In crystallo screening for proline analog inhibitors of the proline cycle enzyme PYCR1

Abstract

Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the biosynthetic half-reaction of the proline cycle by reducing Δ¹-pyrroline-5-carboxylate (P5C) to proline through the oxidation of NAD(P)H. Many cancers alter their proline metabolism by up-regulating the proline cycle and proline biosynthesis, and knockdowns of PYCR1 lead to decreased cell proliferation. Thus, evidence is growing for PYCR1 as a potential cancer therapy target. Inhibitors of cancer targets are useful as chemical probes for studying cancer mechanisms and starting compounds for drug discovery; however, there is a notable lack of validated inhibitors for PYCR1. To fill this gap, we performed a small-scale focused screen of proline analogs using X-ray crystallography. Five inhibitors of human PYCR1 were discovered: l-tetrahydro-2-furoic acid, cyclopentanecarboxylate, l-thiazolidine-4-carboxylate, l-thiazolidine-2-carboxylate, and N-formyl l-proline (NFLP). The most potent inhibitor was NFLP, which had a competitive (with P5C) inhibition constant of 100 μm The structure of PYCR1 complexed with NFLP shows that inhibitor binding is accompanied by conformational changes in the active site, including the translation of an α-helix by 1 Å. These changes are unique to NFLP and enable additional hydrogen bonds with the enzyme. NFLP was also shown to phenocopy the PYCR1 knockdown in MCF10A H-RAS^V12 breast cancer cells by inhibiting de novo proline biosynthesis and impairing spheroidal growth. In summary, we generated the first validated chemical probe of PYCR1 and demonstrated proof-of-concept for screening proline analogs to discover inhibitors of the proline cycle.

Keywords: X-ray crystallography; breast cancer; enzyme inhibitor; enzyme kinetics; tumor metabolism.

Figure 1

The proline cycle and inhibitors of proline cycle enzymes.A, the enzymes and reactions of the proline cycle. B, chemical structures of THFA, CPC, l-T4C, l-T2C, and NFLP.

Figure 2

Structures of the proline analogs screened in crystallo against PYCR1.1, (S)-(−)-tetrahydro-2-furoic acid; 2, pyrrole-2-carboxylic acid; 3, 4-oxo-l-proline, 4, cis-l-3-hydroxyproline; 5, α-methyl-l-proline; 6, trans-4-hydroxy-l-proline; 7, cis-4-hydroxy-l-proline; 8, cis-4-hydroxy-d-proline; 9, trans-4-hydroxy-d-proline; 10, d-proline; 11, R-(−)-2-pyrrolidinemethanol; 12, (S)-α-allyl-proline; 13, thiazolidine-2-carboxylate; 14, l-thiazolidine-4-carboxylate; 15, 1,3-thiazolidine-2,4-dicarboxylate; 16, dimethyl 1,3-thiazolidine-2,4-dicarboxylate; 17, l-proline methyl ester; 18, l-4-hydroxyproline methyl ester; 19, cis-4-hydroxy-d-proline methyl ester; 20, N-methyl l-proline; 21, N-formyl l-proline; 22, N-acetyl l-proline; 23, trans-1-acetyl-4-hydroxyl-l-proline; 24, l-(+)-mandelic acid; 25, sodium-l-lactate; 26, d-cycloserine; 27, cyclopentanecarboxylate.

Figure 3

Inhibition kinetics of PYCR1 with proline analogs.A, L-proline. B, THFA. C, CPC. D, T4C. E, T2C. F, NFLP. The data were measured in 50 mm Tris (pH 7.5) with 1 mm EDTA disodium salt while holding NADH fixed at 175 μm and varying d,l-P5C (0–2000 μm). The concentration of the substrate l-P5C was considered to be half the total d,l-P5C concentration added to the assays. Curves represent global fits of the data to a competitive inhibition model showing initial velocity in units of µm [NAD⁺] s⁻¹ as a function of l-P5C concentration (µm).

Figure 4

Location of the inhibitor binding site.A, dimer of PYCR1 complexed with NFLP. The box indicates the inhibitor binding site. The two chains of the dimer have different colors. B, close-up view of the binding site. The two chains of the dimer have different colors.

Figure 5

The structures of PYCR1 complexed with proline analog inhibitors.A, l-proline (PDB code 5UAU). B, THFA. C, CPC. D, l-T4C. E, l-T2C. F, NFLP. The blue cages represent polder omit maps contoured at 4σ. In the schematic diagrams on the right, blue dashes denote hydrogen bonds unique to the thiazolidine complexes, and red dashes indicate those unique to the NFLP complex.

Figure 6

Conformational changes needed to accommodate the formyl group of NFLP.A, superposition of the PYCR1-NFLP complex (pink) and the PYCR1-proline complex (gray) (PDB 5UAU). The arrows denote the directions of conformational changes needed to accommodate the steric bulk of the formyl group of NFLP. Red and black dashes denote the His-223–Asp-229 ion pairs in the NFLP and proline complexes, respectively. B, the location of αK in the decamer. Left, the decamer viewed down the 5-fold axis with each chain colored differently. Right, side view of the decamer with αK in red.

Figure 7

NFLP targets proline metabolism in breast cancer spheroids.A, abundance of proline M + 0 and M + 5 labeling in MCF10A hRAS^V12 spheroids upon ¹³C₅-glutamine incubation and treatment without (n = 3) or with NFLP (5 mm; n = 3). Analysis was performed at 5th day of treatment. B, relative abundance of intracellular proline levels in MCF10A hRAS^V12 spheroids upon treatment (5 days) without (n = 6), with THFA (n = 3) or with NFLP (n = 6). C, protein content in MCF10A hRAS^V12 spheroids treated for 5 days without (n = 6), with THFA (n = 3) or with NFLP (n = 6). Bar graphs show mean ± S.D. from biological independent samples and P-values were obtained with Mann-Whitney tests.

Figure 8

Activity of a bacterial PRODH in the presence of NFLP.A, activity of the E. coli PutA PRODH domain with either 100 mm l-proline or 100 mm NFLP as the substrate. B, activity of the E. coli PutA PRODH domain with l-proline as the substrate (100 mm) at various NFLP concentrations. The rates have been normalized to the rate obtained in the absence of NFLP.