Estimating the Extinction Coefficient for a Protein

Program year: 1990/1991Digitized from print original stored in HDRA new method has been developed for estimating the extinction coefficient for a protein. The only information that is needed is the amino acid composition of the protein, and this is readily available if the gene coding for the protein has been sequenced. This method will allow extinction coefficients to estimated with an accuracy of better than 4% for most proteins. This will allow much more quantitative studies than had been possible in the past with the many proteins that can be obtained in only small amounts. A nonlinear least squares analysis was used to calculate the "best" extinction coefficients over the wavelength range 272 nanometers (nm) to 286 nm for the chromophores tyrosine (Tyr), tryptohan (Trp) and cystine (-S-S-). These values were then used to estimate the extinction coefficients for 17 proteins, yielding estimates that were on average within 3.4% of the experimental values. For comparison, the extinction coefficients for the model compounds N-acetyl-Tyr-ethyl ester (N-Ac-Tyr-OEE), N-acetyl-Trp-ethyl ester (N-Ac-Trp-OEE), and oxidized glutathione were determined over the same wavelength range in the following solvents: water, 6 M guanidinium hydrochloride (GdnHCl), 8 M urea, 1-propanol, and formamide. It was found that Trp, Tyr, and -S-S-in proteins resemble these model compounds in 6 M GdnHCl and 8 M urea rather than in water or in the more nonpolar solvents 1-propanol and formamide. The estimates for protein extinction coefficients based on model compounds in 6 M GdnHCl and 8 M urea were on average within 3.8% and 3.9%, respectively, of the experimental values

Suppressor of Fused Regulates Gli Activity through a Dual Binding Mechanism

The Hedgehog pathway drives proliferation and differentiation by activating the Gli/Ci family of zinc finger transcription factors. Gli/Ci proteins form Hedgehog signaling complexes with other signaling components, including the kinesin-like protein Costal-2, the serine-threonine kinase Fused, and Suppressor of Fused [Su(fu)]. In these complexes Gli/Ci proteins are regulated by cytoplasmic sequestration, phosphorylation, and proteolysis. Here we characterize structural and functional determinants of Su(fu) required for Gli regulation and show that Su(fu) contains at least two distinct domains: a highly conserved carboxy-terminal region required for binding to the amino-terminal ends of the Gli proteins and a unique amino-terminal domain that binds the carboxy-terminal tail of Gli1. While each domain is capable of binding to different Gli1 regions independently, interactions between Su(fu) and Gli1 at both sites are required for cytoplasmic tethering and repression of Gli1. Furthermore, we have solved the crystal structure of the amino-terminal domain of human Su(fu)(27-268) at 2.65 Å resolution. This domain forms a concave pocket with a prominent acidic patch. Mutation at Asp(159) in the acidic patch disrupts Gli1 tethering and repression while not strongly disrupting binding, indicating that the amino-terminal domain of Su(fu) likely impacts Gli binding through a mechanism distinct from that for tethering and repression. These studies provide a structural basis for understanding the function of Su(fu)

Deconstruction of Activity-Dependent Covalent Modification of Heme in Human Neutrophil Myeloperoxidase by Multistage Mass Spectrometry (MS⁴)

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1<i>H</i>-purin-6­(9<i>H</i>)-one (<b>1</b>), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by <b>1</b> in a labile peptide–heme–peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS⁴) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that <b>1</b> was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of <b>1</b>. The final orientation of <b>1</b> perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of <b>1</b> by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain

Discovery of Potent and Selective Periphery-Restricted Quinazoline Inhibitors of the Cyclic Nucleotide Phosphodiesterase PDE1

We disclose the discovery and X-ray cocrystal data of potent, selective quinazoline inhibitors of PDE1. Inhibitor (<i>S</i>)-<b>3</b> readily attains free plasma concentrations above PDE1 IC₅₀ values and has restricted brain access. The racemic compound <b>3</b> inhibits >75% of PDE hydrolytic activity in soluble samples of human myocardium, consistent with heightened PDE1 activity in this tissue. These compounds represent promising new tools to probe the value of PDE1 inhibition in the treatment of cardiovascular disease

Discovery of Potent and Selective Periphery-Restricted Quinazoline Inhibitors of the Cyclic Nucleotide Phosphodiesterase PDE1

We disclose the discovery and X-ray cocrystal data of potent, selective quinazoline inhibitors of PDE1. Inhibitor (<i>S</i>)-<b>3</b> readily attains free plasma concentrations above PDE1 IC₅₀ values and has restricted brain access. The racemic compound <b>3</b> inhibits >75% of PDE hydrolytic activity in soluble samples of human myocardium, consistent with heightened PDE1 activity in this tissue. These compounds represent promising new tools to probe the value of PDE1 inhibition in the treatment of cardiovascular disease

The 2.0 Å crystal structure of the ERα ligand-binding domain complexed with lasofoxifene

Lasofoxifene is a new and potent selective estrogen receptor modulator (SERM). The structural basis of its interaction with the estrogen receptor has been investigated by crystallographic analysis of its complex with the ligand-binding domain of estrogen receptor α at a resolution of 2.0 Å. As with other SERMs, lasofoxifene diverts the receptor from its agonist-bound conformation by displacing the C-terminal AF-2 helix into the site at which the LXXLL motif of coactivator proteins would otherwise be able to bind. Lasofoxifene achieves this effect by occupying the space normally filled by residue Leu 540, as well as by modulating the conformation of residues of helix 11 (His 524, Leu 525). A well-defined salt bridge between lasofoxifene and Asp 351 suggests that charge neutralization in this region of the receptor may explain the some of the antiestrogenic effects of lasofoxifene. The results suggest general features of ERα/SERM recognition, and add a new dimension to efforts to rationalize differences between the biological activity profiles exhibited by these important pharmacological agents

Decreasing the Rate of Metabolic Ketone Reduction in the Discovery of a Clinical Acetyl-CoA Carboxylase Inhibitor for the Treatment of Diabetes

Acetyl-CoA carboxylase (ACC) inhibitors offer significant potential for the treatment of type 2 diabetes mellitus (T2DM), hepatic steatosis, and cancer. However, the identification of tool compounds suitable to test the hypothesis in human trials has been challenging. An advanced series of spirocyclic ketone-containing ACC inhibitors recently reported by Pfizer were metabolized in vivo by ketone reduction, which complicated human pharmacology projections. We disclose that this metabolic reduction can be greatly attenuated through introduction of steric hindrance adjacent to the ketone carbonyl. Incorporation of weakly basic functionality improved solubility and led to the identification of <b>9</b> as a clinical candidate for the treatment of T2DM. Phase I clinical studies demonstrated dose-proportional increases in exposure, single-dose inhibition of de novo lipogenesis (DNL), and changes in indirect calorimetry consistent with increased whole-body fatty acid oxidation. This demonstration of target engagement validates the use of compound <b>9</b> to evaluate the role of DNL in human disease

Aminomethyl-Derived Beta Secretase (BACE1) Inhibitors: Engaging Gly230 without an Anilide Functionality

A growing subset of β-secretase (BACE1) inhibitors for the treatment of Alzheimer’s disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug–drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound <b>16</b>, which exhibits robust in vivo efficacy as a BACE1 inhibitor