4 research outputs found
Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action
A range of acyl-lysine (acyl-Lys)
modifications on histones and
other proteins have been mapped over the past decade but for most,
their functional and structural significance remains poorly characterized.
One limitation in the study of acyl-Lys containing proteins is the
challenge of producing them or their mimics in site-specifically modified
forms. We describe a cysteine alkylation-based method to install hydrazide
mimics of acyl-Lys post-translational modifications (PTMs) on proteins.
We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys,
and ubiquityl-Lys that could be recognized selectively by relevant
acyl-Lys modification antibodies. The acyl-Lys modified histone H3
proteins were reconstituted into nucleosomes to study nucleosome dynamics
and stability as a function of modification type and site. We also
installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin
analog, both of which could be cleaved by deubiquitinating enzymes.
Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study
the SAGA deubiquitinating moduleās molecular recognition. These
results suggest that acyl-Lys mimics offer a relatively simple and
promising strategy to study the role of acyl-Lys modifications in
the function, structure, and regulation of proteins and protein complexes
Pyridinylquinazolines Selectively Inhibit Human Methionine Aminopeptidaseā1 in Cells
Methionine
aminopeptidases (MetAPs), which remove the initiator
methionine from nascent peptides, are essential in all organisms.
While MetAP2 has been demonstrated to be a therapeutic target for
inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell
proliferation. Our earlier efforts identified two structural classes
of human MetAP1 (<i>Hs</i>MetAP1)-selective inhibitors (<b>1</b>ā<b>4</b>), but all of them failed to inhibit
cellular <i>Hs</i>MetAP1. Using MnĀ(II) or ZnĀ(II) to activate <i>Hs</i>MetAP1, we found that <b>1</b>ā<b>4</b> could only effectively inhibit purified <i>Hs</i>MetAP1
in the presence of physiologically unachievable concentrations of
CoĀ(II). In an effort to seek CoĀ(II)-independent inhibitors, a novel
structural class containing a 2-(pyridin-2-yl)Āquinazoline core has
been discovered. Many compounds in this class potently and selectively
inhibited <i>Hs</i>MetAP1 without CoĀ(II). Subsequently,
we demonstrated that <b>11j</b>, an auxiliary metal-dependent
inhibitor, effectively inhibited <i>Hs</i>MetAP1 in primary
cells. This is the first report that an <i>Hs</i>MetAP1-selective
inhibitor is effective against its target in cells
Rapamycin-inspired macrocycles with new target specificity
Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.</p
Optimization of the Potency and Pharmacokinetic Properties of a Macrocyclic Ghrelin Receptor Agonist (Part I): Development of Ulimorelin (TZP-101) from Hit to Clinic
High-throughput screening of Tranzyme Pharmaās
proprietary
macrocycle library using the aequorin Ca<sup>2+</sup>-bioluminescence
assay against the human ghrelin receptor (GRLN) led to the discovery
of novel agonists against this G-protein coupled receptor. Early hits
such as <b>1</b> (<i>K</i><sub>i</sub> = 86 nM, EC<sub>50</sub> = 134 nM) though potent in vitro displayed poor pharmacokinetic
properties that required optimization. While such macrocycles are
not fully rule-of-five compliant, principally due to their molecular
weight and clogP, optimization of their pharmacokinetic properties
proved feasible largely through conformational rigidification. Extensive
SAR led to the identification of <b>2</b> (<i>K</i><sub>i</sub> = 16 nM, EC<sub>50</sub> = 29 nM), also known as ulimorelin
or TZP-101, which has progressed to phase III human clinical trials
for the treatment of postoperative ileus. X-ray structure and detailed
NMR studies indicated a rigid peptidomimetic portion in <b>2</b> that is best defined as a nonideal type-Iā² Ī²-turn.
Compound <b>2</b> is 24% orally bioavailable in both rats and
monkeys. Despite its potency, in vitro and in gastric emptying studies, <b>2</b> did not induce growth hormone (GH) release in rats, thus
demarcating the GH versus GI pharmacology of GRLN