There are at least three additional cancer-associated extracellular proteases as potential candidates for the activation of 12-ADT-based prodrugs. These are Human Glandular Kallikrein 2 (HK2 also known as KLK2), Fibroblast Activation Protein (FAP), and Prostate Specific Membrane Antigen (PSMA also known as FOLH1). HK2 is a trypsin-like protease, uniquely secreted into the extracellular fluid at high enzymatically active levels only by normal and malignant prostate epithelial cells. Like PSA, once in the blood, its enzymatic activity is inhibited by serum protease inhibitors, making it an alternative candidate for prostate-targeted prodrug activation. Another alternative candidate is the serine protease, FAP. This is based upon the studies of W. Nathaniel Brennen, initiated while a graduate student with Sam Denmeade, and then, as a post-doctoral fellow with John Isaacs. Subsequently, Dr. Brennen continued this collaboration when he became an independent faculty investigator at Hopkins. His studies focused on the tumor-promoting activity of the influx within sites of metastatic prostate cancer of tumor-infiltrating host-derived fibroblasts that have a highly increased plasma membrane expression of FAP. FAP is a type II integral membrane serine prolyl protease of the dipeptidyl peptidase IV family, which is characterized by a unique post-prolyl cleavage specificity. Based on its restricted expression and unique substrate requirements, FAP is an ideal potential candidate for prodrug activation. PSMA is highly expressed on the extracellular plasma membranes of prostate cancer cells and, as originally discovered by Warren D. W. Heston, has folate hydrolase enzymatic activity. PSMA expression is also upregulated after ADT in resistant metastatic prostate cancer.

There are at least three additional cancer-associated extracellular proteases as potential candidates for the activation of 12-ADT-based prodrugs. These are Human Glandular Kallikrein 2 (HK2 also known as KLK2), Fibroblast Activation Protein (FAP), and Prostate Specific Membrane Antigen (PSMA also known as FOLH1). HK2 is a trypsin-like protease, uniquely secreted into the extracellular fluid at high enzymatically active levels only by normal and malignant prostate epithelial cells. Like PSA, once in the blood, its enzymatic activity is inhibited by serum protease inhibitors, making it an alternative candidate for prostate-targeted prodrug activation. Another alternative candidate is the serine protease, FAP. This is based upon the studies of W. Nathaniel Brennen, initiated while a graduate student with Sam Denmeade, and then, as a post-doctoral fellow with John Isaacs. Subsequently, Dr. Brennen continued this collaboration when he became an independent faculty investigator at Hopkins. His studies focused on the tumor-promoting activity of the influx within sites of metastatic prostate cancer of tumor-infiltrating host-derived fibroblasts that have a highly increased plasma membrane expression of FAP. FAP is a type II integral membrane serine prolyl protease of the dipeptidyl peptidase IV family, which is characterized by a unique post-prolyl cleavage specificity. Based on its restricted expression and unique substrate requirements, FAP is an ideal potential candidate for prodrug activation. PSMA is highly expressed on the extracellular plasma membranes of prostate cancer cells and, as originally discovered by Warren D. W. Heston, has folate hydrolase enzymatic activity. PSMA expression is also upregulated after ADT in resistant metastatic prostate cancer.