Zachary F. Walls

Specificity of a Prodrug-Activating Enzyme hVACVase: the Leaving Group Effect

Human valacyclovirase (hVACVase) is a prodrug-activating enzyme for amino acid prodrugs including the antiviral drugs valacyclovir and valganciclovir. In hVACVase-catalyzed reactions, the leaving group of the substrate corresponds to the drug moiety of the prodrug, making the leaving group effect essential for the rational design of new prodrugs targeting hVACVase activation. In this study, a series of valine esters, phenylalanine esters, and a valine amide were characterized for the effect of the leaving group on the efficiency of hVACVase-mediated prodrug activation. Except for phenylalanine methyl and ethyl esters, all of the ester substrates exhibited a relatively high specificity constant (k(cat)/K(m)), ranging from 850 to 9490 mM(-1)·s(-1). The valine amide Val-3-APG exhibited significantly higher K(m) and lower k(cat) values compared to the corresponding ester Val-3-HPG, indicating poor specificity for hVACVase. In conclusion, the substrate leaving group has been shown to affect both binding and specific activity of hVACVase-catalyzed activation. It is proposed that hVACVase is an ideal target for α-amino acid ester prodrugs with relatively labile leaving groups while it is relatively inactivate toward amide prodrugs.

A Generalizable Strategy for Imaging pre-mRNA Levels in Living Subjects Using Spliceosome-Mediated RNA Trans-Splicing

Molecular imaging of gene expression is currently hindered by the lack of a generalizable platform for probe design. For any gene of interest, a probe that targets protein levels must often be generated empirically. Targeting gene expression at the level of mRNA, however, would allow probes to be built on the basis of sequence information alone. Presented here is a class of generalizable probes that can image pre-mRNA in a sequence-specific manner, using signal amplification and a facile method of delivery. Methods: Pre–trans-splicing molecules (PTMs) were engineered to capitalize on the phenomenon of spliceosome-mediated RNA trans-splicing. Using a modular binding domain that confers specificity by base-pair complementarity to the target pre-mRNA, PTMs were designed to target a chimeric target mini gene and trans-splice the Renilla luciferase gene onto the end of the target. PTMs and target genes were transfected in cell culture and assessed by luciferase assay, reverse-transcriptase polymerase chain reaction, Western blot, and rapid analysis of 5′ cDNA ends. PTMs and target genes were also assessed in vivo by hydrodynamic delivery in mice. Results: Efficiency and specificity of the trans-splicing reaction were found to vary depending on the binding domain length and structure. Specific trans-splicing was observed in living animals (P = 0.0862, Kruskal–Wallis test). Conclusion: Described here is a model system used to demonstrate the feasibility of spliceosome-mediated RNA trans-splicing for imaging gene expression at the level of pre-mRNA using optical imaging techniques in living animals. The experiments reported here show proof of principle for a generalizable imaging probe against RNA that can amplify signal on detection and be delivered using existing gene delivery methodology.

Mutants of listeriolysin O for enhanced liposomal delivery of macromolecules.

Delivery of macromolecules into the cytosolic space of eukaryotic cells is a pressing challenge in biopharmaceutics. Macromolecules are often encapsulated into liposomes for protection and improved distribution, but the their size often induces endocytosis of the vehicle at the target site, leading to degradation of the cargo. Listeriolysin O is a key virulence factor of Listeria monocytogenes that forms pores in the endosomal membrane, ultimately allowing the bacterium to escape into the cytosol. This function of LLO has been used to improve cytosolic delivery of liposomally encapsulated macromolecules in a number of instances, but its innate toxicity and immunogenicity have prevented it from achieving widespread acceptance. Through site-directed mutagenesis, this study establishes a mutant of LLO (C484S) with enhanced activity, allowing for a reduction in the amount of LLO used for future applications in liposomal drug delivery.

Liposomal Coencapsulation of Doxorubicin with Listeriolysin O Increases Potency via Subcellular Targeting

Liposomal doxorubicin is a clinically important drug formulation indicated for the treatment of several different forms of cancer. For doxorubicin to exert a therapeutic effect, it must gain access to the nucleus. However, a large proportion of the liposomal doxorubicin dose fails to work because it is sequestered within endolysosomal organelles following endocytosis of the liposomes due to the phenomenon of ion trapping. Listeriolysin O (LLO) is a pore-forming protein that can provide a mechanism for endosomal escape. The present study demonstrates that liposomal coencapsulation of doxorubicin with LLO enables a significantly larger percentage of the dose to colocalize with the nucleus compared to liposomes containing doxorubicin alone. The change in intracellular distribution resulted in a significantly more potent formulation of liposomal doxorubicin as demonstrated in both the ovarian carcinoma cell line A2780 and its doxorubicin-resistant derivative A2780ADR.

Synthesis and characterization of valyloxy methoxy luciferin for the detection of valacyclovirase and peptide transporter.

An amino acid ester derivative of luciferin (valoluc) was synthesized to mimic the transport and activation of valacyclovir. This molecule was characterized in vitro for specificity and enzymatic constants, and then assayed in two different, physiologically-relevant conditions. It was demonstrated that valoluc activation is sensitive to the same cellular factors as valacyclovir and thus has the potential to elucidate the dynamics of amino acid ester prodrug therapies in a functional, high-throughput manner.

Development and evaluation of the Medication-based Index of physical Function (MedIP)

Background the development of an objective and comprehensive drug-based index of physical function for older adults has the potential to more accurately predict fall risk. Design the index was developed using 862 adults (ages 57-85) from the National Social Life, Health, and Aging Project (NSHAP) Wave 1 study. The index was evaluated in 70 adults (ages 51-88) from a rehabilitation study of dizziness and balance. Methods the prevalence among 601 drugs for 1,694 side effects was used with fall history to determine the magnitude of each side effects contribution towards physical function. This information was used to calculate a Medication-based Index of Physical function (MedIP) score for each individual based on his or her medication profile. The MedIP was compared to the timed up and go (TUG) test as well as drug counts using receiver operating characteristic (ROC) analysis. The associations between various indices of physical function and MedIP were calculated. Results within the NSHAP data set, the MedIP was better than drug counts or TUG at predicting falls based on ROC analysis. Using scores above and below the cutpoint, the MedIP was a significant predictor of falls (OR = 2.61 [95% CI 1.83, 3.64]; P < 0.001). Using an external data set, it was shown that the MedIP was significantly correlated with fall number (P = 0.044), composite physical function (P = 0.026) and preferred gait speed (P = 0.043). Conclusion the MedIP has the potential to become a useful tool in the healthcare and fall prevention of older individuals.

Using scientific inquiry to increase knowledge of vaccine theory and infectious diseases.

Background: The aim of this study was to design and evaluate a laboratory activity based on scientific inquiry to educate first-year pharmacy students in the U.S. about vaccination theory and the attributes of common pathogens. Methods: The laboratory activity had two principal sections. The first consisted of an interactive game during which students rolled a die to determine outcomes based on a set of pre-determined criteria. In the second section, students generated and tested hypotheses about vaccine theory using a computer simulation that modeled disease transmission within a large population. In each section students were asked to evaluate epidemiological data and make inferences pertinent to vaccination effectiveness. Results: Mean scores on a knowledge-based assessment given immediately before and immediately after the activity increased from 46% to 71%. Discussion: A laboratory activity designed to stimulate scientific inquiry within pharmacy students enabled them to increase their knowledge of common vaccines and infectious diseases.

Engineering Luciferases for Assays and Imaging

Luciferases have served a number of purposes in biomedical applications, including within reporter gene and split reporter complementation assays. These proteins, however, have not evolved for the purpose of biomedical research, and it is not surprising that the utility and robustness of these assays can be improved by protein engineering of the luciferase. In this chapter, we provide an overview of luciferases, protein engineering, and how protein engineering is applied to luciferases.