Additional file 2: Table S1. of New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag

List of proteins examined for expression and solubility. IDP# Identification protein number relative to CSGID targets (http://csgid.org/); Locus ID for NCBI; Expression: expressed proteins are highlighted in yellow (1-Low expression, 2-Medium expression, 3-High expression). Non-expressed proteins are highlighted in white; Solubility: soluble proteins are highlighted in yellow (1-Low solubility, 2-Medium solubility, 3-High solubility). Non-soluble proteins are highlighted in white (0-undetectable solubility); Purified: proteins purified by single step on Ni-NTA resin and desalting. Purified proteins are highlighted in yellow (1-Low solubility, 2-Medium, 3-High). Non-purified proteins are highlighted in white (0-undetectable); InsP6 cleavage: proteins cleaved by InsP6 CPD autoprocessing activation and purified by “negative” Ni-NTA purification step. Soluble proteins after the cleavage are highlighted in yellow (1-Low solubility, 2-Medium, 3-High). Proteins lost after InsP6-cleavage/Ni-NTA purification are highlighted in white (0-undetectable). (XLSX 16 kb

Additional file 1: Figure S1. of New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag

Sixteen representative samples of proteins expressed from vector pCPD show diversity of expression, solubility and recovery after InsP6 induced autoprocessing. Numbers at top indicate CSGID IDP designation as listed in Additional file 2. Lanes for each sample represent Whole cell lysate (L), Soluble fraction from lysate (S), Purified fusion protein (P), and purified protein after CPD autoprocessing (P’). Figure was assembled from multiple SDS-polyacrylamide gels stained with Coomassie Blue. The scores at each stage and the total amount of purified protein after autoprocessing are shown below the gel figure. The amounts of total purified proteins from 1 mL cultures were determined from absorbance at 260 nm using a NanoDrop. (TIFF 830 kb

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 CH₂ 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 pyrrolo­[2,3-<i>d</i>]­pyrimidines 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 CH₂ 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 pyrrolo­[2,3-<i>d</i>]­pyrimidines related to <b>1</b> provide targeted antifolates that warrant further evaluation as anticancer agents