HUMAN BUTYRYLCHOLINESTERASE MUTANTS FOR COCAINE DETOXIFICATION

Cocaine is one of the most reinforcing drugs of abuse and has caused serious medical and social problems. There is no FDA-approved medication specific for cocaine. It is of a high priority to develop an effective therapeutic treatment for cocaine abuse. Human butyrylcholinesterase (BChE) has been recognized as a promising candidate of enzyme therapy to metabolize cocaine into biologically inactive metabolites and prevent it from reaching central nervous system (CNS). However, the catalytic activity of wide-type human BChE against cocaine is not sufficiently high for treatment of cocaine abuse. Dr. Zhan’s lab has successfully designed and discovered a series of high-activity mutants of human BChE specific for cocaine metabolism. This dissertation is mainly focused to address the possible concerns in further development of promising human BChE mutants for cocaine detoxification, including whether the administration of this exogenous enzyme will affect the cholinergic system, whether it can efficiently hydrolyze cocaine’s toxic metabolites, and whether the commonly used therapeutic agents will significantly affect the catalytic activity of the BChE mutants against cocaine when they are co-administered. According to the results obtained, all of the examined BChE mutants have a considerably improved catalytic efficiency against (-)-cocaine, without significantly improving the catalytic efficiency against any of the other examined substrates, including neurotransmitter acetylcholine. Two representative mutants (including E12-7) also have a considerably improved catalytic activity against cocaethylene (formed from combined use of cocaine and alcohol) compared to wild-type BChE, and E12-7 can rapidly metabolize cocaethylene, in addition to cocaine, in rats. Further evaluation of possible drug-drug interactions between E12-7 and some other commonly used therapeutic agents revealed that all of the examined agents, except some tricyclic antidepressants, do not significantly inhibit E12-7. In addition, an effort to discover new mutants with further improved activity against cocaine led to the discovery of a new BChE mutant, denoted as E20-7, according to both the in vitro and in vivo assays. The encouraging outcomes of the present investigation suggest that it is possible to develop a more effective enzyme therapy for cocaine abuse treatment using one of the most promising BChE mutants, such as E12-7 or E20-7

Structural Mechanism of Substrate Specificity In Human Cytidine Deaminase Family APOBEC3s

APOBEC3s (A3s) are a family of human cytidine deaminases that play important roles in both innate immunity and cancer. A3s protect host cells against retroviruses and retrotransposons by deaminating cytosine to uracil on foreign pathogenic genomes. However, when mis-regulated, A3s can cause heterogeneities in host genome and thus promote cancer and the development of therapeutic resistance. The family consists of seven members with either one (A3A, A3C and A3H) or two zinc-binding domains (A3B, A3D, A3D and A3G). Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA-bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination. Using a combination of computational molecular modeling and parallel molecular dynamics (pMD) simulations followed by experimental verifications, I investigated the roles of active site residues and surrounding loops in determining the substrate specificity and RNA versus DNA binding among A3s. Starting with A3B, I revealed the structural basis and gatekeeper residue for DNA binding. I also identified a unique auto-inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity compared to the highly similar A3A. Besides, I investigated the structural mechanism of substrate specificity and ssDNA binding conformation in A3s. I found an interdependence between substrate conformation and specificity. Specifically, the linear DNA conformation helps accommodate CC dinucleotide motif while the U-shaped conformation prefers TC. I also identified the molecular mechanisms of substrate sequence specificity at -1’ and -2’ positions. Characterization of substrate binding to A3A revealed that intra-DNA interactions may be responsible for the specificity in A3A. Finally, I investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods. Overall, the comprehensive analysis of A3s in this thesis shed light into the structural mechanism of substrate specificity and broaden the understanding of molecular interactions underlying the biological function of these enzymes. These results have implications for designing specific A3 inhibitors as well as base editing systems for gene therapy

High Activity Mutants of Butyrylcholinesterase for Cocaine Hydrolysis

Butyrylcholinesterase (BChE) polypeptide variants of the presently-disclosed subject matter have enhanced catalytic efficiency for (−)-cocaine, as compared to wild-type BChE. Pharmaceutical compositions of the presently-disclosed subject matter include a BChE polypeptide variant having an enhanced catalytic efficiency for (−)-cocaine. A method of the presently-disclosed subject matter for treating a cocaine-induced condition includes administering to an individual an effective amount of a BChE polypeptide variant, as disclosed herein, to lower blood cocaine concentration

High Activity Mutants of Butyrylcholinesterase for Cocaine Hydrolysis

Butyrylcholinesterase (BChE) polypeptide variants of the presently-disclosed subject matter have enhanced catalytic efficiency for (−)-cocaine, as compared to wild-type BChE. Pharmaceutical compositions of the presently-disclosed subject matter include a BChE polypeptide variant having an enhanced catalytic efficiency for (−)-cocaine. A method of the presently-disclosed subject matter for treating a cocaine-induced condition includes administering to an individual an effective amount of a BChE polypeptide variant, as disclosed herein, to lower blood cocaine concentration

High Activity Mutants of Butyrylcholinesterase for Cocaine Hydrolysis

Butyrylcholinesterase (BChE) polypeptide variants of the presently-disclosed subject matter have enhanced catalytic efficiency for (−)-cocaine, as compared to wild-type BChE. Pharmaceutical compositions of the presently-disclosed subject matter include a BChE polypeptide variant having an enhanced catalytic efficiency for (−)-cocaine. A method of the presently-disclosed subject matter for treating a cocaine-induced condition includes administering to an individual an effective amount of a BChE polypeptide variant, as disclosed herein, to lower blood cocaine concentration

Cocaine Hydrolase-Fc Fusion Proteins for Cocaine and Methods for Utilizing the Same

The presently-disclosed subject matter includes isolated polypeptides that comprise a butyrylcholinestrase (BChE) polypeptide and a second polypeptide. The BChE polypeptide as well as the second polypeptide can be variants and/or fragments thereof. The presently-disclosed subject matter also includes a pharmaceutical composition that comprises the present isolated polypeptide and a suitable pharmaceutical carrier. Further still, methods are provided for treating cocaine-induced conditions, and comprise administering the isolated polypeptide and/or pharmaceutical compositions thereof to an individual

Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s

The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC0 motif, A3A can accommodate the CCC0 motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy

Plant Expression of Cocaine Hydrolase-Fc Fusion Protein for Treatment of Cocaine Abuse

BACKGROUND: A recently reported cocaine hydrolase (CocH3) fused with fragment crystallizable (Fc) region of human immunoglobulin G1, denoted as CocH3-Fc, is known as a promising therapeutic candidate for the treatment of cocaine overdose and addiction. A challenge for practical therapeutic use of this enzyme exists in the large-scale protein production and, therefore, it is interesting to identify a low-cost and feasible, sustainable source of CocH3-Fc production. RESULTS: CocH3-Fc was transiently expressed in plant Nicotiana benthamiana leaves. The plant-expressed protein, denoted as pCocH3-Fc, was as active as that expressed in mammalian cells both in vitro and in vivo. However, compared to the mammalian-cell expressed CocH3-Fc protein, pCocH3-Fc had a shorter biological half-life, probably due to the lack of protein sialylation in plant. Nevertheless, the in vivo half-life was significantly extended upon the PEGylation of pCocH3-Fc. The Fc fusion did not prolong the biological half-life of the plant-expressed enzyme pCocH3-Fc, but increased the yield of the enzyme expression in the plant under the same experimental conditions. CONCLUSIONS: It is feasible to express pCocH3-Fc in plants. Further studies on the pCocH3-Fc production in plants should focus on the development of vectors with additional genes/promoters for the complete protein sialylation and for a better yield

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies [preprint]

Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses