Scientists at NCATS have developed an analog of Methotrexate (MTX) that incorporates the proteasome-targeting properties of E3-ubiquitin ligase small molecule ligands (MTX-PROTACs) to directly bind to the MTX target dihydrofolate reductase (DHFR) and mark the protein for proteasomal degradation. This unique property may dramatically lower the therapeutic dose required in a treatment setting. The initial series of molecules that utilized differing E3 ligase ligands and linkers to MTX demonstrated dramatically differential effects on the cellular turnover of DHFR in a lymphoma B-cell (HBL-1) line. These results suggested that it was possible to achieve targeted tissue selectivity with specific analog designs. Further, in comparative cell culture studies with the parent MTX, scientists observed that MTX appeared to lead to an increase in cellular DHFR, a mechanism that may allow some cancer cells to continue to proliferate once MTX administration is discontinued. The disappearance of DHFR from the HBL-1 cells treated with the MTX-PROTACs analog could have a significant positive effect on overcoming chemotherapy resistance that results from drug discontinuance.

The MTX-PROTACs of this invention result in the degradation of the DHFR in the cell, lowering the levels dramatically, in direct contrast to MTX which by stabilizes DHFR from turnover, while binding tightly to it. When MTX is withdrawn or its plasma levels decrease, DHFR bound MTX may eventually dissociate from DHFR and diffuse from a cancer cell allowing the remaining high levels of DHFR to function again, resulting in proliferation of any remaining cancer cells. By utilizing MTX-PROTAC, it is possible to achieve the efficacy of MTX, without the danger of chemotherapy resistance.