Screening and In Vitro Testing of Antifolate Inhibitors of Human Cytosolic Serine Hydroxymethyltransferase

Abstract

Metabolic reprogramming of tumor cells toward serine catabolism is now recognized as a hallmark of cancer. Serine hydroxymethyltransferase (SHMT), the enzyme providing one-carbon units by converting serine and tetrahydrofolate (H4PteGlu) to glycine and 5,10-CH2-H4PteGlu, therefore represents a target of interest in developing new chemotherapeutic drugs. In this study, 13 folate analogues under clinical evaluation or in therapeutic use were in silico screened against SHMT, ultimately identifying four antifolate agents worthy of closer evaluation. The interaction mode of SHMT with these four antifolate drugs (lometrexol, nolatrexed, raltitrexed, and methotrexate) was assessed. The mechanism of SHMT inhibition by the selected antifolate agents was investigated in vitro using the human cytosolic isozyme. The results of this study showed that lometrexol competitively inhibits SHMT with inhibition constant (Ki) values in the low micromolar. The binding mode of lometrexol to SHMT was further investigated by molecular docking. These results thus provide insights into the mechanism of action of antifolate drugs and constitute the basis for the rational design of novel and more potent inhibitors of SHMT.

Structural basis of inhibition of the human serine hydroxymethyltransferase SHMT2 by antifolate drugs

“…3D). These interactions actually more closely represent the hSHMT2-LTX structure, and were also predicted from the previously mentioned molecular docking studies involving hSHMT1 and LTX [18]. Furthermore, the predicted binding of the residue Asn387 with the N11 and N18 atoms of PTX [17], does not exist in the hSHMT2-PTX crystal structure, where Glu98 and Tyr106 perform this function (Fig.…”

“…In the hSHMT1 model, Tyr82 makes a pi‐stacking interaction with the p‐ aminobenzoate of LTX, which is also observed in our hSHMT2 structure where Tyr105 performs the same function. However, the predicated interactions in hSHMT1 of the main chain nitrogen of Ala395 and Tyr82 with the α‐ and γ‐carboxylates of LTX do not exist in the hSHMT2 crystal structure .…”

“…Prior to this study there were no crystal structures of hSHMT in complex with antifolates, however, there were molecular docking studies which aimed to provide insights into their binding modes. In 2015, Paiardini et al [18] performed an in silico screen of antifolate analogues against hSHMT1. The most potent antifolate was LTX, which was shown to be a competitive low micromolar inhibitor of hSHMT1 with an almost identical K d value to the compound leucovorin [18].…”

“…In 2015, Paiardini et al [18] performed an in silico screen of antifolate analogues against hSHMT1. The most potent antifolate was LTX, which was shown to be a competitive low micromolar inhibitor of hSHMT1 with an almost identical K d value to the compound leucovorin [18]. The molecular docking analysis of LTX showed notable differences in the binding mode to what was observed in our hSHMT2-LTX crystal structure.…”

The catalytic activity of serine hydroxymethyltransferase is essential for de novo nuclear dTMP synthesis in lung cancer cells

“…Tumour cells recycle one-carbon units from different amino acids within the folate and methionine cycles to produce the building blocks needed to sustain high proliferation rates [1][2][3]. Given its pivotal role in OCM, it is not surprising that SHMT is considered a potential drug target for cancer therapy [4][5][6][7]. Serine fuels the folate cycle through the reaction catalysed by serine hydroxymethyltransferase (SHMT) that reversibly interconverts serine and tetrahydrofolate (THF) to glycine and 5,10-methylene-THF (Fig.…”