Marcus J. C. Long

Using supramolecular hydrogels to discover the interactions between proteins and molecular nanofibers of small molecules

Here we report the first example of the use of supramolecular hydrogels to discover the protein targets of aggregates of small molecules.

Inhibitor mediated protein degradation.

The discovery of drugs that cause the degradation of their target proteins has been largely serendipitous. Here we report that the tert-butyl carbamate-protected arginine (Boc(3)Arg) moiety provides a general strategy for the design of degradation-inducing inhibitors. The covalent inactivators ethacrynic acid and thiobenzofurazan cause the specific degradation of glutathione-S-transferase when linked to Boc(3)Arg. Similarly, the degradation of dihydrofolate reductase is induced when cells are treated with the noncovalent inhibitor trimethoprim linked to Boc(3)Arg. Degradation is rapid and robust, with 30%-80% of these abundant target proteins consumed within 1.3-5 hr. The proteasome is required for Boc(3)Arg-mediated degradation, but ATP is not necessary and the ubiquitin pathways do not appear to be involved. These results suggest that the Boc(3)Arg moiety may provide a general strategy to construct inhibitors that induce targeted protein degradation.

Glutathione (GSH)-decorated magnetic nanoparticles for binding glutathione-S-transferase (GST) fusion protein and manipulating live cells

Iron oxide-based magnetic nanoparticles (MNP) surface-decorated with glutathione (GSH) via a dopamine anchor bind to human α1-glutathione S-transferase (GST) with high affinity and specificity and are able to separate GST fusion proteins from cell lysates. Both the purified GST and the protein of interest (POI) preserve their innate properties. The conjugate of MNP and the GST fusion protein also enables magnetic manipulation of cells.

Mechanistic Studies of Semicarbazone Triapine Targeting Human Ribonucleotide Reductase in Vitro and in Mammalian Cells TYROSYL RADICAL QUENCHING NOT INVOLVING REACTIVE OXYGEN SPECIES

Background: Diferric-tyrosyl radical [(FeIII2-Y·)(FeIII2)] cofactor-bearing subunit (β2) of ribonucleotide reductase is targeted by a Phase-II cancer drug, Triapine (3-AP). Results: Y· loss precedes iron loss without reactive oxygen species formation. Conclusion: Fe(II)-(3-AP) inhibits β2 catalytically resulting in iron-loaded β2 with a reduced Y·. Significance: Susceptibility of β2 to inhibition via Y· reduction by metal complexes implicates a new avenue to develop RNR inhibitors. Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the β2 subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(FeIII2-Y·)(FeIII2)] cofactor for de novo DNA biosynthesis. Several mechanisms for 3-AP inhibition of β2 have been proposed; one involves direct iron chelation from β2, whereas a second involves Y· destruction by reactive oxygen species formed in situ in the presence of O2 and reductant by Fe(II)-(3-AP). Inactivation of β2 can thus arise from cofactor destruction by loss of iron or Y·. In vitro kinetic data on the rates of 55Fe and Y· loss from [(55FeIII2-Y·)(55FeIII2)]-β2 under aerobic and anaerobic conditions reveal that Y· loss alone is sufficient for rapid β2 inactivation. OxyblotTM and mass spectrometric analyses of trypsin-digested inhibited β2, and lack of Y· loss from H2O2 and O2̇̄ treatment together preclude reactive oxygen species involvement in Y· loss. Three mammalian cell lines treated with 5 μm 3-AP reveal Y· loss and β2 inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo- over [(FeIII2-Y·)(FeIII2)]-β2 in lysates, similar iron-content in β2 immunoprecipitated from 3-AP-treated and untreated [55Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited β2 are most consistent with Y· loss being the predominant mode of inhibition, with β2 remaining iron-loaded and stable. A model consistent with in vitro and cell-based biochemical studies is presented in which Fe(II)-(3-AP), which can be cycled with reductant, directly reduces Y· of the [(FeIII2-Y·)(FeIII2)] cofactor of β2.

Cell Compatible Trimethoprim-Decorated Iron Oxide Nanoparticles Bind Dihydrofolate Reductase for Magnetically Modulating Focal Adhesion of Mammalian Cells

On the basis of the high affinity binding of trimethoprim (TMP) to Escherichia coli dihydrofolate reductase (eDHFR), TMP-decorated iron oxide nanoparticles bind to eDHFR with high affinity and specificity, which allows magnetic modulation of focal adhesion of mammalian cells adhered to a surface. Besides being the first example of nanoparticles that selectively bind to eDHFR, the biocompatibility of the conjugate of TMP-iron oxide nanoparticles renders a convenient and versatile platform for investigating the cellular responses to specific, mechanical perturbation of proteins via a magnetic force.

Prion-like nanofibrils of small molecules (PriSM) selectively inhibit cancer cells by impeding cytoskeleton dynamics.

Background: Small molecules form prion-like nanofibrils termed as PriSM. Results: PriSM accumulate selectively in cancer cells and impede cytoskeleton dynamics. Conclusion: PriSM is selectively toxic to cancer cells. Significance: We hope this work will inspire the exploration of PriSM, formed by hydrophobic peptides, as a new paradigm of polypharmacological agents. Emerging evidence reveals that prion-like structures play important roles to maintain the well-being of cells. Although self-assembly of small molecules also affords prion-like nanofibrils (PriSM), little is known about the functions and mechanisms of PriSM. Previous works demonstrated that PriSM formed by a dipeptide derivative selectively inhibiting the growth of glioblastoma cells over neuronal cells and effectively inhibiting xenograft tumor in animal models. Here we examine the protein targets, the internalization, and the cytotoxicity pathway of the PriSM. The results show that the PriSM selectively accumulate in cancer cells via macropinocytosis to impede the dynamics of cytoskeletal filaments via promiscuous interactions with cytoskeletal proteins, thus inducing apoptosis. Intriguingly, Tau proteins are able to alleviate the effect of the PriSM, thus protecting neuronal cells. This work illustrates PriSM as a new paradigm for developing polypharmacological agents that promiscuously interact with multiple proteins yet result in a primary phenotype, such as cancer inhibition

Temporally Controlled Targeting of 4-Hydroxynonenal to Specific Proteins in Living Cells

In-depth chemical understanding of complex biological processes hinges upon the ability to systematically perturb individual systems. However, current approaches to study impacts of biologically relevant reactive small molecules involve bathing of the entire cell or isolated organelle with excess amounts, leading to off-target effects. The resultant lack of biochemical specificity has plagued our understanding of how biological electrophiles mediate signal transduction or regulate responses that confer defense mechanisms to cellular electrophilic stress. Here we introduce a target-specific electrophile delivery platform that will ultimately pave the way to interrogate effects of reactive electrophiles on specific target proteins in cells. The new methodology is demonstrated by photoinducible targeted delivery of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent conjugation of the HNE-precursor to HaloTag fused to the target proteins enables directed HNE delivery upon photoactivation. The strategy provides proof of concept of selective delivery of reactive electrophiles to individual electrophile-responsive proteins in mammalian cells. It opens a new avenue enabling more precise determination of the pathophysiological consequences of HNE-induced chemical modifications on specific target proteins in cells.

Clofarabine Targets the Large Subunit (α) of Human Ribonucleotide Reductase in Live Cells by Assembly into Persistent Hexamers

Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α(2))(m)(β(2))(n), regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to ClF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-α(6). Our data validate hRNR as an important target of ClF, provide evidence that in vivo αs quaternary structure can be perturbed by a nonnatural ligand, and suggest small-molecule-promoted, persistent hexamerization as a strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell.

On-Demand Targeting: Investigating Biology with Proximity-Directed Chemistry

Proximity enhancement is a central chemical tenet underpinning an exciting suite of small-molecule toolsets that have allowed us to unravel many biological complexities. The leitmotif of this opus is “tethering”—a strategy in which a multifunctional small molecule serves as a template to bring proteins/biomolecules together. Scaffolding approaches have been powerfully applied to control diverse biological outcomes such as protein–protein association, protein stability, activity, and improve imaging capabilities. A new twist on this strategy has recently appeared, in which the small-molecule probe is engineered to unleash controlled amounts of reactive chemical signals within the microenvironment of a target protein. Modification of a specific target elicits a precisely timed and spatially controlled gain-of-function (or dominant loss-of-function) signaling response. Presented herein is a unique personal outlook conceptualizing the powerful proximity-enhanced chemical biology toolsets into two paradigms: “multifunctional scaffolding” versus “on-demand targeting”. By addressing the latest advances and challenges in the established yet constantly evolving multifunctional scaffolding strategies as well as in the emerging on-demand precision targeting (and related) systems, this Perspective is aimed at choosing when it is best to employ each of the two strategies, with an emphasis toward further promoting novel applications and discoveries stemming from these innovative chemical biology platforms.

Magnetic nanoparticles for direct protein sorting inside live cells

This work reports the first example of the biofunctional magnetic nanoparticles as a “magnetic dock” for directly sorting proteins inside live cells. We decorate the iron oxide nanoparticles with the ligands that bind selectively to fusion proteins consisting of the proteins of interest (POIs) and the ligand receptors. Similar to protein sorting processes on vesicles, the clusters of these functionalized magnetic nanoparticles only bind to the fusion proteins via the interaction with the receptors, but exhibit little interaction to other proteins. This work demonstrates new applications of magnetic nanoparticles and may ultimately contribute to the exploration of the functions of proteins via the selective, spatiotemporal control of the proteins by a magnetic force.