Malignant neoplasms often display metabolic defects that make them extremely dependent upon external sources of metabolites such as amino-acids. These defects can range from loss of de novo synthesis capacity to inactive salvage pathways and represent potentially drug-able targets. To target a metabolic tumor defect we use a variety of protein engineering techniques to create enzymes displaying the requisite catalytic and pharmacological properties to deplete the required factor and thus specifically starve the tumor.                  For example: L-cysteine is not normally an essential amino acid, however certain tumors overexpress the xCT(-) L-cystine/glutamate antiporter in order to maintain sufficient cysteine levels needed for protein and glutathione production. This suggests 1) that either the demand for L-cys outstrips what they can make internally or 2) they have lost or down-regulated their native cysteine biosynthetic capacity or their growth making L-cysteine a tumor essential amino acid. In support of these observations the use of small molecule xCT(-) inhibitors such as sulfasalazine have been shown to retard the growth of prostate and small cell lung cancer tumor xenografts. Although halting cystine import is promising it does not eliminate the transport of free L-Cys by the Na+-dependent ASC transporter and/or Na+-independent transporters, allowing tumoral escape. To capitalize on these promising observations, we engineered human cystathionine-?-lyase (hCGL) as a bio-therapeutic to deplete both L-cystine and L-cysteine for the treatment of L-cysteine dependent tumors.  Screening of saturation mutagenesis libraries of hCGL led to a prototype molecule with greatly enhanced L-cystine/L-cysteine degrading activity, a very long activity half-life in biological fluids which after pharmacological optimization was capable of depleting serum L-cystine/L-cysteine levels for over three days in mice.  This molecule was able to drastically effect the growth of prostate carcinoma allografts without any apparent toxicity in the mouse model.

Engineering an L-cyst(e)ine degrading therapeutic for treating dependent tumors

247th National Meeting (2014)

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