Summary /

Extensive SAR explorations yielded a probe molecule ML246 that is able to reduce the PNC population in PC3M cells at sub-micromolar concentrations with cytotoxicity observed at ~10μM (25-fold selectivity). The probe shows ~10-fold selectivity between growth inhibition and cytotoxicity in the NCI-60 panel. Thus the chemical series represents first-in-class molecules that reduce the prevalence of the PNC and do not operate via known cytotoxic mechanisms.

Figure 1. NCI-60 panel: Dose response growth inhibition curves for ML246 in 60 cancer cell lines. The curves are grouped according to the type of cancer. Concentrations able to produce a percentage of growth below zero indicate loss of cell density and can be correlated to cell death.

Summary /

After activity optimization in the PNC assay, it was necessary to examine if these compounds had an effect on migration. Table 1 summarizes preliminary results obtained in an invasion assay with ML246.

Table 1. BellBrook Labs Proliferation and Migration Data for ML246 and an inactive control.

Summary /

An examination of the number of cells that have invaded through the 3D collagen channel reflects their migratory aptitude. Gratifyingly, the novel PNC-reducer probe ML246 showed dose dependent inhibition of migration with IC s 3–4 μM. The numbers of cells in the output port after the assay duration were also counted as a measurement of proliferation. Here too, the probe showed dose-dependent inhibition activity, suggesting that the anti-invasive effect of the compound is concomitant with an anti-proliferative effect.

Figure 2.  (A) Histogram representing number and size (μm) of PC3M soft agar colonies after 14 days treatment with ML246 at 3.8 and 18.6 nM. (B) Representative images of colonies at these concentrations.

Summary /

ML246 is a novel chemical entity and has no previously disclosed interactions with biological targets. The probe has been screened against 44 GPCR and CNS relevant targets in the PDSP comprehensive binding panel. With the exception of the sigma 2 receptor, ML246 was mostly inactive against all these targets. Notably, this includes dopaminergic activity, as the original hit was found to be an allosteric D1 agonist in PubChem AID 504660.

Table 2. Profiling of the probe, ML246, in the PDSP comprehensive panel.

Summary /

ML246 has good stability in PBS buffer. It also has good stability in mouse plasma and is not rapidly metabolized by mouse liver microsomes in the presence of NADPH. At a 50 mpk high dose, the probe provided significant in vivo exposure in mice, with a C max of 20.3 μM @ 30 min and a C last of 1.3 μM @ 48 hrs. Some mortality was observed (3/18), but this is expected, as the exposure is well beyond the expected therapeutic levels. Subsequent experiments with 25 mpk intraperitoneal (IP) dosing provided very good exposure without any sign of toxicity.

Table 3. In vitro ADME data for ML246.

Table 4. Comparison of Mouse Pharmacokinetics Parameters of Probe after IP Dosing of 50 or 25 mpk in Male C57BL/6 Mice.