In vitro activity: Src homology region 2 domain-containing phosphatase 1 (SHP-1) has been implicated as a potential cancer therapeutic target by its negative regulation of immune cell activation and the activity of the SHP-1 inhibitor sodium stibogluconate that induced IFN-gamma(+) cells for anti-tumor action. To develop more potent SHP-1-targeted anti-cancer agents, inhibitory leads were identified from a library of 34,000 drug-like compounds. Among the leads and active at low nM for recombinant SHP-1, tyrosine phosphatase inhibitor-1 (TPI-1) selectively increased SHP-1 phospho-substrates (pLck-pY394, pZap70, and pSlp76) in Jurkat T cells but had little effects on pERK1/2 or pLck-pY505 regulated by phosphatases SHP-2 or CD45, respectively. TPI-1 induced mouse splenic-IFN-gamma(+) cells in vitro, approximately 58-fold more effective than sodium stibogluconate, and increased mouse splenic-pLck-pY394 and -IFN-gamma(+) cells in vivo. TPI-1 also induced IFN-gamma(+) cells in human peripheral blood in vitro. Significantly, TPI-1 inhibited ( approximately 83%, p < 0.002) the growth of B16 melanoma tumors in mice at a tolerated oral dose in a T cell-dependent manner but had little effects on B16 cell growth in culture. TPI-1 also inhibited B16 tumor growth and prolonged tumor mice survival as a tolerated s.c. agent. TPI-1 analogs were identified with improved activities in IFN-gamma(+) cell induction and in anti-tumor actions. In particular, analog TPI-1a4 as a tolerated oral agent completely inhibited the growth of K1735 melanoma tumors and was more effective than the parental lead against MC-26 colon cancer tumors in mice. These results designate TPI-1 and the analogs as novel SHP-1 inhibitors with anti-tumor activity likely via an immune mechanism, supporting SHP-1 as a novel target for cancer treatment.

Kinase Assay: A rapid SHP-1 PTP assay was developed for screening the compounds in a commercial library of 34,000 drug-like small chemicals (Chembridge, MA) and for evaluating lead compounds and analogs. Briefly, compounds were placed in 96-well plates (Falcon, 353072) and mixed with recombinant SHP-1 protein (0.1 μg/well) in 90 μl of HEPES buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.2 mM DTT and 0.1 mg/ml BSA). The plates were incubated at room temperature for 10 minutes prior to the addition of fluorescence substrate DIFMUP (40 μM stock in HEPES buffer, 10 μl/well) to initiate PTP reaction. Upon completion of PTP reaction at room temperature for 1hr in darkness, fluorescence signal of individual wells were recorded using a Victor2 Multilabel Counter (Victor, CA). They were compared to that of control SHP-1 PTP reaction (~ 10,000 units of fluorescence signal) in the absence of any compound (100%) for calculating relative SHP-1 inhibition induced by the compounds after subtracting the background signal (~ 500 units of fluorescence signal) of the substrate. The activities of lead compounds on recombinant SHP-2 or MKP1 were evaluated similarly. Analogs of lead compound TPI-1 from the library were identified via searching pubchem databases of ~ 1 million compounds based on structural similarities and purchased from commercial sources (Chembridge, MA). Their chemical features of druggability were extracted from pubchem databases.

Cell Assay: Jurkat cells in culture medium (3 × 106 cells/ml) were treated with agents for designated times at room temperature. After brief centrifuging in a microfuge (4,000 rpm, 2 min), the cell pellet was lysed on ice for 30 min in 100 μl of cold lysis buffer (1% NP40, 50 mM Tris, pH 7.4, 150 mM NaCl, 20 mM NaF, 0.2 mM Na3VO4 and 1 mM Na3MO4) containing a cocktail of proteinase inhibitors (Sigma, 1 tablet/10 ml). The lysates were cleared by centrifuging (14,000 rpm, 10 min) in a microfuge at 4°C to remove insoluble parts, mixed with equal volume of 2 × SDS-PAGE sample buffer, boiled for 5 min and analyzed (~ 3 × 105cells/well) by SDS-PAGE/Western blotting as described previously. Relative intensities of phosphotyrosine bands were quantified through densitometry analysis.

For assessing the anti-tumor activities of lead compounds, mice (6~8-week old, female, Taconic Farms, Germantown, NY) were inoculated (s.c.) at the flanks with tumor cells. Four days post-inoculation, the mice were treated with vehicle control or the lead compounds as indicated. Tumor volumes were measured during the study period and calculated using the formula for a prolate spheroid (V= 4/3 πa2b). Student’s t test was used for assessing the significance of tumor volume differences among differential treatment groups. Mouse viability and body weights were also recorded during the study period. Internal organs of the mice were inspected visually upon their termination at the end of the experiment. All studies involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the Cleveland Clinic.