Glycinamide Ribonucleotide Research Articles

Molecular Factors Associated with Pemetrexed Sensitivity According to Histological Type in Non-small Cell Lung Cancer

This study was designed to investigate potential molecules that predict chemosensitivity to pemetrexed (Alimta®) in surgically resected non-small cell lung cancer (NSCLC). Chemosensitivity to ALM and other drugs was assessed by succinate dehydrogenase inhibition (SDI) test in 69 NSCLC samples (55 adenocarcinomas, and 14 squamous cell carcinomas). The mRNA expression levels of Alimta®-target enzymes [thymidylate synthase (TYMS); dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT)], Alimta®-metabolizing enzymes [γ-glutamyl hydrase (GGH) and folylpolyglutamate synthase] and an Alimta® transporter [reduce folate carrier (RFC)] were measured and examined for potential correlations to chemosensitivity. The squamous cell carcinoma samples showed higher TYMS expression and lower RFC expression than did the adenocarcinoma samples. In the adenocarcinoma sample analyses, GGH expression was inversely correlated to sensitivity. The histology-dependent differences in chemosensitivity to Alimta® may be attributed to the histology-dependent differences in TYMS and RFC expression. In adenocarcinomas, GGH potentially represents a marker for chemosensitivity to Alimta®.

GART mediates the renewal of intestinal epithelial barrier via p38/p53/PUMA cascade in colitis.

Glycinamide ribonucleotide formyltransferase (GART) has been established as a pivotal enzyme in de novo purine synthesis, and mediates cellular apoptosis in many diseases. We aimed to investigate the role of GART in the pathogenesis of Crohn's disease (CD). In our study, we demonstrated for the first time that GART expression is up-regulated in patients with active CD and in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis model. Moreover, the inhibition of GART induced cellular apoptosis and suppressed the migration of IECs through the activation of the MEKK3-MKK3-p38 mitogen-activated protein kinase (MAPK) pathway, following with the dys-regulation of p53 and p53 up-regulated modulator of apoptosis (PUMA). Taken together, GART plays a critical role in the protection of cellular apoptosis and migration of intestinal epithelial cells to maintain the integrity of the epithelial barrier, thus providing a new potential approach in designing a novel therapy for CD.

Tumor Targeting with Novel 6-Substituted Pyrrolo [2,3-d] Pyrimidine Antifolates with Heteroatom Bridge Substitutions via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis

Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in 1 by N (4), O (8), or S (9), or with N-substituted formyl (5), acetyl (6), or trifluoroacetyl (7) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro antiproliferative activity toward engineered Chinese hamster ovary cells expressing FRs by 4-9 over the CH2 analogue 1. Compounds 4-9 inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for 4 with FRα and GARFTase showed that the bound conformations of 4 required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, 4 was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo[2,3-d]pyrimidines related to 1 provide targeted antifolates that warrant further evaluation as anticancer agents.

The effect of inhibiting glycinamide ribonucleotide formyl transferase on the development of neural tube in mice.

BackgroundFolate deficiency is closely related to the development of neural tube defects (NTDs). However, the exact mechanism is not completely understood. This study aims to induce murine NTDs by inhibiting one of the folate metabolic pathways, de novo purine synthesis and preliminarily investigate the potential mechanisms. The key enzyme, glycinamide ribonucleotide formyl transferase (GARFT) was inhibited by a specific inhibitor, lometrexol (DDATHF) in the pregnant mice.MethodsPregnant mice were intraperitoneally injected with various doses of DDATHF on gestational day 7.5 and embryos were examined for the presence of NTDs on gestational day 11.5. GARFT activity and levels of ATP, GTP, dATP and dGTP were detected in embryonic brain tissue. Proliferation and apoptosis was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemical assay and western blotting.Results40 mg kg−1 body weight (b/w) of DDATHF caused the highest incidence of NTDs (30.8 %) and therefore was selected as the optimal dose to establish murine NTDs. The GARFT activity and levels of ATP, GTP, dATP and dGTP in embryonic brain tissue were significantly decreased after DDATHF treatment. Furthermore, Levels of proliferation-related genes (Pcna, Foxg1 and Ptch1) were downregulated and apoptosis-related genes (Bax, Casp8 and Casp9) were upregulated. Expression of phosphohistone H3 was significantly decreased while expression of cleaved caspase-3 was greatly increased.ConclusionsResults indicate that DDATHF induced murine NTDs by disturbing purine metabolism and further led to abnormal proliferation and apoptosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-016-0114-x) contains supplementary material, which is available to authorized users.

Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase.

Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition.

Abstract 4791: Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor á and the proton-coupled folate transporter

Abstract The proton coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum, while folate receptor (FR) á delivers folate substrates into the cell via endocytosis. FRá and PCFT are highly expressed in ovarian cancer. PCFT transport is favored by acidic pH, including pHs associated with the acidic tumor microenvironment. We previously synthesized a novel class of 6-substituted pyrrolo[2,3-d]pyrimidine thiophene antifolates with 4- and 3-carbon bridge lengths, designated AGF117 and AGF150, targeted to FRá and PCFT, but not the ubiquitously expressed reduced folate carrier (RFC). To increase antitumor specificity and activity, reflecting multi-polar interactions with cellular targets, we synthesized fluorinated analogs of AGF117 and AGF150, designated AGF278 and AGF283. In the presence of a physiologic concentration of reduced folate, AGF278 and AGF283 potently inhibited proliferation of Chinese hamster ovary (CHO) cells engineered to express PCFT, but not RFC or FRá (R2/PCFT4) (IC50s of 6 and 1.5 nM, respectively), or FRá without PCFT or RFC (RT16) (IC50s of 4.8 and 5.4 nM). AGF278 and AGF283 also inhibited growth of IGROV1 (IC50s of 1.5 and 11 nM, respectively) and SKOV3 (IC50s of 37.8 and 30.2 nM, respectively) ovarian cancer cells. We measured [3H]methotrexate uptake in R2/PCFT4 CHO cells in the presence of AGF278 and AGF283; AGF278 and AGF283 potently inhibited PCFT transport with increased binding over AGF117 and AGF150, respectively, as reflected in Ki values. Analogous to AGF117 and AGF150, glycinamide ribonucleotide (GAR) formyltransferase (GARFTase), the first folate-dependent enzyme in de novo purine biosynthesis, was implicated as the cellular target for AGF278 and AGF283 as adenosine (10 ìM) and 5-aminoimidazole-4-carboxamide (320 ìM) protected IGROV1 cells from growth inhibition. GARFTase inhibition by AGF278 and AGF283 was confirmed by an in situ GARFTase assay which measures incorporation of [14C]glycine into formyl GAR, the GARFTase product, with potencies similar to those of the parent molecules. Preliminary in vivo studies with AGF278 using SKOV3 tumor xenografts in SCID mice established antitumor efficacy. Our results suggest that novel fluorinated 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating ovarian cancer, reflecting their selective cellular uptake by PCFT and FRá and potent inhibition of de novo purine biosynthesis. Additional utility of our fluorine-substituted analogs lies in their potential for positron emission tomography imaging of PCFT-expressing tumors. Citation Format: Mike R. Wilson, Manasa P. Ravindraa, Adrianne Wallace-Povirk, Lisa Polin, Zhanjun Hou, Aleem Gangjee, Larry H. Matherly. Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor á and the proton-coupled folate transporter. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4791.

Abstract 1365: Pseudo-cis and pseudo-trans amide conformations lead to potent targeted antifolates that are selectively transported by FR over RFC

Abstract Drugs usually bind to different targets in different conformations. As antitumor agents, antifolates first need to be selectively transported by tumor cells via the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) or folate receptors (FRs). The second step is the binding of antifolates with the intracellular targets. We are systematically developing novel folate analogs with selective membrane transport for FRs and PCFT over the ubiquitously expressed RFC. These 6-substituted pyrrolo[2,3-d]pyrimidine antifolates are principally inhibitors of de novo purine biosynthesis at the steps catalyzed by glycinamide ribonucleotide formyl transferase (GARFTase) and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase). We previously demonstrated that classical antifolates bind to FRα and GARFTase in different conformations. An amide in the bridge region of classical antifolates has two interchangeable lowest energy conformers, pseudo-cis and pseudo-trans. We reported that a novel antifolate (AGF238) with an amide in the bridge region binds to FRα in a pseudo-trans conformation and binds to GARFTase in a pseudo-cis conformation. As part of our continued efforts to pursue targeted antifolates as antitumor agents, we designed an expand series of amide-bridged classical antifolates with different energy barriers between the pseudo-cis and pseudo-trans conformers. Among these, AGF266 was exclusively transported by Chinese hamster ovary (CHO) cells engineered to express human FRα (IC50 = 0.42 nM) or FRβ (IC50 = 0.88 nM) over CHO expressing human RFC (IC50 > 1000 nM). AGF266 was also found to exhibit highly potent antitumor activity (IC50 = 0.79 nM in KB human tumor cells). AGF266 inhibited GARFTase as its principal cellular target, based on patterns of protection from growth inhibition by adenosine and AICA. Our in vitro findings of antitumor activity associated with FR selectivity suggest that further preclinical evaluation of AGF266 as a potential antitumor agent is warranted. Citation Format: Aleem Gangjee, Weiguo Xiang, Aamod Dehkne, Mike R. Wilson, Adrianne Wallace-Povrik, Carrie O’Connor, Zhanjun Hou, Larry H. Matherly. Pseudo-cis and pseudo-trans amide conformations lead to potent targeted antifolates that are selectively transported by FR over RFC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1365.

Abstract 1359: Design, synthesis and evaluation of fluorinated antifolates for improved selectivity and potency against tumor cells

Abstract Folates are important single carbon transferring cofactors required for the biosynthesis of purine and pyrimidine nitrogen bases and DNA. Antifolates inhibit synthesis of these nitrogenous bases and function as antineopastic agents. The use of currently marketed antifolates such as methotrexate (MTX and pemetrexed (PMX) is limited by dose-limiting toxicity due to non-selective transport by the ubiquitously expressed reduced folate carrier (RFC). We previously reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine classical antifolates that are selectively transported by folate receptors (FR) and/or by the proton-coupled folate transporter (PCFT) over RFC and inhibit FR/PCFT expressing tumor cells (KB and IGROV1) at sub-nanomolar IC50 values. These antifolates are comparatively more potent and circumvent the important limitation of currently marketed clinical antifolates that lack selective uptake into tumors. As an extension of the SAR, we now report on the role of fluorine atom(s) in improving selectivity as well potency. Fluorine was incorporated into our 6-substituted pyrrolo[2,3-d]pyrimidine antifolates to improve pharmacodynamics by improving binding affinity to target proteins via a number of well documented electrostatic and hydrophobic interactions of fluorine. In addition, fluorines were strategically placed on the side chain of our targeted antifolates for improved potency and selectivity through induced conformational restriction due to a plausible intramolecular fluorine-hydrogen bonding interaction. The fluorinated analogs had IC50s in the subnanomolar range (IC50 = 0.33 to 0.29 nM) for KB tumor cells and nanomolar (IC50 = 2.77 to 3.47 nM) for IGROV-1 cells. In addition, these analogs showed 600- to 1000-fold selectivity for FRα expressing cells over RFC. Protection studies suggest that glycinamide ribonucleotide formyl transferase (GARFTase) is the intracellular targets. In vivo activity was established toward the SKOV3 tumor xenograft model. These preclinical studies suggest that these fluorinated analogs are candidates for possible clinical trial as antitumor agents. Citation Format: Aleem Gangjee, Manasa P. Ravindra, Mike R. Wilson, Adrianne W. Povrik, Lisa P. Hou, Carrie O’Connor, Zhanjun Hou, Larry H. Matherly. Design, synthesis and evaluation of fluorinated antifolates for improved selectivity and potency against tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1359.

Abstract 2358: KHK2805, a novel ADCC- and CDC-enhanced anti-FOLR1 antibody with AccretaMab® technology, shows a potent anti-tumor activity in combination with pemetrexed

Abstract Introduction: Folate receptor 1 (FOLR1, FR alpha) is a folate transporter which is expressed in many cancers including ovarian cancer (OvC) and non-small cell lung cancer (NSCLC), and which is an attractive target for cancer therapy, is currently the subject of ongoing studies. We established KHK2805, a novel anti-FOLR1 monoclonal antibody with AccretaMab® technology to enhance both the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities. It was demonstrated that KHK2805 exhibits markedly high ADCC and CDC activity levels in clinical samples from ovarian cancer patients, while a tolerable safety profile has been observed in preclinical models using cynomolgus monkeys (100 mg/kg weekly for 4 weeks, intravenously). A greater understanding of the biology of FOLR1 is important for providing a novel therapeutic option for KHK2805 for patients with cancer. Pemetrexed (PEM), a second-generation anti-folate which inhibits thymidylate synthase, glycinamide ribonucleotide transformylase and dihydrofolate reductase, is used as a standard therapy for patients with cancers such as NSCLC. It is not fully understood how PEM treatment affects the expression of FOLR1 on cancer cells. We therefore examined the level of FOLR1 expression in cancer cells after PEM treatment, and the anti-tumor activity of KHK2805 in combination with PEM. Materials and Methods: The FOLR1 expression levels after PEM treatment were examined in OvC, NSCLC, and endometrial cancer cells by flow cytometry. The ADCC activity of KHK2805 against PEM-treated cells was evaluated. The anti-tumor activity of KHK2805 in combination with PEM was investigated in SCID mice. Results: Flow cytometry showed that PEM treatment increased the FOLR1 expression of various cancers such as OvC (IGROV1, SKOV3, MCAS), NSCLC (NCI-H1437, NCI-H2228), and endometrial cancer (MESSA, HEC1A, HEC1B) cells. In addition, PEM treatment enhanced the ADCC activity of KHK2805 against MCAS, NCI-H1437, and NCI-H2228, in comparison to cells that did not receive PEM treatment. Furthermore, the anti-tumor activity of KHK2805 in SCID mice bearing subcutaneous MCAS tumors was enhanced by PEM treatment. Conclusions: PEM induced further FOLR1 expression in OvC, NSCLC, and endometrial cancer, resulting in the enhancement of the ADCC activity of KHK2805. The use of KHK2805 with PEM might therefore be a potent therapeutic option. Citation Format: Munetoshi Ando, Keiko Nagata, Toshihiko Ishii, Ryuichiro Nakai, Takeshi Takahashi. KHK2805, a novel ADCC- and CDC-enhanced anti-FOLR1 antibody with AccretaMab® technology, shows a potent anti-tumor activity in combination with pemetrexed. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2358.

Abstract 2263: Prognostic significance of REV3like and TYMS gene expression in blood samples from non-small cell lung patients treated with platinum-based chemotherapy plus pemetrexed

Abstract BACKGROUND: Pemetrexed is an effective antineoplastic agent as monotherapy (second line and maintenance setting) or in platinum-based combination regimes (first-line). Pemetrexed inhibits three key enzymes in the folate metabolic pathway: thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT). As a consequence, pemetrexed interferes with the synthesis of both pyrimidine and purine, thereby effectively inhibiting both DNA and RNA synthesis. The aim of this study was to investigate the impact of the PBMC (peripheral blood mononuclear cells) expression levels of AEG1, BRCA1, REV3like, ant TYMS mRNA in the clinical outcome in NSCLC patients treated with Pemetrexed-based therapy. MATERIAL AND METHODS: This is a prospective, observational cohort study, of consecutively selected patients. We included patients with non-squamous NSCLC, stage IV, treated with platinum-based chemotherapy plus Pemetrexed. Blood samples from each patient were collected at the beginning of treatment. Quantitative reverse transcription (qRT)-PCR was performed with the use of the TaqMan gene expression assay (Applied Biosystems) according to the manufacturer's instructions. Amplifications were carried out on the Applied Biosystem 7500 RT-PCR system, and relative gene expression values were calculated by the ΔΔCt method (Sequence Detection System 2.0.5). Results from analysis, done in triplicate, are expressed relative to expression levels of GAPDH. RESULTS: We here present preliminary results of 16 patients included in the study: median age was 63.4 (range 52-82); 12 were males. Histological subtypes were 13 adenocarcinomas, and 3 large cell carcinomas. Pemetrexed was combined with Carboplatin in 9 patients and Cisplatin in 7 patients. Best response obtained was partial response in 7 patients, stable disease in 3 patients, and progression in 5 patients. We found significant differences in the REV3like expression levels between patients with PR&SD and patients with PD (0.69 ± 0.14 vs 0.44 ± 0.07; P = 0.003). In addition there were statistically significant differences in disease free survival (3 ± 0.74 vs 13 ± 1.89, P = 0.035; 12 ± 2.32 vs 3 ± 2.19, P = 0.003, months), and overall survival (10 ± 5.21 vs not reached, P = 0.026; not reached vs 12 ± 2.38, P = 0.007, months) between patients with mRNA levels of REV3like and TYMS under mean value of the cohort and patients with levels over mean value of the our cohort. CONCLUSIONS: This study supports the value of the PBMC expression profiling of the REV3like and TYMS genes in the prognosis of NSCLC treated with platinum-based chemotherapy plus Pemetrexed. Citation Format: Maria Teresa Agullo-Ortuño, C.Vanessa Díaz-García, José A. López-Martín, Elena Prieto-García, Santiago Ponce, Jon Zugazagoitia, Lara Iglesias-Docampo, Ana B. Enguita, Luis Paz-Ares, Juan A. Nuñez-Sobrino. Prognostic significance of REV3like and TYMS gene expression in blood samples from non-small cell lung patients treated with platinum-based chemotherapy plus pemetrexed. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2263.

Targeting Nonsquamous Nonsmall Cell Lung Cancer via the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]Pyrimidine Thienoyl Antifolates.

Pemetrexed (PMX) is a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate used for therapy of nonsquamous nonsmall cell lung cancer (NS-NSCLC). PMX is transported by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). Unlike RFC, PCFT is active at acidic pH levels characterizing the tumor microenvironment. By real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, PCFT transcripts and proteins were detected in primary NS-NSCLC specimens. In six NS-NSCLC cell lines (A549, H1437, H460, H1299, H1650, and H2030), PCFT transcripts and proteins were detected by real-time RT-PCR and western blots, respectively. 6-Substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates related to PMX [compound 1 (C1) and compound 2 (C2), respectively] are selective substrates for PCFT over RFC. In the NS-NSCLC cell lines, both [(3)H]PMX and [(3)H]C2 were transported by PCFT. C1 and C2 inhibited proliferation of the NS-NSCLC cell lines; A549, H460, and H2030 cells were more sensitive to C1 than to PMX. C1 and C2 inhibited glycinamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis. When treated at pH 6.8, which favors PCFT uptake, C1 and C2 inhibited clonogenicity of H460 cells greater than PMX; PMX inhibited clonogenicity more than C1 or C2 at pH 7.2, which favors RFC transport over PCFT. Knockdown of PCFT in H460 cells resulted in decreased [(3)H]PMX and [(3)H]C2 transport and decreased growth inhibition by C1 and C2, and to a lesser extent by PMX. In vivo efficacy of C1 was seen toward H460 tumor xenografts in severe-combined immunodeficient mice. Our results suggest that 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates offer significant promise for treating NS-NSCLC by selective uptake by PCFT.

6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors.

2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or β (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and β over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.

Abstract 4481: Tumor-targeting with novel 6-substituted thienoyl[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis

Abstract Recent interest has focused on folate receptor (FR) α in delivering folate-based therapeutics to solid tumors, as FRα is expressed in human tumors, notably ovarian and non-small cell lung cancers. While FRα is present in normal epithelial tissues, this occurs at apical membranes where it is inaccessible to the circulation. Selectivity of FR-targeted agents would be enhanced if these were poor substrates for the ubiquitous reduced folate carrier (RFC). We previously described 6-substituted thieno[2,3-d]pyrimidine benzoyl antifolates with varying bridge lengths (n = 2-8 carbons) as potent and selective inhibitors of tumor cells that express FRs. The most potent compound (AGF50) included a 4-carbon bridge and showed FRα selectivity and inhibition of de novo purine biosynthesis. To investigate the role of the side-chain aromatic ring in transport and pathway inhibition, we synthesized AGF50 analogs with a variety of aryl side-chain modifications (AGF102, AGF103, AGF55, AGF65, and AGF132). The various 6-substituted thienoyl[2,3-d]pyrimidine antifolates were tested in proliferation assays with isogenic Chinese hamster ovary (CHO) sublines expressing RFC, proton-coupled folate transporter (PCFT), or FRα, and with KB tumor cells. The analogs were inert toward RFC- and PCFT-expressing cells. Toward FRα-expressing CHO and KB cells, inhibitory potencies were in the order, AGF102 = AGF132 = AGF103 &amp;gt; AGF50 &amp;gt; AGF64 &amp;gt;&amp;gt; AGF55, with IC50s ranging from ∼ 1 nM to 182 nM. Drug effects were abolished with excess folic acid, confirming FR uptake. Inhibition of cell proliferation was reflected in binding affinities to FRα, measured by competition with [3H]folic acid. While PCFT-targeted activity was nominal, all drugs still bound to PCFT, as reflected in inhibition of [3H]methotrexate uptake at pH 5.5 and 6.8. By protection experiments with thymidine or adenosine, de novo purine biosynthesis was confirmed as the principal targeted pathway for the active analogs. While protection by 5-aminoimidazole-4-carboxamide (AICA) was complete with AGF64 (suggesting glycinamide ribonucleotide formyltransferase (GARFTase) as the primary target), for AGF50, AGF102, AGF103 and AGF132, protection was incomplete, implying a dual targeting of GARFTase and AICA ribonucleotide formyltransferase (AICARFTase). Our studies establish an important structure-activity relationship for FRα-targeted antifolates, involving modifications of the side-chain aromatic group that are well tolerated for FRα binding and transport. 6-Substituted thienoyl[2,3-d]pyrimidine analogs were all inhibitors of de novo purine biosynthesis, although relative targeting of GARFTase versus AICARFTase varied with the nature of the aromatic side-chain. Citation Format: Larry H. Matherly, Xin Zhang, Adrianne Wallace, Zhanjun Hou, Christina George, Xilin Zhou, Aleem Gangjee. Tumor-targeting with novel 6-substituted thienoyl[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2015-4481

Abstract 4488: Folate receptor targeted therapy using small molecule drug conjugates constructed with high affinity antifolate ligands

Abstract Folate receptor (FR)-targeted small molecule drug conjugates (SMDC) have shown promising clinical results. To date, all related SMDCs have been constructed with folic acid. Whereas folic acid displays very high affinity for the FR (kd ∼ 0.1 nM), antifolates with similar high binding affinity and specificity have also been reported. This knowledge prompted our investigation towards substituting the folic acid targeting moiety in our SMDCs with high affinity antifolates, such as CB3717 (a thymidylate synthase inhibitor), and the glycinamide ribonucleotide formyltransferase (GARFTase) inhibitors, AG94 and AG147, thereby testing the hypothesis that greater antitumor activity should result from the combination of inhibitors. Using mice bearing FR-positive human tumor xenografts, we observed curative activity with a CB3717-based vinca alkaloid SMDC under conditions where no cures resulted with vintafolide (EC145) treatment. Similarly, tubulysin SMDCs constructed with AG94 and AG147 were found to produce curative activity using a dosing regimen where a folic acid-tubulysin conjugate did not. Of note, treatment of mice with simple co-mixtures of the antifolates and untargeted drugs was found to be ineffective, thereby confirming the need for linking the two moieties together to produce maximal activity. Taken together, these studies demonstrate that high affinity antifolates can be used in an SMDC format serving as both a high affinity targeting ligand and as a second drug having different, and perhaps a complementary mechanism of action to yield greater antitumor activity. Citation Format: Christopher P. Leamon, Joseph A. Reddy, Iontcho R. Vlahov, Fei You, Hanna F. Klein, Paul J. Kleindl, Melissa Nelson, Marilynn Vetzel, Patrick J. Klein, Larry H. Matherly, Aleem Gangjee. Folate receptor targeted therapy using small molecule drug conjugates constructed with high affinity antifolate ligands. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4488. doi:10.1158/1538-7445.AM2015-4488

Abstract 4393: Design and synthesis of novel conjugates for targeting the folate receptor: Exploiting cytotoxic GARFTase inhibitors for delivering additional chemotherapeutic payloads to cancer cells

Abstract Background: Antifolates targeting glycinamide ribonucleotide formyltransferase (GARFTase) disrupt cell division (mitosis) by inhibiting the de novo purine biosynthesis pathway. Recently, novel GARFTase inhibitors, exhibiting high folate receptor (FR) binding affinity and low affinity for the reduced folate carrier (RFC), have been explored as chemotherapeutic agents. Theoretically, these GARFTase inhibitors could also be used as FR-targeting ligands for delivering additional payloads to tumor types over-expressing the FR. In this poster, we will present our practical approach to explore the dual role of these new GARFTase inhibitors as cytotoxic agents and as targeting ligands for delivering tubulysins. Tubulysins are natural products isolated from myxobacterial species and are highly potent mitotic poisons that inhibit tubulin polymerization. Methods: We developed a general method for preparing dual drug/ligand conjugates possessing two disulfide-based self-immolative linker systems. In our synthetic protocol we relied on consecutive introduction of two heterosubstituted disulfide bonds. A GARFTase inhibitor was first connected to a hydrophilic spacer unit through the first disulfide-based linker system. The obtained conjugate contains also an S-9-fluorenylmethyl (Fm)-protected thiol in its molecular architecture. Treatment with a strong organic base resulted in spontaneous removal of the Fm moiety. The deprotected thiol reacted in situ with a propreatary thiophilic tubulysin derivative, present in the reaction mixture, and a second self-immolative disulfide-based linker system was formed. Results: Novel conjugates of GARFTase inhibitor with tubulysins were prepared using newly designed synthetic strategies. In a high-yielding chemoselective reaction we introduced a second disulfide bond into a molecular framework without destroying the pre-existing disulfide linker system. The versatility of this synthetic protocol was demonstrated by synthesizing several related drug conjugates. Conclusion: In the poster to be presented, we will disclose, for the first time, the structure and synthesis of novel conjugates of GARFTase inhibitors with tubulysins. The target compounds contain two self-immolative disulfide linker systems. The observed excellent in vivo activity in tumor xenografts demonstrates the dual role of GARFTase inhibitors as therapeutic agents and as FR-targeting ligands for simultaneously delivering two highly potent payloads. Citation Format: Iontcho R. Vlahov, Fei You, Hanna F. Klein, Paul J. Kleindl, Melissa Nelson, Marilynn Vetzel, Joseph A. Reddy, Christopher P. Leamon, Larry H. Matherly, Aleem Gangjee. Design and synthesis of novel conjugates for targeting the folate receptor: Exploiting cytotoxic GARFTase inhibitors for delivering additional chemotherapeutic payloads to cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4393. doi:10.1158/1538-7445.AM2015-4393

Abstract 5403: Targeted therapy of non-small cell lung cancer (NSCLC) by novel 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective transport by the proton-coupled folate transporter (PCFT)

Abstract Pemetrexed (PMX) is a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate used for first-line and maintenance therapy of NSCLC. Whereas correlations were reported between clinical responses in NSCLC and levels of folate receptor (FR) α, FRα is not a pharmacologically relevant PMX transporter. Rather, PMX is primarily transported by the reduced folate carrier (RFC) and PCFT. RFC is ubiquitously expressed, whereas PCFT shows more limited expression in normal tissues, although both are broadly expressed in tumor cells. Unlike RFC, PCFT is active at acidic pH, with significant levels at pH 6.8. Using patient cDNAs from normal lung (n = 8) and NSCLC samples (n = 37), we measured transcripts for RFC and PCFT, and for intracellular targets for PMX, including thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFTase), by real-time RT-PCR (qPCR). PCFT was detected at similar levels in normal lung and NSCLC specimens, while RFC was decreased in tumors compared to normal lung. Expression of TS and GARFTase were increased in NSCLC samples, compared to normal tissue. In NSCLC cell lines A549, CRL5872, H460, H1650 and H2030, PCFT and RFC, but not FRα, were detected by qPCR. We synthesized novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers related to PMX [AGF94 (2′,5′) and AGF154 (2′,4′)] as potential antitumor agents. Based on patterns of inhibition of proliferation of engineered Chinese hamster ovary cells expressing human RFC or PCFT, AGF94 and AGF154, unlike PMX, are not transported by RFC but are excellent substrates for PCFT. AGF94 and AGF154 showed potencies comparable to PMX toward all the NSCLC cell lines. We found that [3H]PMX and [3H]AGF154 were transported by PCFT into the NSCLC cell lines at pH 5.5 and pH 6.8 and uptake could be blocked by non-radioactive AGF94. AGF154 accumulation over sustained exposure at pH 5.5 was greater than that of PMX, whereas accumulations of AGF154 and PMX were nearly identical at pH 6.8. In H460 cells treated with PMX, thymidine (10 μM) partially protected cells from growth inhibition, whereas neither adenosine (10 μM) nor 5-aminoimidazole-4-carboxamide (AICA) (320 μM) were protective. In cells treated with AGF94 or AGF154, adenosine and AICA, but not thymidine, were completely protective, consistent with inhibition of de novo purine biosynthesis at GARFTase. Potent GARFTase inhibition by AGF94 and AGF154 was confirmed by an in situ assay which measures incorporation of [14C]glycine into formyl GAR (FGAR), the GARFTase product. Our results suggest that novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating NSCLC, reflecting their selective membrane transport by PCFT over RFC and potent inhibition of de novo purine biosynthesis. Citation Format: Mike R. Wilson, Zhanjun Hou, Lei Wang, Si Wang, Manohar Ratnam, Aleem Gangjee, Larry Matherly. Targeted therapy of non-small cell lung cancer (NSCLC) by novel 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective transport by the proton-coupled folate transporter (PCFT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5403. doi:10.1158/1538-7445.AM2015-5403

Novel 5-substituted pyrrolo[2,3-d]pyrimidines as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and as potential antitumor agents.

A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6-11 with varying chain lengths (n = 1-6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds 6-11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8-10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure-activity relationship for transport and dual target inhibition.

Synthesis and antitumor activity of a novel series of 6-substituted pyrrolo[2,3-d]pyrimidines as potential nonclassical antifolates targeting both thymidylate and purine nucleotide biosynthesis

A novel series of 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines were designed and synthesized as potential nonclassical antifolates targeting both thymidylate and purine nucleotide biosynthesis. Condensation of 2,4-diamino-6-hydroxypyrimidine with ethyl-4-chloroacetoacetate and subsequent hydrolysis afforded the key intermediate, 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid. Coupling with various amino acid methyl esters followed by saponification and condensation with 3-(aminomethyl)pyridine provided target compounds 1–9. The new compounds exhibited micromolar to submicromolar antiproliferative potencies against a panel of tumor cell lines including KB, A549 and HepG2. Growth inhibition of compound 2 toward KB cells resulted in cytotoxicity and G1/G2-phase accumulation, and was partially protected by excess thymidine and adenosine, but was completely reversed in the combination of thymidine and adenosine, indicating both thymidylate and de novo purine nucleotide synthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both glycinamide ribonucleotide formyltransferase (GARFTase) and AICA ribonucleotide formyltransferase (AICARFTase). The results of the docking studies show that 2 could bind and inhibit both thymidylate synthase (TS) and the two folate-dependent purine biosynthetic enzymes (GARFTase and AICARFTase), which is consistent with the results of in vitro metabolic assays. Our studies establish that compound 2 is an excellent lead analog as a multitargeted antifolate for further structure optimization.

Analysis of Antifolate Drugs with Disease Tissue Specificity

Antifolates, analogues of the essential vitamin folic acid, are used in the clinic to treat cancers and inflammatory diseases. Antifolates are primarily transported into cells via the endogenously expressed reduced folate carrier (RFC). Conversely, our collaborators in Aleem Gangjee's group at Duquesne University have synthesized antifolates (AG antifolates) that transport poorly by the RFC, but are efficiently transported by the folate receptor (hFR). The GPI-anchored hFR is lowly expressed on the apical surface in a subset of normal epithelial lineages, but is highly expressed in many cancers of epithelial origin and on activated macrophages in inflammatory disease. Therefore, AG antifolate molecules have specificity for transport into disease cells over healthy cells. These newly developed AG antifolates cause cell death via inhibition of an enzyme involved in de novo purine synthesis, glycinamide ribonucleotide (GAR) transformylase.We analyzed a series of AG antifolates using biophysical and biochemical techniques to understand both the specificity for transport by the folate receptor as well as the inhibition of the GAR transformylase in order to drive informed, hypothesis-based drug design. Our data, including pH-dependent binding profiles, enzyme inhibition data, and crystallographic models of protein in complex with AG molecules will be presented in the context of drug design and development.

In silico analysis of glycinamide ribonucleotide transformylase inhibition by PY873, PY899 and DIA.

In humans, purine de novo synthesis pathway consists of multi-functional enzymes. Nucleotide metabolism enzymes are potential drug targets for treating cancer and autoimmune diseases. Glycinamide ribonucleotide transformylase (GART) is one of the most important trifunctional enzymes involved in purine synthesis. Previous studies have demonstrated the role of folate inhibitors against tumor activity. In this present study, three components of GART enzyme were targeted as receptor dataset and in silico analysis was carried out with folate ligand dataset. To accomplish the task, Autodock 4.2 was used for determining the docking compatibilities of ligand and receptor dataset. Taken together, it has been suggested that folate ligands could be potentially used as inhibitors of GART.