Selective antifolates for chemically labeling proteins in mammalian cells.

Pairs of small molecule ligands and receptor proteins that associate to the exclusion of all other biological components (i.e., orthogonal interaction pairs) are powerful tools for analyzing, modulating or otherwise perturbing protein function in living cells and organisms.[1] Trimethoprim (TMP, 1, Scheme 1) is a selective inhibitor of bacterial forms of dihydrofolate reductase (DHFR). TMP strongly inhibits Escherischia coli DHFR (eDHFR) (KI = ca. 1 nM), yet weakly inhibits mammalian forms of the enzyme (KI > 1 uM).[2] The orthogonality of the TMP-eDHFR interaction to eukaryotic cells has been exploited to develop a general method for labeling eDHFR fusion proteins in wild-type mammalian cells with cell-permeable TMP-fluorophore conjugates.[3, 4] A heterodimeric conjugate of TMP with synthetic ligation factor (SLF) has been developed as a chemical inducer of dimerization (CID) and used to activate transcription in a yeast three-hybrid assay as well as modulate glycosylation in mammalian cells.[5, 6] Scheme 1 Schematic representation of antifolates under study. The common numbering scheme for 5-substituted benzyl pyrimidines is given for 1 (trimethoprim, TMP). Compounds 2b and 3b are heterodimeric conjugates of 2 and 3 to acetylated 5(6)-carboxy fluorescein ... We sought to determine whether selective DHFR inhibitors (antifolates) besides TMP could be utilized for biotechnological applications in mammalian cells. DHFR has been the focus of intense drug discovery efforts for several decades,[7] and in recent years there has been considerable focus put on developing selective inhibitors against DHFR’s from pathogenic organisms such as Pneumocystis carinii, Plasmodium falciparum, and Staphylococcus aureus.[8] Hundreds of TMP analogs (substituted 5-benzyl pyrimidines) have been prepared in efforts to analyze binding modes and to develop improved therapies against resistant organisms.[9-16] We chose to focus our efforts on compounds 2 and 3 (Scheme 1, Table 1) which exhibit ca. nanomolar inhibitory activities against P. falicparum DHFR (pfDHFR) and P. carinii DHFR (pcDHFR), respectively while exhibiting minimal or no activity against mammalian DHFR’s.[9, 15, 16] Table 1 DHFR enzyme inhibition (KI, nM) for selected (Scheme 1).[a] Conjugates of 2 and 3 to a hydrophobic, acetylated fluorescein were prepared (Scheme 1, 2b, 3b). The conjugation strategy was informed by previously reported structural analyses that revealed the binding modes of each compound to their respective targets. Yuthavong and co-workers used semiempirical methods to model the binding of a close analog of 2 to the known structure of pfDHFR.[15] The 4′-trimethoxy-benzyl substituent was modeled to form a π--π stacking interaction with Phe116 of the wild-type pfDHFR. The para-methoxy is in van der waals contact with cys50 near the entrance to the binding pocket, and we surmised that alkyl linkage at this position would be the least disruptive to binding. The structure of pcDHFR in complex with 3 and NADPH was reported by Cody and co-workers.[17] The 5′-(5-carboxy-1-pentynyl side chain interacts with Arg75, contributing strongly to the high affinity. The 4′-methoxy group interacts with the hydrophobic side chains of Leu25 and Ile33 near the entrance to the binding pocket, and we chose to conjugate at this position. We first sought to validate our conjugation strategy by directly characterizing the ability of the non-fluorescent analogs 2a and 3a to inhibit Toxoplasma gondii DHFR (tgDHFR), a protozoan DHFR used as a model for biochemical characterization of pfDHFR inhibitors[18] and pcDHFR, respectively. Using an assay based on measurement of the change in absorbance at 340 nm when dihydrofolate is reduced to tetrahydrofolate in the presence of NADPH, inhibition constants for each analog were determined (Table 2).[12] Methotrexate (MTX) and TMP were included in the assay as controls. 2a potently inhibited tgDHFR (IC50 = 0.032 μM), while poorly inhibiting rat liver DHFR (IC50 = 6 μM). 3a also exhibited excellent potency and selectivity, strongly inhibiting pcDHFR (IC50 = 0.025 μM), while showing ca. 350-fold lower activity against mammalian DHFR. The inhibition data suggested that heterodimers of 2 and 3 linked at the indicated positions would retain effective potency against their putative targets, while maintaining sufficient selectivity for applications in mammalian cell lines. Table 2 DHFR enzyme inhibition (IC50, μM) of substituted inhibitor analogs. The linker-substituted analogs, 2a and 3a were further characterized by determining their ability to inhibit E. coli growth. The minimum inhibitory concentrations (MIC) of TMP, 2a and 3a were ca. 0.2 μM, 20 μM and >100 μM, respectively. In the case of 2a, we were interested in knowing its affinity for eDHFR relative to that of TMP. Compound 2 is ca. 30-fold and ca. 100-fold more potent than TMP against wild-type pfDHFR and the TMP-resistant double mutant (C59R, S108N), respectively (Table 1). That an analog of 2 is substantially less potent against eDHFR than TMP suggests that 2b or other heterodimers of 2 could be used simultaneously with TMP for assays in mammalian cells. In the case of 3a, we expected that it would strongly inhibit eDHFR, as a 5′-(5-carboxyalkyloxy) TMP analog was shown to be ca. 50-fold more potent against eDHFR than TMP itself.[10] The fact that 3a does not inhibit E. coli growth up to the concentrations tested suggests the molecule cannot cross the cell membrane. This is not entirely surprising as the carboxyl group of 3a is likely deprotonated, and localized charges typically lower the membrane permeability of small molecules.[3] In order to be useful as a research tool for chemical biology applications, orthogonal ligand-receptor pairs must meet, at minimum, three criteria: 1) The protein acceptor must be soluble and expressible in mammalian cells; 2) the ligand and its conjugates must be cell-permeable; and 3) the ligand must diffuse freely within cells and not partition to particular organelles or sub-cellular domains. We used epi-fluorescence microscopy to determine the cell permeability and sub-cellular distribution of 2b and its selective binding to a soluble, TMP-resistant mutant (K27E, C59R, S108N) of the DHFR domain of P. falciparum DHFR-thymidylate synthetase.[19] An expression vector that targeted pfDHFR to the nucleus was prepared. Targeting was achieved by encoding pfDHFR soluble domain with a N-terminal fusion of three copies of the canonical simian virus 40 large T-antigen nuclear localization sequence (DPKKKRKV).[4] We then transiently transfected NIH 3T3 fibroblast cells with the vector. Approximately 24 hours after transfection, the cells were incubated with low (500 nM) concentrations of 2b and imaged microscopically (Fig. 1). Figure 1 Selective chemical labelling of sub-cellularly targeted pfDHFR in living mammalian cells. Bright field (left) and fluorescence (right) micrographs show adherent NIH3T3 fibroblast cells transiently expressing nucleus-localized pfDHFR soluble domain (K27E, ... Diffuse fluorescence was observed in all cells incubated with 2b, and some of the cells exhibited distinct nuclear fluorescence with more brightly fluorescent nucleoli, characteristic of the nucleus-targeting sequence.[4] The diffuse fluorescence indicates that 2b readily enters cells where the fluorescein moiety is hydrolyzed by intracellular esterases, yielding the fluorescent fluorescein dianion. We attribute nuclear staining to the specific binding of 2b to nucleus-targeted pfDHFR soluble domain. We performed analogous imaging experiments with 3b and cells transfected with a vector encoding pcDHFR fused to the N-terminal nucleus localization sequence. However, we did not observe any intracellular fluorescence or nuclear staining with 3b, providing further evidence that analogs of 3 cannot passively diffuse into cells due to the presence of the 5′-(5-carboxy-1-pentynyl) moiety. Substituted analogs of the established antifolates 2 and 3 retain similar potency and selectivity of the parent compounds when assessed in an inhibition assay. Compound 2a, a heterodimeric conjugate of 2 to a hydrophobic, acetylated fluorescein passively diffused into mammalian cells and selectively labeled a recombinantly expressed fusion of the soluble domain of pfDHFR. These results demonstrate that the considerable efforts devoted to finding selective inhibitors of pathogenic DHFR′s can be leveraged to identify and develop new tools for chemical biology. Further studies are underway to comprehensively characterize the interaction between substituted analogs of 2 and soluble mutants of pfDHFR, and to further exploit this interaction for chemically labeling proteins in living mammalian cells.