Fluorine-Substituted Pyrrolo[2,3-<i>d</i>]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of <i>de Novo</i> Purine Nucleotide Biosynthesis

Ravindra, Manasa; Wilson, Mike R.; Tong, Nian; O’Connor, Carrie; Karim, Mohammad A.; Polin, Lisa; Wallace-Povirk, Adrianne; White, Kathryn; Kushner, Juiwanna; Hou, Zhanjun; Matherly, Larry H.; Gangjee, Aleem

doi:10.1021/acs.jmedchem.8b00408

Cited by 22 publications

(47 citation statements)

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“…Moreover, PCFT transport is further enhanced by a replacement of the side-chain phenyl ring by a 2′,3′- and 2′,4′-thiophene, as in AGF94 and AGF154, respectively ( Figure 2 B) [ 41 , 147 , 148 ]. Side-chain 3′-fluorinated thienoyl analogs of this series (AGF278, AGF283; Figure 2 B) were also synthesized and show high levels of FRα- and PCFT-targeted activity [ 149 ].…”

Section: Targeting Eoc Via Targeted Antifolates: C1 Metabolism As a Unique Vulnerability For Eocmentioning

confidence: 99%

“…Panel ( A ): structures are shown for the dual SHMT1 and SHMT2 inhibitors SHIN1 and SHIN2 [ 150 , 151 ], the MTHFD2 inhibitor DS18561882 [ 152 ], as well as pemetrexed [ 137 ] and CT900 (BGC945, ONX0801) [ 26 ] (both principally thymidylate synthase inhibitors). Panel ( B ): structures are shown for pyrrolo[2,3- d ]pyrimidine GARFTase inhibitors, AGF17 [ 145 ], AGF23 [ 145 ], AGF94 [ 147 ], AGF154 [ 148 ], AGF278 [ 149 ], and AGF283 [ 149 ], as well as the pyrrolo[3,2- d ]pyrimidine antifolate AGF347 [ 153 ], which acts as a multitargeted inhibitor of SHMT2 in the mitochondria and of SHMT1, GARFTase, and ATIC in the cytosol.…”

Section: Targeting Eoc Via Targeted Antifolates: C1 Metabolism As a Unique Vulnerability For Eocmentioning

confidence: 99%

“…AGF94, AGF154, AGF278, and AGF283 are potent (nanomolar) inhibitors of EOC cell lines (IGROV1, SKOV3, A2780) with a wide (~3000-fold) range of FRα levels and relatively constant PCFT, although the uptake by FRα predominated when both systems were present [ 14 , 149 ]. In IGROV1 cells, FRα knockdown (~95%) had a modest impact on in vitro drug efficacy, establishing transport redundancy for pyrrolo[2,3- d ]pyrimidine antifolates such as AGF94 by the PCFT and FRα [ 14 , 149 ]. For AGF94, this finding was extended in vivo [ 14 ].…”

Section: Targeting Eoc Via Targeted Antifolates: C1 Metabolism As a Unique Vulnerability For Eocmentioning

confidence: 99%

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Folate Transport and One-Carbon Metabolism in Targeted Therapies of Epithelial Ovarian Cancer

Wallace-Povirk

Hou

Nayeen

et al. 2021

Cancers

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New therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. To identify new approaches for targeting EOC, metabolic vulnerabilities must be discovered and strategies for the selective delivery of therapeutic agents must be established. Folate receptor (FR) α and the proton-coupled folate transporter (PCFT) are expressed in the majority of EOCs. FRβ is expressed on tumor-associated macrophages, a major infiltrating immune population in EOC. One-carbon (C1) metabolism is partitioned between the cytosol and mitochondria and is important for the synthesis of nucleotides, amino acids, glutathione, and other critical metabolites. Novel inhibitors are being developed with the potential for therapeutic targeting of tumors via FRs and the PCFT, as well as for inhibiting C1 metabolism. In this review, we summarize these exciting new developments in targeted therapies for both tumors and the tumor microenvironment in EOC.

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Section: Targeting Eoc Via Targeted Antifolates: C1 Metabolism As a Unique Vulnerability For Eocmentioning

confidence: 99%

Section: Targeting Eoc Via Targeted Antifolates: C1 Metabolism As a Unique Vulnerability For Eocmentioning

confidence: 99%

Section: Targeting Eoc Via Targeted Antifolates: C1 Metabolism As a Unique Vulnerability For Eocmentioning

confidence: 99%

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Folate Transport and One-Carbon Metabolism in Targeted Therapies of Epithelial Ovarian Cancer

Wallace-Povirk

Hou

Nayeen

et al. 2021

Cancers

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“…To identify the individual (RFC versus PCFT) transport fluxes, assays were performed in the presence of 10 μM of the RFC [N α -(4-amino-4-deoxypteroyl)-N δ -hemiphthaloyl-L-ornithine (PT523)] 37 or PCFT (AGF94) 28 inhibitors. PT523 selectively inhibits RFC transport 38 and AGF94 selectively inhibits PCFT transport 13 , 32 , 33 . Results for R1-11/Tet-on-RFC/PCFT cells are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Folate transporter dynamics and therapy with classic and tumor-targeted antifolates

O’Connor

Wallace-Povirk

Ning

et al. 2021

Sci Rep

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There are three major folate uptake systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antifolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antifolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular folates were determined by the type and levels of the major folate transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antifolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major folate transport systems that depends on transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antifolates.

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“…Membrane Transport Assays. Plasma membrane transport measurements were performed at 37°C as previously described (Ravindra et al, 2018). Buffers used were MES [2-(N-morpholino) ethanesulfonic acid]-buffered saline at pH 5.5 (20 mM MES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , and 5 mM glucose) and HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]-buffered saline at pH 6.8 and pH 7.2 (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , and 5 mM glucose).…”

Section: Methodsmentioning

confidence: 99%

Cellular Pharmacodynamics of a Novel Pyrrolo[3,2-d]pyrimidine Inhibitor Targeting Mitochondrial and Cytosolic One-Carbon Metabolism

et al. 2019

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Folate-dependent one-carbon (C1) metabolism is compartmentalized in the mitochondria and cytosol and is a source of critical metabolites for proliferating tumors. Mitochondrial C1 metabolism including serine hydroxymethyltransferase 2 (SHMT2) generates glycine for de novo purine nucleotide and glutathione biosynthesis and is an important source of NADPH, ATP, and formate, which affords C1 units as 10-formyl-tetrahydrofolate and 5,10-methylene-tetrahydrofolate for nucleotide biosynthesis in the cytosol. We previously discovered novel first-in-class multitargeted pyrrolo[3,2-d]pyrimidine inhibitors of SHMT2 and de novo purine biosynthesis at glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase with potent in vitro and in vivo antitumor efficacy toward pancreatic adenocarcinoma cells. In this report, we extend our findings to an expanded panel of pancreatic cancer models. We used our lead analog AGF347 [(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl) butyl)-2-fluorobenzoyl)-L-glutamic acid] to characterize pharmacodynamic determinants of antitumor efficacy for this series and demonstrated plasma membrane transport into the cytosol, uptake from cytosol into mitochondria, and metabolism to AGF347 polyglutamates in both cytosol and mitochondria. Antitumor effects of AGF347 downstream of SHMT2 and purine biosynthesis included suppression of mammalian target of rapamycin signaling, and glutathione depletion with increased levels of reactive oxygen species. Our results provide important insights into the cellular pharmacology of novel pyrrolo[3,2-d]pyrimidine inhibitors as antitumor compounds and establish AGF347 as a unique agent for potential clinical application for pancreatic cancer, as well as other malignancies. SIGNIFICANCE STATEMENT This study establishes the antitumor efficacies of novel inhibitors of serine hydroxymethyltransferase 2 and of cytosolic targets toward a panel of clinically relevant pancreatic cancer cells and demonstrates the important roles of plasma membrane transport, mitochondrial accumulation, and metabolism to polyglutamates of the lead compound AGF347 to drug activity. We also establish that loss of serine catabolism and purine biosynthesis resulting from AGF347 treatment impacts mammalian target of rapamycin signaling, glutathione pools, and reactive oxygen species, contributing to antitumor efficacy.

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Fluorine-Substituted Pyrrolo[2,3-d]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis

Cited by 22 publications

References 70 publications

Folate Transport and One-Carbon Metabolism in Targeted Therapies of Epithelial Ovarian Cancer

Folate Transport and One-Carbon Metabolism in Targeted Therapies of Epithelial Ovarian Cancer

Folate transporter dynamics and therapy with classic and tumor-targeted antifolates

Cellular Pharmacodynamics of a Novel Pyrrolo[3,2-d]pyrimidine Inhibitor Targeting Mitochondrial and Cytosolic One-Carbon Metabolism

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