DHFR and breast carcinoma: Although a previous study suggested that Pyr is also a direct binder and inhibitor of TP [34] in biochemical experiments (Biacore and differential scanning fluorimetry), CETSA experiments with Pyr, MTX, Cyc, and Cyc analogues could not confirm binding to TP in breast cancer cell lysates at relevant concentrations for DHFR engagement and phenotypic responses in breast cancer cell lines.