349563 Use of Peptoids As a Method to Increase Proteolytic Stability of Therapeutic Proteins

Monday, November 4, 2013
Grand Ballroom B (Hilton)
Florencio Serrano Castillo, Chemical Engineering, University of Arkansas, Fayetteville, AR

Pharmaceutical development is one of the most active and involved industries. The process of developing a new drug, from it early stages to when it reaches patients, can take several years and significant capital investment. These obstacles are further highlighted with protein-based therapeutic drugs. Due to our bodies’ own defense mechanisms, the practical use of protein-based drugs is very limited. For example, they are almost exclusively administered through direct injection into the target area; this, however, causes low delivery efficiency due to permeability issues and proteolytic degradation both at the site and in the surrounding serum. These obstacles are further highlighted when we consider that many diseases that could be targeted by protein biotherapeutics are associated with the over expression of different proteases. These deficits in their bioavailability, causes protein-based drugs to have very short in vivo circulating half-lives, and to be susceptible to rapid renal clearance. 

The most widely used of these methods, so far, is PEGylation, the conjugation of polyethylene glycol to proteins. This process has been shown to increase the protease stability, water solubility, and half-life of circulating biomolecules. In spite of all of these advantages, PEGylation tends to increase the cost of pharmaceutical production due to the cost of its multi-step modification procedure, as well as making drug approval difficult due to the heterogeneous nature of the polymers it forms. More recently a promising alternative to PEGylation has emerged, NMEGylation. NMEGylation consists of the conjugation of methoxyethamine, which has a chemically similar backbone structure to the PEGmonomer. However, since NMEG peptoids are synthesized in a sequence-specific manner they are much more homogeneous and easily characterized than their PEG equivalents. This method has been successfully applied to an antiviral peptide, increasing its serum protease resistance and water solubility.

This project focused on optimizing the properties (e.g. length and secondary structure) of NMEG peptoids, in order to find the structure that will best modify the biophysical properties of protein-based pharmaceuticals, as well as optimizing the condition for its linkage to potential protein therapeutics.

We have synthesized linear peptoids with 3, 5, 7 and 10 NMEG to investigate the effect of NMEG length on the ability to reduce proteolytic degradation while maintaining function. We measured the peptoid’s ability to inhibit protein degradation by using a surrogate protein, beta-lactamase (TEM-1). We will also use this protein to performed the peptoid conjugation procedures, and assess protein activity before and after conjugation with the NMEG peptoids. TEM-1 is an ideal protein for this studies due to its availability, its display of first order kinetics, and because it is easily purified by metal affinity chromatography . Enzyme activity will be monitored at all stages of the modification process by the hydrolysis of penicillin-G at 240 nm on a spectrophotometer. Results for this step show that penicillinase activity is maintained at dilution ratios of 1:1600 (conjugated TEM-1: PBS buffer) for reactions with a 4-fold and a 12-fold peptoid molar excess. Lastly both the conjugated and unconjugated beta-lactamase will be exposed to a variety of proteases, such as trypsin, aminopeptidase, and elastase. This incubation will take place over time, and penicillinase activity will be measured before and after. Preliminary results show that our first NMEG peptoid/beta-lactamase conjugation still retained activity in the presence of a 4-fold and a 12-fold excess of peptoid. After confirming conjugation with MALDI we will confirm protease resistance and enzyme activity by running penicillinase assays in presence of proteases. Analysis on an SDS-PAGE should further verify proteolysis resistance.

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