Pomper. exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We exhibited these brokers selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively HIV-1 integrase inhibitor toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells. for 10?min and loaded into an AKTAprime Plus FPLC System (GE 11001313) using a washed HiLoad Superdex 200 PG (GE 28989335) column. One mL fractions were collected and monitored using A280 spec. HIV-1 integrase inhibitor Fractions made up of protein were then run on SDS PAGE. All fractions made up of primarily the band specific for PE35 were pooled and concentrated to less than 1?mL. This method was also used to purify Fluorescein-labeled PE35-MU2. To assess protein-MU2 conjugation, we used either the thiol-reactive chromogenic reagent DNTB (Ellmans reagent), the fluorescent substrate ABDF, or a non-reducing SDS PAGE gel based assay. For the DNTB assay, thiol concentrations must be above 50?M. BSA solutions plus or minus Protein-MU2 were mixed 1:1 with a 2?mM solution of DNTB in DMSO. The reaction was then incubated at room heat for 10? min shaking and read at 412?nm. Concentration of thiol was decided using the molar extinction coefficient of DNTB. For the gel-based assay, GZMB solutions plus or minus conjugate were purified and dialyzed as described above which allowed any free thiol to form disulfides. Reactions were run on a non-reducing SDS PAGE gel using a BioRad Mini-Protean gelcast system (BioRad 1658005) with Mini-Protean 4C15% pre-cast gels. Gels were run at 150?V for 45?min, washed once with water, stained with SimplyBlue [Thermo LC6060] protein stain, and de-stained in water. The ABDF assay was performed using a Sensolyte ABDF Assay HIV-1 integrase inhibitor Kit (Anaspec AS-72137)] according to manufacturers recommendations. The plate was read at 389/513 ex/em. Free thiol concentrations of PE35 plus or minus MU2 were determined using a GSH standard curve. Fluorescein conjugation of protein-MU2 constructs Fluorescein-labeled protein-MU2 constructs were generated by incubating proteins with NHS-fluorescein (Thermo 46410) for 1 to 2 2?h at room temperature in the dark. Free NHS-Fluorescein was removed using either Pierce? Dye Removal Columns (Thermo 22858) according to manufacturers recommendations or via FPLC. Conjugation efficiency was decided using the ratio between A280 and A495 using the respective molar extinction coefficient for each protein, and the fluorescein conjugate. PSMA enzymatic assay Lysate from LNCaP cells (robustly PSMA positive) was generated by pelleting cells from culture, lysing them in a solution of 50?mM Tris HCl pH?=?7.5, 140?mM NaCl, and 1% Triton X-100 to a concentration of 1 1??107 per mL. Cells were then incubated on ice for 15? min and then spun at 18,000??for 15?min. Supernatant was then harvested and stored at???80?C or used immediately as a source of PSMA. The lysate was then diluted 1:10 in PBS and incubated at 37?C for 4?h with the PSMA specific substrate N-acetyl-aspartyl-glutamate [NAAG (4?M)] and either buffer, protein-MU2 conjugate, an unconjugated protein, or a control urea-based PSMA inhibitor, ZJ43, to validate the assay. To determine the amount of NAAG converted to N-acetyl-aspartate and free glutamate by PSMA, a fluorescent enzyme-coupled Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (Thermo A-12221) was used according to the protocol provided by the manufacturer. This reaction was done in Costar? 96-Well Half-Area Plates (Fischer Corning 3694) in 100 uL total volume and was incubated for 1?h at 37?C in the dark. The plate was then read at 530/590?nm ex/em. Activity was measured in natural RFUs and inhibition curves were based off a variable 4 parameter nonlinear regression. Unconjugated and conjugated protein-MU2 PSMA inhibition was measured in terms of % untreated control activity. GZMB functional assay GZMB-specific enzymatic assay ([Biovision K168-100) was assayed according to manufacturers suggestions. The plate was read at 380/500 ex/em following incubation. This reaction with GZMB or GZMB-MU2 was compared to a standard curve of free AMC to calculate the amount of substrate released. In vitro characterization of protein-urea conjugates Human prostate cancer cell lines were obtained from ATCC and produced in RPMI made up of 10% FBS with supplemental l-glutamine at 5?mM and were passaged weekly. PSMA[+] PIP-PC3 and PSMA[?] Flu-PC3 cells were provided by Dr. Pomper. Cells were treated with either vehicle [PBS pH?=?7.4] or the protein drug conjugate of interest at doses up to 250?nM. For GZMB-MU2, F2r doses did not exceed 100?nM due to GZMBs ability to affect cell growth via.