Novel SHP-1 inhibitors tyrosine phosphatase inhibitor-1 and analogs with preclinical anti-tumor activities as tolerated oral agents

Abstract

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.

Fig 1

TPI-1 inhibits recombinant and cellular SHP-1 and had little cytotoxicity in vitro and in mice. A) Jurkat cells in culture were treated with vehicle control or lead compounds #1 – 5 (10 µg/ml) for 10 min; total cell lysates (TCL) of the cells were prepared and analyzed by SDS-PAGE/Western blotting with antibodies as indicated. B) Chemical structures of lead compound TPI-1. C) Jurkat cells were cultured in the absence or presence of TPI-1 (A) for 6 days prior to quantification of cell growth by MTT assays. Data represent mean ± SD of triplicate samples. D) Viability of Balb/c mice treated with TPI-1 (~10 mg/kg, s. c., daily, 5d/wk) for two weeks. E) Relative activities of recombinant SHP-1, SHP-2 or MKP1 in the absence or presence of escalating doses of TPI-1 (mean ± SD of triplicates).

Fig 2

TPI-1 selectively increases SHP-1 phospho-substrates in Jurkat cells at low nM levels. Jurkat cells were untreated or treated with TPI-1 (A–C) or non-specific phosphatase inhibitor PV (D) at various doses for 10 min. TCL of the cells were prepared and analyzed by SDS-PAGE/Western blotting using antibodies as indicated.

Fig 3

TPI-1 induces IFNγ⁺ cells in mouse splenocytes and human peripheral blood in vitro. Relative numbers of IFNγ⁺ cells in mouse splenocytes (A and B) or human peripheral blood (C and D) cultured in the absence or presence of TPI-1 (A and C) or SSG (B and D) for 16 hrs as quantified by ELISPOT assays. Data present the mean ± SD of duplicate samples.

Fig 4

TPI-1 increases spleen pLck-pY394 and IFNγ⁺ cells in mice. Mice were treated with vehicle control or TPI-1 (1 or 3 mg/kg, s.c., daily). A/B), Splenocytes from the mice were processed into total cell lysates (TCL) and analyzed by SDS-PAGE/Western blotting to quantify pLck-pY394 levels (A) for calculating induction by densitometry analysis (B). C, The splenocytes were also used in ELISPOT assays to quantify IFNγ⁺ cells (mean ± SD of duplicate samples).

Fig 5

TPI-1 inhibits B16 tumor growth as a tolerated single agent. A) Growth of B16 cells cultured with TPI-1 for 5 days (MTT, mean ± SD of triplicates). B) Tumor volumes in C57BL/B6 mice (n=5) bearing 4-day established B16 tumors (5 10⁴ cells/inoculation) and treated with oral TPI-1 (3 mg/kg, daily, 5 d/wk). C) B16 tumor volumes in nude mice (n=5) treat as in B. D) Tumor volumes of C57BL/B6 mice (n=5) bearing 4-day established B16 tumors (10⁵ cells/inoculation) and treated with TPI-1 (1 mg/kg, daily, 5 d/wk, s.c.). E) B16 tumor volumes in C57BL/B6 mice (n=5) bearing 4-day established tumors (5 × 10⁴ cells/inoculation) and treated with SSG (12 mg, daily, 5 d/wk, s.c.). F) Body weights of the mice in B. G) Tumor volumes of C57BL/B6 mice (n = 5) bearing 4-day established tumors (10⁵ cells/inoculation) and treated with vehicle control or TPI-1 (1 mg/kg, daily, 5d/wk, s.c.). H) Tumor volumes of C57BL/B6 mice (n=5) bearing 4-day established B16 tumors (10⁵ cells/inoculation) and treated with vehicle control IL-2 (3 × 10⁵ IU, bid, 5d/wk, ip). I) Viability of C57BL/B6 mice (n = 5) bearing 4-day established tumors (10⁵ cells/inoculation) and treated with vehicle control or TPI-1 (1 mg/kg, daily, 5 d/wk, s.c.).

Fig 6

TPI-1 analogs inhibit rSHP-1 in correlation with activity to increase SHP-1 phospho-substrate pLck-pY394 in Jurkat cells and induce IFNγ+ cells in mouse splenocytes. A) Chemical structures of TPI-1 analogs. B) pLck-pY394 levels in Jurkat cells treated with vehicle control, TPI-1 or selective analogs ( 10 µg/ml, 10 min) were quantified by Western blotting with antibodies as indicated. C) Activities of recombinant SHP-1 (rSHP-1) in the absence or presence of TPI-1 and selective analogs (10 µg/ml) as quantified by in vitro phosphatase assays (mean ± SD of triplicates). D) Numbers of IFNγ+ cells in mouse splenocytes cultured in the presence of vehicle control, TPI-1 or analogs for 16 hrs as quantified by ELISPOT assays (mean ± SD of duplicates).

Fig 7

TPI-1 analogs TPI-1a2 and TPI-1a4 have anti-tumors in mice at tolerated oral doses. A/B) C57BL/6 mice bearing 4-day-established B16 tumors (s. c.) were treated with vehicle control TPI-1a2 (A) or TPI-1a4 (B) (3 mg/kg/daily, oral, 5d/week). Tumor volumes on day 22 were recorded (n = 5). C) Tumor volumes (n=5) in mice bearing 4-day established B16 tumors and treated with TPI-1a10 (3 mg/kg/day, oral, 5d/wk). D/E) Tumor volumes (n = 5) in Balb/c mice bearing 4-day established MC-26 tumors were treated with vehicle control, TPI-1 (3 mg/kg/daily, oral, 5 d/week) (D) or TPI-1a4 (3 mg/kg/daily, oral, 5d/week) (E). Data in D and E were from a single experiment and are presented in two panels for comparison. F) Body weights of the mice in D and E at the end of the experiment. G/H) C3H/HeJ mice (female, 8 wks, 5/group) bearing 4-day established K1735 tumors (s.c.) were treated with vehicle control or a4 (1 mg/kg, daily, oral, 5d/wk). Tumor volumes (G, n = 5) and mouse body weights (H, n = 5) were recorded. I) Growth of K1735 cells in the presence of vehicle control or a4 in culture for 3 days as quantified by MTT assays (mean ± SD of triplicates).