Enzyme purification and sustained enzyme activity for pharmaceutical biocatalysis by fusion with phase-separating intrinsically disordered protein.

In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time-consuming and cost-prohibitive to purify enzymes using conventional chromatographic processes at scale.

A fragment-based selection approach for the discovery of peptide macrocycles targeting protein kinases.

Protein kinases are implicated in diverse signaling cascades and have been targeted with small molecules that typically bind the conserved ATP-binding active site. These inhibitors are often promiscuous and target multiple protein kinases, which has led to the development of alternate strategies to discover selective ligands. We have recently described a fragment-based selection approach, where a small-molecule warhead can be non-covalently tethered to a phage-displayed library of cyclic peptides.

Small molecule gated split-tyrosine phosphatases and orthogonal split-tyrosine kinases.

Protein kinases phosphorylate client proteins, while protein phosphatases catalyze their dephosphorylation and thereby in concert exert reversible control over numerous signal transduction pathways. We have recently reported the design and validation of split-protein kinases that can be conditionally activated by an added small molecule chemical inducer of dimerization (CID), rapamycin. Herein, we provide the rational design and validation of three split-tyrosine phosphatases (PTPs) attached to FKBP and FRB, where catalytic activity can be modulated with rapamycin.

Ligand-gated split-kinases.

The activity of protein kinases are naturally gated by a variety of physiochemical inputs, such as phosphorylation, metal ions, and small molecules. In order to design protein kinases that can be gated by user-defined inputs, we describe a sequence dissimilarity based approach for identifying sites in protein kinases that accommodate 25-residue loop insertion while retaining catalytic activity. We further demonstrate that the successful loop insertion mutants provide guidance for the dissection of protein kinases into two fragments that cannot spontaneously assemble and are thus inactive but can be converted into ligand-gated catalytically active split-protein kinases.

Self-assembled cation transporters made from lipophilic 8-phenyl-2'-deoxyguanosine derivatives.

We have previously reported that 8-phenyl-2'-deoxyguanosine derivatives (8PhGs) are able to extract metal cations from an aqueous phase into an organic phase. Herein we report on the ability of 8PhGs to transport metal cations across a bulk lipophilic liquid membrane. The experiments were performed using lithium, sodium, potassium, and strontium picrate salts with the parent lipophilic Gi, two isomeric 8PhG derivatives, cis-dicyclohexano-18-crown-6 (CD18C6) and [2•2•2] cryptand as reference compounds.