Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface.

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.

Quantum mechanical origin of the conformational preferences of 4-thiaproline and its S-oxides.

The saturated ring and secondary amine of proline spawn equilibria between pyrrolidine ring puckers as well as peptide bond isomers. These conformational equilibria can be modulated by alterations to the chemical architecture of proline. For example, C(γ) in the pyrrolidine ring can be replaced with sulfur, which can be oxidized either stereoselectively to yield diastereomeric S-oxides or completely to yield a sulfone. Here, the thiazolidine ring and peptide bond conformations of 4-thiaproline and its S-oxides were analyzed in an Ac-Xaa-OMe system using NMR spectroscopy, X-ray crystallography, and hybrid density functional theory. The results indicate that the ring pucker of the S-oxides is governed by the gauche effect, and the prolyl peptide bond conformation is determined by the strength of the n → π* interaction between the amide oxygen and the ester carbonyl group. These findings, which are consistent with those of isologous 4-hydroxyprolines and 4-fluoroprolines, substantiate the importance of electron delocalization in amino acid conformation.

IMPDH2 Is an Intracellular Target of the Cyclophilin A and Sanglifehrin A Complex.

Natural products have demonstrated utility in the clinic and can also act as probes to understand complex cellular pathways. Sanglifehrin A (SFA) is a mixed polyketide and non-ribosomal peptide synthase natural product with sub-nano-molar affinity for its receptor cyclophilin A (PPIA). It has been shown to behave in vitro as an immune suppressant. Here, we identify inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) as an intracellular target of the PPIA-SFA binary complex. The formation of this ternary complex does not inhibit the enzymatic activity of IMPDH2. Rather, ternary complex formation modulates cell growth through interaction with the cystathionine-ß-synthase (CBS) domain of IMPDH2. We further demonstrate that the SFA complex is highly isoform selective for IMPDH2 (versus IMPDH1). This work reveals a role for the CBS domains of IMPDH2 in cellular proliferation, suggesting a more complex role than previously suspected for IMPDH2 in T cell activation and proliferation.

Stringency of the 2-His-1-Asp active-site motif in prolyl 4-hydroxylase.

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His-1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C-H bonds. Prolyl 4-hydroxylase (P4H) is an alpha-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His-1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.