8 research outputs found
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 pyrroloÂ[2,3-<i>d</i>]Âpyrimidine 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 <i>in vitro</i> toward FR- and PCFT-expressing tumor cells and <i>in vivo</i> 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 <i>de novo</i> synthesis of purine
nucleotides. This study used purified human GARFTase (formyltransferase
domain) to assess <i>in vitro</i> inhibition by eight novel
thieno- and pyrroloÂ[2,3-<i>d</i>]Âpyrimidine antifolates.
Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and
AGF147) inhibited GARFTase with <i>K</i><sub>i</sub> 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
6‑Substituted Pyrrolo[2,3‑<i>d</i>]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-pyrroloÂ[2,3-<i>d</i>]Âpyrimidine
antifolate thiophene regioisomers of AGF94 (<b>4</b>) with a
thienoyl side chain and three-carbon bridge lengths [AGF150 (<b>5</b>) and AGF154 (<b>7</b>)] were synthesized as potential
antitumor agents. These analogues inhibited proliferation of Chinese
hamster ovary (CHO) sublines expressing folate receptors (FRs) α
or β (IC<sub>50</sub>s < 1 nM) or the proton-coupled folate
transporter (PCFT) (IC<sub>50</sub> < 7 nM). Compounds <b>5</b> and <b>7</b> inhibited KB, IGROV1, and SKOV3 human tumor cells
at subnanomolar concentrations, reflecting both FRα and PCFT
uptake. AGF152 (<b>6</b>) and AGF163 (<b>8</b>), 2,4-diamino-5-substituted-furoÂ[2,3-<i>d</i>]Âpyrimidine 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 <b>5</b> and <b>7</b> were
reported. In severe combined immunodeficient mice bearing SKOV3 tumors, <b>7</b> 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
Tumor Targeting with Novel 6‑Substituted Pyrrolo [2,3‑<i>d</i>] 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 <b>1</b> by N (<b>4</b>), O (<b>8</b>), or S (<b>9</b>), or with N-substituted formyl (<b>5</b>), acetyl (<b>6</b>), or trifluoroacetyl (<b>7</b>) 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 <b>4</b>–<b>9</b> over the CH<sub>2</sub> analogue <b>1</b>. Compounds <b>4</b>–<b>9</b> inhibited de novo purine biosynthesis and glycinamide ribonucleotide
formyltransferase (GARFTase). X-ray crystal structures for <b>4</b> with FRα and GARFTase showed that the bound conformations
of <b>4</b> required flexibility for attachment to both FRα
and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, <b>4</b> was highly efficacious. Our results establish that heteroatom
substitutions in the 3-atom bridge region of 6-substituted pyrroloÂ[2,3-<i>d</i>]Âpyrimidines related to <b>1</b> provide targeted
antifolates that warrant further evaluation as anticancer agents
Tumor Targeting with Novel 6‑Substituted Pyrrolo [2,3‑<i>d</i>] 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 <b>1</b> by N (<b>4</b>), O (<b>8</b>), or S (<b>9</b>), or with N-substituted formyl (<b>5</b>), acetyl (<b>6</b>), or trifluoroacetyl (<b>7</b>) 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 <b>4</b>–<b>9</b> over the CH<sub>2</sub> analogue <b>1</b>. Compounds <b>4</b>–<b>9</b> inhibited de novo purine biosynthesis and glycinamide ribonucleotide
formyltransferase (GARFTase). X-ray crystal structures for <b>4</b> with FRα and GARFTase showed that the bound conformations
of <b>4</b> required flexibility for attachment to both FRα
and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, <b>4</b> was highly efficacious. Our results establish that heteroatom
substitutions in the 3-atom bridge region of 6-substituted pyrroloÂ[2,3-<i>d</i>]Âpyrimidines related to <b>1</b> provide targeted
antifolates that warrant further evaluation as anticancer agents