Structure-Based Design and Discovery of a Long-Acting Cocaine Hydrolase Mutant with Improved Binding Affinity to Neonatal Fc Receptor for Treatment of Cocaine Abuse.

Despite decades of efforts to develop a pharmacotherapy for cocaine abuse treatment, there is still no FDA-approved treatment of diseases associated with this commonly abused drug. Our previously designed highly efficient cocaine hydrolases (CocHs) and the corresponding Fc-fusion proteins (e.g., CocH3-Fc) are recognized as potentially promising therapeutic enzyme candidates for cocaine abuse treatment, but all with limited biological half-lives. In order to prolong the biological half-life and, thus, decrease the required frequency of the enzyme administration for cocaine abuse treatment, we have modeled the Fc-fusion CocH binding with neonatal Fc receptor (FcRn) in the present study. This approach led to the design and testing of CocH3-Fc(M6), a CocH3-Fc mutant with nearly 100-fold increased binding affinity: from Kd = ~ 4 µM to Kd = 43 nM. As a result, CocH3-Fc(M6) indeed revealed a markedly prolonged biological half-life (t1/2 = 206 ± 7 h or ~ 9 days) in rats, longer than other known Fc-fusion protein drugs such as abatacept and alefacept (for other therapeutic purposes) in the same species (rats). It has been demonstrated that a single dose of 3 mg/kg CocH3-Fc(M6) effectively blocked 20 mg/kg cocaine-induced hyperactivity on day 18 after CocH3-Fc(M6) administration. This is the first attempt to rationally design long-acting Fc-fusion enzyme mutant based on combined computational modeling and experimental measurement of the Fc-fusion CocH binding with FcRn. The similar structure-based design strategy may be used to prolong the biological half-lives of other Fc-fusion protein drugs.

Clinical Potential of an Enzyme-based Novel Therapy for Cocaine Overdose.

It is a grand challenge to develop a truly effective medication for treatment of cocaine overdose. The current available, practical emergence treatment for cocaine overdose includes administration of a benzodiazepine anticonvulsant agent (e.g. diazepam) and/or physical cooling with an aim to relieve the symptoms. The inherent difficulties of antagonizing physiological effects of drugs in the central nervous system have led to exploring protein-based pharmacokinetic approaches using biologics like vaccines, monoclonal antibodies, and enzymes. However, none of the pharmacokinetic agents has demonstrated convincing preclinical evidence of clinical potential for drug overdose treatment without a question mark on the timing used in the animal models. Here we report the use of animal models, including locomotor activity, protection, and rescue experiments in rats, of drug toxicity treatment with clinically relevant timing for the first time. It has been demonstrated that an efficient cocaine-metabolizing enzyme developed in our previous studies can rapidly reverse the cocaine toxicity whenever the enzyme is given to a living rat, demonstrating promising clinical potential of an enzyme-based novel therapy for cocaine overdose as a successful example in comparison with the commonly used diazepam.