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A. Desai et al.
Results
(+)-SW209415 inhibits 15-PGDH and induces prostaglandin E2 in vivo
We first examined the potency of (+)-SW209415 in inhibiting enzymatic activity of recombinant 15-PGDH protein in vitro in a titration experiment in which (+)- SW209415 was added to ~5 nM 15-PGDH protein. The half minimal inhibitory concentration (IC50) of (+)- SW209415 was 1.1 nM, consistent with this compound acting as a tight-binding inhibitor of 15-PGDH (and sug- gesting approximately 50% activity of the recombinant protein) (Figure 1A). A Morrison design experiment esti- mated that the Kiapp for (+)-SW209415 is 0.06 nM (Figure 1B). (+)-SW209415 thus retains the tight-binding inhibitor characteristics of the parent SW033291 compound. To test the potency in inhibiting 15-PGDH in cells, (+)-SW209415 was added to cultures of A549 cells stimulated with inter- leukin-1β, and increases in levels of PGE2 secreted into the culture media were determined. In this cell-based assay, (+)-SW209415 showed a half maximal effective concen- tration (EC50) of approximately 10 nM, demonstrating improved potency compared to SW033291 which had an EC50 of 40 nM (Figure 1C, arrows denote approximate EC50 values). Furthermore, (+)-SW209415 retained high poten- cy in inducing PGE2 in vivo in the bone marrow of treated mice: mice treated in vivo with a single intraperitoneal dose of 2.5 mg/kg showing a 2-fold induction of bone marrow PGE2 at 2 and 3 h following treatment, with lev- els falling at 6 h and returning to baseline at 12 h after
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treatment (Figure 1D).21 This PGE2 response matches the persistent 2-fold elevation of PGE2 observed in the 15- PGDH knockout mouse. Last, in vivo administration of (+)- SW209415 recapitulated the induction of two cytokines, stem cell factor and CXCL12, in the CD45– bone marrow stromal cell population, which was previously demon- strated by SW03329120 (Online Supplementary Figure S2).
(+)-SW209415 promotes hematopoietic homing, engraftment and recovery after hematopoietic stem cell transplantation
We next investigated the effects of (+)-SW209415 on steps in hematopoietic recovery following murine HSCT. We studied four distinct biological steps in recovery from HSCT: (i) homing of transplanted HSC to recipient mar- row; (ii) expansion of transplanted HSC as hematopoietic colonies in the spleen; (iii) expansion of the stem cell- enriched SKL population in the bone marrow; and (iv) pro- duction of mature neutrophils in the peripheral blood. We first investigated the effects of (+)-SW209415 on enhanc- ing efficiency of HSC homing to bone marrow following transplantation. CFSE-labeled cells that successfully homed to the recipient’s bone marrow were quantified 16 h following the transplant. Three doses of (+)-SW209415 induced a 2-fold increase in donor marrow cell homing to the marrow cavity of recipient mice tibiae (P=0.0002), with an activity essentially identical to that of three doses of (+)-SW033291 (Figure 2A). Homing of SKL cells to the bone marrow niche was increased by 1.8-fold in (+)- SW209415-treated mice (P=0.0029) (Online Supplementary
Figure 1. (+)-SW209415 inhibits 15-PGDH and induces prostaglandin E2 (PGE2) in vivo. (A) Increasing concentra- tions of (+)-SW209415 were added to 5 nM of 15-PGDH protein and enzyme activity was measured in duplicate determinations, with percent inhibition at each concentra- tion depicted in the graph. (B) Graph of the relative initial 15- PGDH enzyme reaction veloci- ties Vi/V0 versus concentra- tion of (+)-SW209415. Vi val- ues are the averages of tripli- cate determinations. (C) PGE2 levels in medium of A549 cells following stimulation with inter- leukin-1β and treatment with increasing concentrations of (+)-SW209415. The arrows indicate the approximate EC50 values. The graph shows the means of four independent determinations ± Standard Error of Mean. (D) Eight-week old C57/Bl6J female mice were administered a single intraperitoneal dose of 2.5 mg/kg (+)-SW209415. Animals were sacrificed at the time points indicated after the injection and bone marrow flushed for analysis of PGE2 via enzyme-linked immunosor- bent assay (3 mice per time- point). hr: hour.
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haematologica | 2018; 103(6)