Targeted therapy of pyrrolo[2,3-d]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment

Abstract

Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies that target unique vulnerabilities in the tumor microenvironment (TME) of EOC have largely been unrealized. One strategy to achieve selective drug delivery for EOC therapy involves use of targeted antifolates via their uptake by folate receptor (FR) proteins, resulting in inhibition of essential one-carbon (C1) metabolic pathways. FRα is highly expressed in EOCs, along with the proton-coupled folate transporter (PCFT); FRβ is expressed on activated macrophages, a major infiltrating immune population in EOC. Thus, there is great potential for targeting both the tumor and the TME with agents delivered via selective transport by FRs and PCFT. In this report, we investigated the therapeutic potential of a novel cytosolic C1 6-substituted pyrrolo[2,3-d]pyrimidine inhibitor AGF94, with selectivity for uptake by FRs and PCFT and inhibition of de novo purine nucleotide biosynthesis, against a syngeneic model of ovarian cancer (BR-Luc) which recapitulates high-grade serous ovarian cancer in patients. In vitro activity of AGF94 was extended in vivo against orthotopic BR-Luc tumors. With late-stage subcutaneous BR-Luc xenografts, AGF94 treatment resulted in substantial anti-tumor efficacy, accompanied by significantly decreased M2-like FRβ-expressing macrophages and increased CD3+ T cells, whereas CD4+ and CD8+ T cells were unaffected. Our studies demonstrate potent anti-tumor efficacy of AGF94 in the therapy of EOC in the context of an intact immune system, and provide a framework for targeting the immunosuppressive TME as an essential component of therapy.

Structure-Based Design of Transport-Specific Multitargeted One-Carbon Metabolism Inhibitors in Cytosol and Mitochondria

“…The mitochondrial matrix has an alkaline pH (pH 7.7−7.9) 49,50 compared to the cytosol (pH 7.0−7.4); 50,51 for docking studies, the protein and our proposed ligands were prepared accordingly. From the docking studies, the 5-atom-bridged compounds showed preferential binding (−13.3 kcal/mol for 11, −10.2 kcal/mol for 9, and −10.0 kcal/mol for 3) and the 3-to 4-atombridged compounds (1,2,4,12,13,14,15,16) showed slightly decreased docked scores (∼−9 kcal/mol). Analogues with heteroatoms in the bridge (5−8) were found to have poorer docked scores (−7.5 to −8.6 kcal/mol).…”

“…RFC is ubiquitously expressed in normal and malignant cells in humans and is the major tissue folate transporter . PCFT is the principal transporter of folates in the upper gastrointestinal tract but shows a more limited tissue distribution than RFC. − PCFT is widely expressed in human tumors and is active under acidic conditions characterizing the tumor microenvironment. , FRα is expressed in normal epithelial tissues (e.g., mammary ducts, lungs, kidneys, choroid plexus) and a wide range of tumors (e.g., breast, cervical, renal, ovarian, endometrial cancers); FRβ is expressed in hematopoietic tissues, activated myeloid cells, and tumor-associated macrophages (TAMs). ,− …”

Multitargeted 6-Substituted Thieno[2,3-d]pyrimidines as Folate Receptor-Selective Anticancer Agents that Inhibit Cytosolic and Mitochondrial One-Carbon Metabolism

“…Interestingly, for several series, these effects on FR-targeted cell inhibitions were more pronounced for FRβ and were independent of the nature of the aromatic side chain. This suggests a potential utility of these compounds for selectively targeting FRβ-expressing cells including protumor TAMs in the tumor microenvironment. , …”

“…This suggests a potential utility of these compounds for selectively targeting FRβ-expressing cells including protumor TAMs in the tumor microenvironment. 16,49 By metabolite rescue studies, we identified de novo purine biosynthesis as the targeted pathway for the thieno[2,3d]pyrimidine antifolates and discovered that compounds 1, 2, and 6 also inhibited mitochondrial C1 metabolism. By in vitro assays with monoglutamyl thieno[2,3-d]pyrimidine antifolates and isolated GARFTase and AICARFTase, inhibition of these folate-dependent targets in de novo purine biosynthesis was confirmed.…”

Pharmacokinetics, bioavailability, and plasma protein binding study of glytrexate, a novel multitarget antifolate

“…According to reports, the cellular uptake of 6-substituted pyrrolo [2,3-d] pyrimidines, depend on multiple (folate) transporters like folate receptor alpha (FRα), FRβ, reduced folate carrier (RFC), and proton coupled folate transporter (PCFT). And they found that the role of folylpolyglutamate synthetase (FPGS) in the retention of 6-substituted pyrrolo [2,3-d] pyrimidines intracellularly ( Dekhne et al, 2020 ; Golani et al, 2020 ; Wallace-Povirk et al, 2021 ; Wallace-Povirk et al, 2022 ). Thus, this hypersensitivity maybe explained by the fact that KB cells express high amount of FRα, which may serve as cellular internalization route for glytrexate.…”