Design strategy for serine hydroxymethyltransferase probes based on retro-aldol-type reaction

Abstract

Serine hydroxymethyltransferase (SHMT) is an enzyme that catalyzes the reaction that converts serine to glycine. It plays an important role in one-carbon metabolism. Recently, SHMT has been shown to be associated with various diseases. Therefore, SHMT has attracted attention as a biomarker and drug target. However, the development of molecular probes responsive to SHMT has not yet been realized. This is because SHMT catalyzes an essential yet simple reaction; thus, the substrates that can be accepted into the active site of SHMT are limited. Here, we focus on the SHMT-catalyzed retro-aldol reaction rather than the canonical serine-glycine conversion and succeed in developing fluorescent and ¹⁹F NMR molecular probes. Taking advantage of the facile and direct detection of SHMT, the developed fluorescent probe is used in the high-throughput screening for human SHMT inhibitors, and two hit compounds are obtained.

Fig. 1

Biological role of SHMT. a Serine–glycine interconversion catalyzed by SHMT. THF = tetrahydrofolate, CH₂-THF = N-5,N-10-methylenetetrahydrofolate. The red dot highlights the carbon that is transferred from Ser to THF. b Schematic overview of hSHMT function. MTHFD = methylenetetrahydrofolate dehydrogenase-cyclohydrolase, CH₂-THF = N-5,N-10-methylenetetrahydrofolate, CH⁺-THF = 5,10-methenyltetrahydrofolate, CHO-THF = 10-formyltetrahydrofolate, NADP⁺ = Nicotinamide adenine dinucleotide phosphate, NADPH = NADP⁺ reduced form. c SHMT, dihydrofolate reductase (DHFR), and thymidylate synthase (TS) in the folate cycle. THF = tetrahydrofolate, CH₂-THF = 5,10-methylenetetrahydrofolate, DHF = dihydrofolate, FdUMP = fluorodeoxyuridine-5′-monophosphate, dUMP = deoxyuridine monophosphate, dTMP = deoxythymidine monophosphate

Fig. 2

Molecular design of hSHMT probes. a Substrate binding site of SHMT1. Illustration of the SHMT1–5-CHO-THF–Gly-PLP complex from crystal structure data (Mouse SHMT, PDB ID: 1EJI). 5-CHO-THF and Gly-PLP are shown as stick models. Color code: oxygen: red; nitrogen: blue; carbon: cyan; phosphorus: orange. b The proposed mechanism of SHMT. (upper) THF-dependent serine–glycine pathway and (lower) THF-independent retro-aldol reaction catalyzed by SHMT. Pi = -PO₃^2–. c Fluorescent probe 1 and ¹⁹F NMR probe 2 used in this study

Fig. 3

Synthesis of hSHMT fluorescent probe 1 and NMR probe 2. Reagents and conditions for synthesis of probe 1: a) N-Dibenzylglycine tert-butyl ester, LDA, dry THF, –60 °C, 10 min, then 6-(dimethylamino)-2-naphthaldehyde, –60 °C, 30 min; b) N-(p-Toluenesulfonyl)-l-phenylalanyl chloride, DMAP, dry THF, r.t., 14 h; c) 5 M NaOH aq., THF, EtOH, r.t., 5 min; d) Pd/C, H₂, MeOH, r.t., 28 h; e) 4 M HCl/EtOAc, r.t. reagents and conditions for synthesis of probe 2: a) N-Dibenzylglycine tert-butyl ester, LDA, dry THF, –78 °C, 10 min, then 4-fluorobenzaldehyde, –78 °C, 30 min; b) N-(p-Toluenesulfonyl)-l-phenylalanyl chloride, pyridine, dry THF, 60 °C, 19 h; c) 5 M KOH aq., EtOH, 40 °C, 2 h; d) Pd/C, H₂, MeOH, r.t., 16 h; e) TFA, DCM, r.t., 17 h. The lower inset indicates the X-ray crystal structure of probe 1 l-erythro intermediate. Color code: oxygen: red; nitrogen: blue; sulfur: yellow; carbon: black; hydrogen: white

Fig. 4

Fluorescent probe targeting hSHMT. a Schematic illustration of hSHMT fluorescent probe 1. b Fluorescence spectral change of probe 1 (4.6 μM) during the hSHMT1-catalyzed reaction from 0 to 60 min. Excitation at 390 nm. Assay conditions: 5 units/mL hSHMT1, 50 mM HEPES buffer (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol (DTT), with or without inhibitor (±)-SHIN1 10 µM, 0.6% DMSO, 37 °C. The inset shows the fluorescence change of probe 1 (5 μM). c Conversion rate analysis of dl-erythro, dl-threo, or L-erythro probes (4.6 µM) by time-dependent fluorescence analysis at 530 nm (excitation at 390 nm). I₀ at 530 nm is the fluorescence intensity under the condition without hSHMT1. Source data are provided as a Source Data file

Fig. 5

NMR probe targeting hSHMT. a Schematic illustration of hSHMT NMR probe 2. b ¹⁹F NMR spectral change of probe 2 (5 mM) upon the addition of hSHMT1 (5 units/mL). Assay conditions: 5 units/mL hSHMT1, 50 mM HEPES buffer (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 30% D₂O, with or without inhibitor (±)-SHIN1 10 µM, 0.1% DMSO, 37 °C. CF₃COOH (–76.5 ppm) was used as the internal standard for ¹⁹F NMR. c Conversion rate of hSHMT probe 2 by hSHMT1 with (blue square) or without (red circle) hSHMT inhibitor (±)-SHIN1 10 µM. Error bars represent s.d., n = 3. d ¹⁹F NMR spectra of probe 2 (1 mM) in rat liver homogenate (2.42 mg proteins/mL in PBS) with or without inhibitor (±)-SHIN1 10 µM. e ¹H (T₂-weighted) and ¹⁹F chemical-shift-selective imaging (11.7 T) of probe 2 (10 mM). Assay conditions: hSHMT1 (1 unit/mL), with or without inhibitor (±)-SHIN1 10 µM, 37 °C, acquisition time = 17 min. Source data of Fig. 5c are provided as a Source Data file

Fig. 6

hSHMT inhibitor screening by utilizing probe 1. a Schematic illustration for hSHMT1 inhibitor screening. Details are described in the Supplementary Methods for high-throughput screening. b Chemical structures and inhibition properties of hit compounds. The IC₅₀ values for Hits 1 and 2 against hSHMT1 were determined by HPLC analysis of Ser–Gly conversion in the presence of various concentrations of Hits 1 and 2. Error bars represent s.d., n = 3. Thermodynamic parameters of the interactions between hSHMT1 and Hit 1 or Hit 2 were determined by ITC. c Inhibition of Ser–Gly conversion catalyzed by endogenous SHMT by Hits 1 and 2 in rat liver homogenate (250 µg proteins/mL). Ser–Gly conversion by SHMT1 or SHMT2 in the presence of varying concentrations of Hits 1 and 2 was quantified by HPLC analysis. Error bars represent s.d., n = 3. Source data of Fig. 6b, c are provided as a Source Data file