Induced Fit Describes Ligand Binding to Membrane-Associated Cytochrome P450 3A4.

Cytochrome P450 3A4 (CYP3A4) is the dominant P450 involved in human xenobiotic metabolism. Competition for CYP3A4 therefore underlies several adverse drug-drug interactions. Despite its clinical significance, the mechanisms CYP3A4 uses to bind diverse ligands remain poorly understood. Highly monodisperse CYP3A4 embedded in anionic lipoprotein nanodiscs containing an equal mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) were used to determine which of the limiting kinetic schemes that include protein conformational change, conformational selection (CS) or induced fit (IF), best described the binding of four known irreversible inhibitors. Azamulin, retapamulin, pleuromutilin, and mibrefadil binding to CYP3A4 nanodiscs conformed to a single-site binding model. Exponential fits of stopped-flow UV-visible absorption spectroscopy data supported multiple-step binding mechanisms. Trends in the rates of relaxation to equilibrium with increasing ligand concentrations were ambiguous as to whether IF or CS was involved; however, global fitting and consideration of the rate constants favored an IF mechanism. In the case of mibrefadil, a transient complex was observed in the stopped-flow UV-visible experiment, definitively assigning the presence of IF in ligand binding. While these studies only consider a small region of CYP3A4's vast ligand space, they provide kinetic evidence that CYP3A4 can use an IF mechanism. SIGNIFICANCE STATEMENT: CYP3A4 is capable of oxidizing numerous xenobiotics, including many drugs. Such promiscuity could not be achieved without conformational changes to accommodate diverse substrates. It is unknown whether conformational heterogeneity is present before (conformational selection) or after (induced fit) ligand binding. Stopped-flow measurements of suicide inhibitors binding to nanodisc-embedded CYP3A4 combined with sophisticated numerical analyses support that induced fit better describes ligand binding to this important enzyme.

CSF1R inhibitors exhibit antitumor activity in acute myeloid leukemia by blocking paracrine signals from support cells.

To identify new therapeutic targets in acute myeloid leukemia (AML), we performed small-molecule and small-interfering RNA (siRNA) screens of primary AML patient samples. In 23% of samples, we found sensitivity to inhibition of colony-stimulating factor 1 (CSF1) receptor (CSF1R), a receptor tyrosine kinase responsible for survival, proliferation, and differentiation of myeloid-lineage cells. Sensitivity to CSF1R inhibitor GW-2580 was found preferentially in de novo and favorable-risk patients, and resistance to GW-2580 was associated with reduced overall survival. Using flow cytometry, we discovered that CSF1R is not expressed on the majority of leukemic blasts but instead on a subpopulation of supportive cells. Comparison of CSF1R-expressing cells in AML vs healthy donors by mass cytometry revealed expression of unique cell-surface markers. The quantity of CSF1R-expressing cells correlated with GW-2580 sensitivity. Exposure of primary AML patient samples to a panel of recombinant cytokines revealed that CSF1R inhibitor sensitivity correlated with a growth response to CSF1R ligand, CSF1, and other cytokines, including hepatocyte growth factor (HGF). The addition of CSF1 increased the secretion of HGF and other cytokines in conditioned media from AML patient samples, whereas adding GW-2580 reduced their secretion. In untreated cells, HGF levels correlated significantly with GW-2580 sensitivity. Finally, recombinant HGF and HS-5-conditioned media rescued cell viability after GW-2580 treatment in AML patient samples. Our results suggest that CSF1R-expressing cells support the bulk leukemia population through the secretion of HGF and other cytokines. This study identifies CSF1R as a novel therapeutic target of AML and provides a mechanism of paracrine cytokine/growth factor signaling in this disease.

Targeting of colony-stimulating factor 1 receptor (CSF1R) in the CLL microenvironment yields antineoplastic activity in primary patient samples.

In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.