydrolase Activity in Culture Medium Based on Turbidity Reduction.

The development of an efficient screening method for the activity of PET-degrading enzymes represents a significant technological advance in the field of enzyme research, with the potential to facilitate the advancement of enzymes for PET recycling. By examining the stable conditions of PET suspension and enzyme production conditions, we developed a method to quantify PET-degrading enzyme activity in E. coli culture medium using turbidity reduction as an indicator. High PET concentration or ionic strength caused aggregation of PET, and the best condition for activity detection was 0.5 mg mL-1 PET in 50 mM sodium phosphate pH 7.0. Preculture of E. coli increased the purity of enzyme secreted in medium. To evaluate the screening method, 720 colonies of the PET2-7M-H229X-F233X mutant library were analyzed and three candidates of high-activity mutants were obtained. The thermostability of the mutants could also be easily measured by measuring the residual activity after heat treatment. The H229T-F233M mutant showed 3.4 times higher degradation rate against PET film than the template enzyme at the initial time. The molecular dynamics simulation implied that the F233M mutation makes space for making an α helix and that the H229T mutation resolved the steric hindrance with Trp199. These mutations were speculated to change the angle of the Trp199 side chain of PET2 to an angle similar to that of the Trp185 of IsPETase, making it suitable for PET binding to the active center. Screening of activity using PET suspensions is compatible with robotic automation and is expected to be useful for validating computationally predicted mutations.

Response to targeted and immune therapy in patients with NSCLC and BRAF non-V600E mutations: A retrospective, multicenter analysis within the national Network Genomic Medicine Lung Cancer (nNGM) in Germany.

8591 Background: Non-small-cell lung cancer (NSCLC) poses a significant global health challenge, with multiple treatment strategies targeting oncogenic drivers. The BRAF V600E mutation, found in 1-3% of NSCLC patients, is effectively targeted by dabrafenib and trametinib, achieving considerable clinical benefits. However, BRAF non-V600E mutations, constituting half of all BRAF mutations, are challenging to treat due to their heterogeneity and lack of information concerning outcome upon targeted and immune therapies. Methods: In order to investigate outcome of patients with BRAF non-V600E mutations upon targeted and (chemo)immune therapy we conducted a retrospective analysis within the national Network Genomic Medicine (nNGM) lung cancer in Germany and. In addition, we evaluated the MEK/ERK activation potential of the BRAF non-V600E mutations from our cohort and their sensitivity to dabrafenib and/or trametinib in vitro. Results: Twenty patients with 11 different BRAF non-V600E mutations were identified. From these patients, 15% achieved partial remission, 40% stable disease, and 40% progressive disease under TKI-therapy. Progression-free survival (PFS) upon targeted therapy ranged from 0.9 to 53.5 months. Notably, patients with MEK/ERK-activating BRAF non-V600E mutations exhibited persistent responses (mPFS 9 months). Specifically, patients with the high-activity mutants p.V600D, p.V600K and p.K601E showed responses to TKI therapy (mPFS 9.2 months). Similar outcome was achieved in patients with activating p.G466V (PFS 4.9 months), p.G469R (PFS 9 months), p.G469V (mPFS 28.2 months) and p.V600_K601delinsE (mPFS 5.6 months) mutations. Interestingly, outcome upon treatment with (chemo)immune therapy for these patients with activating mutations was similar compared to targeted therapy (mPFS 15.0 and 11.7 months). Expectedly, patients with kinase-dead mutants (p.G466R, p.D594E, p.D594G) were not responding to TKIs (mPFS 2.5 months) and had considerably better outcomes under (chemo)immune therapy (mPFS 11.0 and 11 months). Conclusions: Patients with activating BRAF non-V600E mutations can be treated efficiently with BRAF/MEK TKI or (chemo)immune therapy while patients with non-activating mutations do not benefit from targeted therapy but from (chemo)immune therapy. Hence, it is of key importance to apply preclinical activation assays showing good correlations to clinical outcomes in our study prior to applying targeted therapy in patients with unknown BRAF non-V600E mutations. Altogether, our study helps to guide personalized treatment decisions in this patient population.

A high-throughput visual screening method for p-hydroxybenzoate hydroxylase to increase phenolic compounds biosynthesis

BackgroundGallic acid (GA) and pyrogallol are phenolic hydroxyl compounds and have diverse biological activities. Microbial-based biosynthesis, as an ecofriendly method, has been used for GA and pyrogallol production. In GA and pyrogallol biosynthetic pathways, the low hydroxylation activity of p-hydroxybenzoate hydroxylase (PobA) towards 3,4-dihydroxybenzoic acid (3,4-DHBA) limited the high-level biosynthesis of GA and pyrogallol.ResultsThis work reported a high activity PobA mutant (Y385F/T294A/V349A PobA) towards 3,4-DHBA. This mutant was screened out from a PobA random mutagenesis library through a novel naked eye visual screening method. In vitro enzyme assay showed this mutant has a kcat/Km of 0.059 μM−1 s−1 towards 3,4-DHBA, which was 4.92-fold higher than the reported mutant (Y385F/T294A PobA). Molecular docking simulation provided the possible catalytic mechanism explanation of the high activity mutant. Expression of this mutant in E. coli BW25113 (Fʹ) can generate 840 ± 23 mg/L GA from 1000 mg/L 3,4-DHBA. After that, this mutant was assembled into a de novo GA biosynthetic pathway. Subsequently, this pathway was introduced into a 3,4-DHBA-producing strain (E. coli BW25113 (Fʹ)ΔaroE) to achieve 301 ± 15 mg/L GA production from simple carbon sources. Similarly, assembling this mutant into a de novo pyrogallol biosynthetic pathway enabled 129 ± 15 mg/L pyrogallol production.ConclusionsThis work established an efficient screening method and generated a high activity PobA mutant. Assembling this mutant into de novo GA and pyrogallol biosynthetic pathways achieved the production of these two compounds from glucose. Besides, this mutant has great potential for the production of GA or pyrogallol derivatives. The screening method could be used for other GA biosynthesis-related enzymes.

Directed Evolution Improves the Enzymatic Synthesis of L-5-Hydroxytryptophan by an Engineered Tryptophan Synthase.

L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high-activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) is 4.36 mM-1∙s-1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

Structure-based tailoring of the first coumarins-specific bergaptol O-methyltransferase to synthesize bergapten for depigmentation disorder treatment

Bergapten has long been used in combination with ultraviolet A irradiation to treat depigmentation disorder. However, extremely low bergapten contents in plants and difficulties in synthesizing bergapten have limited its application. Here, we developed an alternative bergapten-production method. We first determined the crystal structures of bergaptol O-methyltransferase from Peucedanum praeruptorum (PpBMT) and the ternary PpBMT–S-adenosyl-L-homocysteine (SAH)–bergaptol complex to identify key residues involved in bergaptol binding. Then, structure-based protein engineering was performed to obtain PpBMT mutants with improved catalytic activity towards bergaptol. Subsequently, a high-activity mutant was used to produce bergapten for pharmacological-activity analysis. Key PpBMT amino acids involved in bergaptol binding and substrate specificity were identified, such as Asp226, Asp246, Ser265, and Val320. Site-directed mutagenesis and biochemical analysis revealed that the V320I mutant efficiently transformed bergaptol to produce bergapten. Pharmacological-activity analysis indicated that bergapten positively affected hair pigmentation in mice and improved pigmentation levels in zebrafish embryos. This report provides the first description of the catalytic mechanism of coumarins-specific O-methyltransferase. The high-activity V320I mutant protein could be used in metabolic engineering to produce bergapten in order to treat depigmentation disorder. This structure–function study provides an alternative synthesis method and important advances for treating depigmentation disorders.

Functional characterizations of polyethylene terephthalate-degrading cutinase-like enzyme Cut190 mutants using bis(2-hydroxyethyl) terephthalate as the model substrate

A mutant of cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190_S226P/R228S, designated as Cut190*, possesses high-thermal stability and has high polyethylene terephthalate (PET)-degrading activity. The functional characterizations of PET-degrading enzymes are generally conducted using accessible substrates such as poly(butylene succinate-co-adipate) (PBSA) and p-nitrophenyl butyrate (pNPB), even though their structures are different from that of PET. Bis(2-hydroxyethyl) terephthalate (BHET) is a component of PET, and the structure is similar to that of PET compared to PBSA and pNPB. Therefore, the analysis using BHET as the substrate is important to evaluate effective PET degradation. In this study, we analyzed the enzymatic activity of Cut190* using BHET under various conditions including Ca2+ concentration and pressure. Although terephthalate was supposed to be the final product, the intermediate product, mono(2-hydroxyethyl) terephthalate (MHET), was the only product generated, possibly due to the low binding affinity of MHET. The Cut190* activity towards BHET was observed even in the absence of Ca2+ and increased with increasing Ca2+ concentration, which is similar to its activity towards pNPB, but different from its activity towards PBSA. The difference can be attributed to the size of substrates. We also analyzed the activities of Cut190* and another high-activity mutant, Cut190*Q138A/D250C-E296C, under high pressures up to 400 MPa. BHET was non-enzymatically hydrolyzed under high pressure at 37 ℃. Enzyme activities were maintained under high pressures, and degradation of BHET in the presence of enzyme was higher than that in the absence of enzyme. Furthermore, the structural analysis of Cut190* under high pressure using Fourier transform infrared spectroscopy showed that most of the enzyme molecules were populated in the native structure below 400 MPa. These results indicate that BHET can contribute to the effective functional analysis of PET-degrading enzyme, and the combination of enzyme and pressure can lead to eco-friendly PET degradation.

Abstract 2976: Engineering next-generation anti-CD25 immunotoxins with improved cytotoxic activity and low immunogenicity

Abstract Objective: CD25 is expressed on many T cell malignancies, including adult T cell leukemia, and is a target for cancer therapy. Immunotoxins against CD25+ cells containing a portion of Pseudomonas exotoxin A (PE38) show extremely high cytotoxic activity. Our lab has previously created a de-immunized PE immunotoxin in which toxin domain II was removed. Removal of domain II confers two major advantages: i) the immunogenic sequences found in domain II are removed, generating a less immunogenic toxin; ii) the non-specific toxicity of domain II-truncated toxins is much lower, allowing much higher doses of immunotoxin to be given safely (7-10-fold higher safe dosage). However, removal of domain II, which does not affect or enhances the cytotoxic activity of immunotoxins targeting the B cell marker CD22 or the mesothelioma marker mesothelin, lowers the activity of anti-CD25 immunotoxins 30-fold. Because lowering toxin immunogenicity and animal toxicity are critical for the success of immunotoxins in a clinical setting, we have engineered domain II truncated immunotoxins with improved activity against CD25+ cells using a novel adaptation of an established screening system. Methods: Eighteen domain II truncation mutants were designed based upon the crystal structure of the toxin. To bypass the resource intensive protein refolding of standard immunotoxins, these domain II mutants were expressed as fusions with the ZZ protein, which is a small (13.6kD) derivative of protein A. Fusion to ZZ allowed easy production of these ZZ-domain II mutant fusion proteins in the E. coli periplasm and fast purification using IgG coated beads. The purified ZZ-domain II mutant toxins were then bound to anti-CD25 IgG by mixing (the ZZ-domain binds human Fc) and tested for cytotoxic activity on the CD25+ T-cell leukemia HUT-102 cell line. The most active domain II mutants were made into standard recombinant immunotoxins and retested. Results: Of the 18 tested ZZ-domain II mutants, 6 were completely inactive, 8 were active at near parental levels, and 4 were as active as the parental toxin. The 4 high-activity and 2 structurally-interesting, medium-activity mutants were then produced as standard recombinant immunotoxins and retested for cytotoxic activity. Two of the 4 high-activity mutants showed a 2-4-fold increase in activity compared to parental domain II truncated immunotoxin. While still being 8-fold less active than immunotoxins containing domain II (PE38), these high-activity mutant immunotoxins will most likely have lower immunogenicity than toxins with PE38 and much less non-specific toxicity, allowing higher doses and more treatment cycles to be given. Our engineered high-activity domain II mutant immunotoxins are improved candidates for treating CD25+ cancers and for eliminating regulatory immunosuppressive T cells. Citation Format: Gilad Kaplan, Fred Lee, Ira Pastan. Engineering next-generation anti-CD25 immunotoxins with improved cytotoxic activity and low immunogenicity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2976.

Augmentation of glycine receptor alpha3 currents suggests a mechanism for glucose-mediated analgesia

The inhibitory glycine receptor (GlyR) mediates rapid synaptic inhibition in the mammalian central nervous system. Recently, glucose was identified as a positive modulator of α1 GlyRs. Here, recombinant human α3GlyRs with and without glucose treatment were studied using patch clamp methods. Similar to α1GlyRs, receptor variants α3L and α3K were potentiated by sugar. Glucose treatment reduced EC50 values of GlyR α3L and α3K by a factor of 4.5 and 3.3, respectively, without affecting maximum currents or desensitization. The high-activity mutant α3L(P185L) was not further potentiated by glucose. Potentiation of glycinergic signalling may underlie some of the analgetic effects of glucose.

Xylenol-orange-assay-of-hydrogen-peroxide for Measuring Uricase Activity and Recognizing High-activity Uricase Mutant

This study investigated xylenol-orange-assay-of-hydrogen-peroxide(XOAHP) for measuring uricase activities in cell lysates and recognizing mutants of higher activity.Four mutants of the intracellular uricase of Bacillus fastidiosus were combined into pairs so that ratios of their catalytic capacities varied from 1.3 to 4.1.The pET28a vector containing mutant gene was transformed into Escherichia coli BL21(DE3).Each of thirty clones was transferred into 1.0 mL liquid medium for amplification,and subject to induced expression for 16 h at 18 ℃.Cells were then harvested and lyzed via sonication treatment to get the supernatant as lysate.In Tris-HCl buffer at pH 8.9,XOAHP was effective to measure uricase activity with uric acid of no more than 0.33 mmol/L.SDS-PAGE showed small differences in expression efficiency of mutants.The activity concentration was positively related to the level of total proteins in lysates of tested mutants.Receiver-operation-curve(ROC) analysis showed the higher the ratio of catalytic capacities of uricase mutants in pairs the chloser to 1.00 of the area-under-thecurve(AUC),with an AUC 0.95 for a pair of mutants at the ratio of about 1.8 between their catalytic capacities.With a threshold of the difference between the activity concentration of a candidate as 1.4 times that of the standard deviation of the starting material,positive mutants of catalytic capacities 1.8 times that of the starting material could be effectively recognized.Therefore,XOAHP plus a proper threshold is effective for high-throughput-screening of mutant library of uricase.

Kinetic characterization of high-activity mutants of human butyrylcholinesterase for the cocaine metabolite norcocaine

It has been known that cocaine produces its toxic and physiological effects through not only cocaine itself, but also norcocaine formed from cocaine oxidation catalysed by microsomal CYP (cytochrome P450) 3A4 in the human liver. The catalytic parameters (kcat and Km) of human BChE (butyrylcholinesterase) and its three mutants (i.e. A199S/S287G/A328W/Y332G, A199S/F227A/S287G/A328W/E441D and A199S/F227A/S287G/A328W/Y332G) for norcocaine have been characterized in the present study for the first time and compared with those for cocaine. On the basis of the obtained kinetic data, wild-type human BChE has a significantly lower catalytic activity for norcocaine (kcat=2.8 min(-1), Km=15 μM and kcat/Km=1.87 × 10(5) M(-1)·min(-1)) compared with its catalytic activity for (-)-cocaine. The BChE mutants examined in the present study have considerably improved catalytic activities against both cocaine and norcocaine compared with the wild-type enzyme. Within the enzymes examined in the present study, the A199S/F227A/S287G/A328W/Y332G mutant (CocH3) is identified as the most efficient enzyme for hydrolysing both cocaine and norcocaine. CocH3 has a 1080-fold improved catalytic efficiency for norcocaine (kcat=2610 min(-1), Km=13 μM and kcat/Km=2.01 × 10(8) M(-1)·min(-1)) and a 2020-fold improved catalytic efficiency for cocaine. It has been demonstrated that CocH3 as an exogenous enzyme can rapidly metabolize norcocaine, in addition to cocaine, in rats. Further kinetic modelling has suggested that CocH3 with an identical concentration with that of the endogenous BChE in human plasma can effectively eliminate both cocaine and norcocaine in a simplified kinetic model of cocaine abuse.

Preparation and in vivo characterization of a cocaine hydrolase engineered from human butyrylcholinesterase for metabolizing cocaine

Cocaine is a widely abused drug without an FDA (Food and Drug Administration)-approved medication. It has been recognized that an ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. human BChE (butyrylcholinesterase)-catalysed hydrolysis. However, the native human BChE has a low catalytic activity against cocaine. We recently designed and discovered a BChE mutant (A199S/F227A/S287G/A328W/Y332G) with a high catalytic activity (kcat=5700 min-1, Km=3.1μM) specifically for cocaine, and the mutant was proven effective in protecting mice from acute cocaine toxicity of a lethal dose of cocaine (180mg/kg of body weight, LD100). Further characterization in animal models requires establishment of a high-efficiency stable cell line for the BChE mutant production at a relatively larger scale. It has been extremely challenging to develop a high-efficiency stable cell line expressing BChE or its mutant. In the present study, we successfully developed a stable cell line efficiently expressing the BChE mutant by using a lentivirus-based repeated-transduction method. The scaled-up protein production enabled us to determine for the first time the invivo catalytic activity and the biological half-life of this high-activity mutant of human BChE in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant (administered to mice 1min prior to cocaine) can quickly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.

Substrate selectivity of high-activity mutants of human butyrylcholinesterase.

Cocaine is one of the most addictive drugs, and there is still no FDA (Food and Drug Administration)-approved medication specific for cocaine abuse. A promising therapeutic strategy is to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma. However, the native BChE has a low catalytic efficiency against the abused cocaine, i.e. (-)-cocaine. Our recently designed and discovered A199S/F227A/S287G/A328W/Y332G mutant and other mutants of human BChE have a considerably improved catalytic efficiency against (-)-cocaine. In the present study, we carried out both computational modeling and experimental kinetic analysis on the catalytic activities of these promising new BChE mutants against other known substrates, including neurotransmitter acetylcholine (ACh), acetylthiocholine (ATC), butyrylthiocholine (BTC), and (+)-cocaine, in comparison with the corresponding catalytic activity against (-)-cocaine. Both the computational modeling and kinetic analysis have consistently revealed that all the examined amino acid mutations only considerably improve the catalytic efficiency of human BChE against (-)-cocaine, without significantly improving the catalytic efficiency of the enzyme against any of the other substrates examined. In particular, all the examined BChE mutants have a slightly lower catalytic efficiency against neurotransmitter ACh compared to the wild-type BChE. This observation gives us confidence in developing an anti-cocaine enzyme therapy by using one of these BChE mutants, particularly the A199S/F227A/S287G/A328W/Y332G mutant.

Human Butyrylcholinesterase–Cocaine Binding Pathway and Free Energy Profiles by Molecular Dynamics and Potential of Mean Force Simulations

In the present study, we have performed combined molecular dynamics and potential of mean force (PMF) simulations to determine the enzyme-substrate (ES) binding pathway and the corresponding free energy profiles for wild-type butyrylcholinesterase (BChE) binding with (-)/(+)-cocaine and for the A328W/Y332G mutant binding with (-)-cocaine. According to the PMF simulations, for each ES binding system, the substrate first binds with the enzyme at a peripheral anionic site around the entrance of the active-site gorge to form the first ES complex (ES1-like) during the binding process. Further evolution from the ES1-like complex to the nonprereactive ES complex is nearly barrierless, with a free energy barrier lower than 1.0 kcal/mol. So, the nonprereactive ES binding process should be very fast. The rate-determining step of the entire ES binding process is the subsequent evolution from the nonprereactive ES complex to the prereactive ES complex. Further accounting for the entire ES binding process, the PMF-based simulations qualitatively reproduced the relative order of the experimentally derived binding free energies (ΔG(bind)), although the simulations systematically overestimated the magnitude of the binding affinity and systematically underestimated the differences between the ΔG(bind) values. The obtained structural and energetic insights into the entire ES binding process provide a valuable base for future rational design of high-activity mutants of BChE as candidates for an enzyme therapy for cocaine overdose and abuse.

Reaction mechanism for cocaine esterase-catalyzed hydrolyses of (+)- and (-)-cocaine: unexpected common rate-determining step.

First-principles quantum mechanical/molecular mechanical free energy calculations have been performed to examine the catalytic mechanism for cocaine esterase (CocE)-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. It has been shown that the acylation of (+)-cocaine consists of nucleophilic attack of the hydroxyl group of Ser117 on the carbonyl carbon of (+)-cocaine benzoyl ester and the dissociation of (+)-cocaine benzoyl ester. The first reaction step of deacylation of (+)-cocaine, which is identical to that of (-)-cocaine, is rate-determining, indicating that CocE-catalyzed hydrolyses of (+)- and (-)-cocaine have a common rate-determining step. The computational results predict that the catalytic rate constant of CocE against (+)-cocaine should be the same as that of CocE against (-)-cocaine, in contrast with the remarkable difference between human butyrylcholinesterase-catalyzed hydrolyses of (+)- and (-)-cocaine. The prediction has been confirmed by experimental kinetic analysis on CocE-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. The determined common rate-determining step indicates that rational design of a high-activity mutant of CocE should be focused on the first reaction step of the deacylation. Furthermore, the obtained mechanistic insights into the detailed differences in the acylation between the (+)- and (-)-cocaine hydrolyses provide indirect clues for rational design of amino acid mutations that could more favorably stabilize the rate-determining transition state in the deacylation and, thus, improve the catalytic activity of CocE. This study provides a valuable mechanistic base for rational design of an improved esterase for therapeutic treatment of cocaine abuse.

Design, preparation, and characterization of high-activity mutants of human butyrylcholinesterase specific for detoxification of cocaine.

Cocaine is a widely abused drug without a U.S. Food and Drug Administration-approved medication. There is a recognized, promising anticocaine medication to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway [i.e., cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma]. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against (-)-cocaine but also certain selectivity for (-)-cocaine over neurotransmitter acetylcholine (ACh), such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. The present study accounting for the mutation-caused changes of the catalytic activities of BChE against both (-)-cocaine and ACh by means of molecular modeling and site-directed mutagenesis has led to identification of three BChE mutants that have not only a considerably improved catalytic efficiency against (-)-cocaine but also the desirable selectivity for (-)-cocaine over ACh. Two representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg). Pretreatment with the BChE mutant (i.e., 1 min before cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. In particular, all mice pretreated with the mutant (e.g., 0.02 mg or more of A199S/F227A/S287G/A328W/E441D BChE) survived. The in vivo data reveal the primary factor (i.e., the relative catalytic efficiency), determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment.

Design of High-Activity Mutants of Human Butyrylcholinesterase against (−)-Cocaine: Structural and Energetic Factors Affecting the Catalytic Efficiency

The present study was aimed to explore the correlation between the protein structure and catalytic efficiency of butyrylcholinesterase (BChE) mutants against (-)-cocaine by modeling the rate-determining transition state (TS1), i.e., the transition state for the first step of chemical reaction process, of (-)-cocaine hydrolysis catalyzed by various mutants of human BChE in comparison with the wild type. Molecular modeling of the TS1 structures revealed that mutations on certain nonactive site residues can indirectly affect the catalytic efficiency of the enzyme against (-)-cocaine through enhancing or weakening the overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. Computational insights and predictions were supported by the catalytic activity data obtained from wet experimental tests on the mutants of human BChE, including five new mutants reported for the first time. The BChE mutants with at least ∼1000-fold improved catalytic efficiency against (-)-cocaine compared to the wild-type BChE are all associated with the TS1 structures having stronger overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. The combined computational and experimental data demonstrate a reasonable correlation relationship between the hydrogen-bonding distances in the TS1 structure and the catalytic efficiency of the enzyme against (-)-cocaine.

Reaction Pathway and Free Energy Profile for Prechemical Reaction Step of Human Butyrylcholinesterase-Catalyzed Hydrolysis of (−)-Cocaine by Combined Targeted Molecular Dynamics and Potential of Mean Force Simulations

Combined targeted molecular dynamics (TMD) and potential of mean force (PMF) simulations have been carried out to uncover the detailed pathway and determine the corresponding free energy profile for the structural transformation from the nonprereactive butyrylcholinesterase (BChE)-(-)-cocaine binding to the prereactive BChE-(-)-cocaine binding associated with the (-)-cocaine rotation in the binding pocket of BChE. It has been shown that the structural transformation involves two transition states (TS1(rot) and TS2(rot)). TS1(rot) is mainly associated with the deformation of the nonprereactive complex, whereas TS2(rot) is mainly associated with the formation of the prereactive complex. It has also been demonstrated that the A328W/Y332G mutation significantly reduces the steric hindrance for (-)-cocaine rotation in the binding pocket of BChE and, thus, decreases the free energy barrier for the structural transformation from the nonprereactive binding to the prereactive binding. The calculated relative free energy barriers are all consistent with available experimental kinetic data. The new mechanistic insights obtained and the novel computational protocol tested in this study should be valuable for future computational design of high-activity mutants of BChE. The general computational strategy and approach based on the combined TMD and PMF simulations may be also valuable in computational studies of detailed pathways and free energy profiles for other similar mechanistic problems involving ligand rotation or another type of structural transformation in the binding pocket of a protein.

Free energy perturbation simulation on transition states and high-activity mutants of human butyrylcholinesterase for (-)-cocaine hydrolysis.

A unified computational approach based on free energy perturbation (FEP) simulations of transition states has been employed to calculate the mutation-caused shifts of the free energy change from the free enzyme to the rate-determining transition state for (-)-cocaine hydrolysis catalyzed by the currently most promising series of mutants of human butyrylcholinesterase (BChE) that contain the A199S/A328W/Y332G mutations. The FEP simulations were followed by Michaelis-Menten kinetics analysis determining the individual k(cat) and K(M) values missing for the A199S/F227A/A328W/Y332G mutant in this series. The calculated mutation-caused shifts of the free energy change from the free enzyme to the rate-determining transition state are in good agreement with the experimental kinetic data, demonstrating that the unified computational approach based on the FEP simulations of the transition states may be valuable for future computational design of new BChE mutants with a further improved catalytic efficiency against (-)-cocaine.

Characterization of a high-activity mutant of human butyrylcholinesterase against (−)-cocaine

Cocaine addiction and overdose are a well-known public health problem. There is no approved medication available for cocaine abuse treatment. Our recently designed and discovered high-activity mutant (A199S/S287G/A328W/Y332G) of human butyrylcholinesterase (BChE) has been recognized to be worth exploring for clinical application in humans as a potential anti-cocaine medication. The catalytic rate constant ( k cat) and Michaelis–Menten constant ( K M) for (−)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE (without fusion with any other peptide) have been determined to be 3060 min −1 and 3.1 μM, respectively, in the present study. The determined kinetic parameters reveal that the un-fused A199S/S287G/A328W/Y332G mutant has a ∼1080-fold improved catalytic efficiency ( k cat/ K M) against (−)-cocaine compared to the wild-type BChE. The ∼1080-fold improvement in the catalytic efficiency of the un-fused A199S/S287G/A328W/Y332G mutant is very close to the previously reported the ∼1000-fold improvement in the catalytic efficiency of the A199S/S287G/A328W/Y332G mutant fused with human serum albumin. These results suggest that the albumin fusion did not significantly change the catalytic efficiency of the BChE mutant while extending the plasma half-life. In addition, we have also examined the catalytic activities of the A199S/S287G/A328W/Y332G mutant against two other substrates, acetylthiocholine (ATC) and butyrylthiocholine (BTC). It has been shown that the A199S/S287G/A328W/Y332G mutations actually decreased the catalytic efficiencies of BChE against ATC and BTC, while considerably improving the catalytic efficiency of BChE against (−)-cocaine.

Recent Progress in Protein Drug Design and Discovery with a Focus on Novel Approaches to the Development of Anticocaine Medications

Cocaine is highly addictive and no anti-cocaine medication is currently available. Accelerating cocaine metabolism, producing biologically inactive metabolites, is recognized as an ideal anti-cocaine medication strategy, especially for the treatment of acute cocaine toxicity. However, currently known wild-type enzymes have either too low a catalytic efficiency against the abused cocaine, in other words (-)-cocaine, or the in vivo half-life is too short. Novel computational strategies and design approaches have been developed recently to design and discover thermostable or high-activity mutants of enzymes based on detailed structures and catalytic/inactivation mechanisms. The structure- and mechanism-based computational design efforts have led to the discovery of high-activity mutants of butyrylcholinesterase and thermostable mutants of cocaine esterase as promising anti-cocaine therapeutics. The structure- and mechanism-based computational strategies and design approaches may be used to design high-activity and/or thermostable mutants of many other proteins that have clear therapeutic potentials and to design completely new therapeutic enzymes.