A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9.

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.

The mechanism of atrial antiarrhythmic action of RSD1235.

INTRODUCTION: RSD1235 is a novel drug recently shown to convert AF rapidly and safely in patients.(1) Its mechanism of action has been investigated in a rat model of ischemic arrhythmia, along with changes in action potential (AP) morphology in isolated rat ventricular myocytes and effects on cloned channels. METHODS AND RESULTS: Ischemic arrhythmias were inhibited with an ED50 of 1.5 micromol/kg/min, and repolarization times increased with non-significant effects on PR and QRS durations. AP prolongation was observed in rat myocytes at low doses, with plateau elevation and a reduction in the AP overshoot at higher doses. RSD1235 showed selectivity for voltage-gated K+ channels with IC50 values of 13 microM on hKv1.5 (1 Hz) versus 38 and 30 microM on Kv4.2 and Kv4.3, respectively, and 21 microM on hERG channels. RSD1235 did not block IK1 (IC50 > 1 mM) nor ICa,L (IC50= 220 microM) at 1 Hz in guinea pig ventricular myocytes (n = 4-5). The drug displayed mild (IC50= 43 microM at 1 Hz) open-channel blockade of Nav1.5 with rapid recovery kinetics after rate reduction (10-->1 Hz, 75% recovery with tau= 320 msec). Nav1.5 blocking potency increased with stimulus frequency from an IC50= 40 microM at 0.25 Hz, to an IC50= 9 microM at 20 Hz, and with depolarization increasing from 107 microM at -120 mV to 31 microM at -60 mV (1 Hz). CONCLUSIONS: These data suggest that RSD1235's clinical selectivity and AF conversion efficacy result from block of potassium channels combined with frequency- and voltage-dependent block of INa.