Design and Synthesis of Active Site Inhibitors of the Human Farnesyl Pyrophosphate Synthase: Apoptosis and Inhibition of ERK Phosphorylation in Multiple Myeloma Cells

Abstract

Human farnesyl pyrophosphate synthase (hFPPS) controls intracellular levels of FPP and post-translational prenylation of small GTPase proteins, which are essential for cell signaling and cell proliferation. Clinical investigations provide evidence that N-BP inhibitors of hFPPS are disease modifying agents that improve survival of multiple myeloma (MM) patients via mechanisms unrelated to their skeletal effects. A new series of N-BPs was designed that interact with a larger portion of the GPP subpocket, as compared to the current therapeutic drugs, and rigidify the (364)KRRK(367) tail of hFPPS in the closed conformation in the absence of IPP. An analogue of this series was used to demonstrate inhibition of the intended biological target, resulting in apoptosis and down-regulation of ERK phosphorylation in human MM cell lines.

Human isoprenoid synthase enzymes as therapeutic targets

“…Gln171 also facilitates binding of the allylic tail via π-stacking interaction involving its planar side chain, but this residue further contributes to substrate binding by providing a polar contact to the pyrophosphate-Mg 2+ -water cluster. In our work, we have shown that these residues participate in similar interactions with bisphosphonate inhibitors that have extended and rigid lipophilic side chains, such as the aminopyridine inhibitors 8a and 8b (Figure 4B) (De Schutter et al, 2012; Lin et al, 2012), and the thienopyrimidine-based inhibitor 10 (Leung et al, 2013b). …”

“…Replacement of the Cα-hydroxyl with a hydrogen (e.g., 2a vs. the corresponding deoxybisphosphonate 2b ) or Cα-halogen substituent (e.g., 2c , d ) has been found to decrease affinity for bone, as well as the potency in inhibiting hFPPS (Marma et al, 2007). Bisphosphonate inhibitors with various heterocyclic lipophilic side chains, including derivatives of zoledronic acid (e.g., 1b , c ) pyridinium deoxybisphosphonates ( 7a–c ) (Zhang et al, 2009), aminopyridines ( 8a–d ) (De Schutter et al, 2012; Lin et al, 2012), azaindoles (Ebetino et al, 2010a,b) and imidazopyridines ( 9a – c , respectively) (Ebetino et al, 2010c), and thienopyrimidines (e.g., 10 ) (Leung et al, 2013a,b) have also been reported.…”

“…Preliminary evidence of improved cell-membrane permeability by these more lipophilic analogs has been deduced mainly from cell-based assays measuring antiproliferation in various cancer cell lines. For example, analog 7c was found to be ~100-fold more potent than 1a in inhibiting the proliferation of breast cancer MCF-7 cells (Zhang et al, 2009), whereas analog 8b was shown to be slightly more potent than 1a in inhibiting the MM cell lines RPMI-8226 and KMS 28PE, in spite of a difference of nearly 10-fold in intrinsic potency (Lin et al, 2012). Inhibition of ERK phosphorylation, a signaling protein down-stream from Ras, was also observed with analog 8b in KMS 28PE cells, providing evidence that this compound inhibits the intended biochemical pathway (Lin et al, 2012).…”

“…For example, analog 7c was found to be ~100-fold more potent than 1a in inhibiting the proliferation of breast cancer MCF-7 cells (Zhang et al, 2009), whereas analog 8b was shown to be slightly more potent than 1a in inhibiting the MM cell lines RPMI-8226 and KMS 28PE, in spite of a difference of nearly 10-fold in intrinsic potency (Lin et al, 2012). Inhibition of ERK phosphorylation, a signaling protein down-stream from Ras, was also observed with analog 8b in KMS 28PE cells, providing evidence that this compound inhibits the intended biochemical pathway (Lin et al, 2012). At the present time, it is difficult to predict whether N-BPs with much higher lipophilicity and lower affinity for bone can provide sufficiently higher systemic exposure in vivo to significantly alter the clinical profile of such compounds in treating non-skeletal diseases.…”

“…Several different assays have been reported in the literature for evaluating hFPPS inhibition in vitro . The most commonly used method was originally developed by Reed and Rilling (1975) and has been adopted by many researchers with only minor modifications (Marma et al, 2007; Dunford et al, 2008; Lin et al, 2012); we refer to this assay as method 1 (M1). As expected, the IC 50 value of any compound can vary considerably, depending on the assay method used for screening.…”