Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

The α-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His ₂ (Asp/Glu) ₁ “facial triad” of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X ⁻ ) coordinates at the vacated site. Halogenation is thought to result from “rebound” of the halogen radical from the X-Fe(III)-OH intermediate produced by hydrogen (H ^• ) abstraction to the substrate radical. The alternative decay pathway for the X-Fe(III)-OH intermediate, rebound of the hydroxyl radical to the substrate radical (as occurs in the hydroxylases), reportedly does not compete. Here we show for the halogenase SyrB2 that positioning of the alkyl group of the substrate away from the oxo/hydroxo ligand and closer to the halogen ligand sacrifices H ^• -abstraction proficiency for halogen-rebound selectivity. Upon replacement of l -Thr, the C4 amino acid tethered to the SyrB1 carrier protein in the native substrate, by the C5 amino acid l -norvaline, decay of the chloroferryl intermediate becomes 130× faster and the reaction outcome switches to primarily hydroxylation of C5, consistent with projection of the methyl group closer to the oxo/hydroxo by the longer side chain. Competing H ^• abstraction from C4 results primarily in chlorination, as occurs at this site in the native substrate. Consequently, deuteration of C5, which slows attack at this site, switches both the regioselectivity from C5 to C4 and the chemoselectivity from hydroxylation to chlorination. Thus, substrate-intermediate disposition and the carboxylate → halide ligand swap combine to specify the halogenation outcome.

Halogenases differ from hydroxylases by coordination of a chloride ion at the reactive iron center, which is taken up by an activated substrate. Biochemical and spectroscopic evidence now show other anions can be used, resulting in the first enzymatic incorporation of nitrogen onto unactivated aliphatic carbons. Iron-dependent halogenases employ cis-halo-Fe(IV)-oxo (haloferryl) complexes to functionalize unactivated aliphatic carbon centers, a capability elusive to synthetic chemists. Halogenation requires (i) coordination of a halide anion (Cl− or Br−) to the enzyme's Fe(II) cofactor, (ii) coupled activation of O2 and decarboxylation of α-ketoglutarate to generate the haloferryl intermediate, (iii) abstraction of hydrogen (H•) from the substrate by the ferryl and (iv) transfer of the cis halogen as Cl• or Br• to the substrate radical. This enzymatic solution to an unsolved chemical challenge is potentially generalizable to installation of other functional groups, provided that the corresponding anions can support the four requisite steps. We show here that the wild-type halogenase SyrB2 can indeed direct aliphatic nitration and azidation reactions by the same chemical logic. The discovery and enhancement by mutagenesis of these previously unknown reaction types suggest unrecognized or untapped versatility in ferryl-mediated enzymatic C-H bond activation.