Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides

Human granulocyte colony-stimulating factor (G-CSF) is an endogenous glycoprotein involved in hematopoiesis. Natively glycosylated and nonglycosylated recombinant forms, lenograstim and filgrastim, respectively, are used clinically to manage neutropenia in patients undergoing chemotherapeutic treatment. Despite their comparable therapeutic potential, the purpose of O-linked glycosylation at Thr133 remains a subject of controversy. In light of this, we have developed a synthetic platform to prepare G-CSF aglycone with the goal of enabling access to native and designed glycoforms with site-selectivity and glycan homogeneity. To address the synthesis of a relatively large, aggregation-prone sequence, we advanced an isonitrile-mediated ligation method. The chemoselective activation and coupling of C-terminal peptidyl Gly thioacids with the N-terminus of an unprotected peptide provide ligated peptides directly in a manner complementary to that with conventional native chemical ligation–desulfurization strategies. Herein, we describe the details and application of this method as it enabled the convergent total synthesis of G-CSF aglycone

Total Chemical Synthesis and Folding of All‑l and All‑d Variants of Oncogenic KRas(G12V)

The Ras proteins are essential GTPases involved in the regulation of cell proliferation and survival. Mutated oncogenic forms of Ras alter effector binding and innate GTPase activity, leading to deregulation of downstream signal transduction. Mutated forms of Ras are involved in approximately 30% of human cancers. Despite decades of effort to develop direct Ras inhibitors, Ras has long been considered “undruggable” due to its high affinity for GTP and its lack of hydrophobic binding pockets. Herein, we report a total chemical synthesis of all-l- and all-d-amino acid biotinylated variants of oncogenic mutant KRas­(G12V). The protein is synthesized using Fmoc-based solid-phase peptide synthesis and assembled using combined native chemical ligation and isonitrile-mediated activation strategies. We demonstrate that both KRas­(G12V) enantiomers can successfully fold and bind nucleotide substrates and binding partners with observable enantio­discrimination. By demonstrating the functional competency of a mirror-image form of KRas bound to its corresponding enantiomeric nucleotide triphosphate, this study sets the stage for further biochemical studies with this material. In particular, this protein will enable mirror-image yeast surface display experiments to identify all-d peptide ligands for oncogenic KRas, providing a useful tool in the search for new therapeutics against this challenging disease target

Measuring H₂¹⁸O Tracer Incorporation on a QQQ-MS Platform Provides a Rapid, Transferable Screening Tool for Relative Protein Synthesis

Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H₂¹⁸O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H₂¹⁸O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment

Measuring H₂¹⁸O Tracer Incorporation on a QQQ-MS Platform Provides a Rapid, Transferable Screening Tool for Relative Protein Synthesis

Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H₂¹⁸O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H₂¹⁸O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment

FGF21 treatment lowers plasma glucose levels while not affecting body weights.

<p>Glucose (A) and body weight (B) measurements are plotted for each mouse model (WT Chow, WT HFD, and dbdb Chow) across each treatment (WT FGF21 and 2 doses of PEG30-FGF21 Q108). Error bars denote standard error. Asterisks denote significant change compared to vehicle treatment within each mouse model and time point (students t-test p value < 0.05).</p

Schematic diagram of FGF21 effects in adipocytes or white adipose tissue.

<p>Schematic diagram of FGF21 effects in adipocytes or white adipose tissue.</p

Acute FGF21 treatment-induced RNA markers in white adipose tissues.

<p>The top 32 RNA markers (Table 1) from IWAT are represented in the clustergram (see Figure S2 for EWAT and RPWAT). Plotted are the logRatio values on a scale of +/- 0.6 (+/- 4 fold) with magenta and cyan signifying up- and down-regulated genes, respectively. Each row is the average of up to 5 animals for that treatment group. Gene names are shown below the clustergram. Native = WT FGF21; PEG-L and PEG-H = PEG30-FGF21 Q108 at 0.75 and 2.5 mg/kg, respectively; and 2d and 5d correspond to number of days of treatment.</p

FGF21 increases phosphorylation of novel peptides in adipocytes and white adipose tissue.

<p>Analyzed extracts were from (A) mouse 3T3L1 adipocytes, (B) human subcutaneous adipocytes, and (C) IWAT or (D) EWAT tissue from C57BL/6 mice on chow diet treated for 15 min, with either vehicle (Veh), WT FGF21 or PEG30-FGF21 Q108 at the doses indicated. Shown in the Western blots are representative samples.</p

Plasma levels of secreted proteins identified in WAT by Affymetrix microarray profiling.

<p>C57BL/6 mice on chow diet were treated with a single dose of vehicle or PEG30-FGF21 Q108 at one of three doses (0.25, 0.75, or 2.5 mg/kg). Plasma Ccl11, Cxcl2, Igf1, Il1rn, and Kitl levels were measured 24 hours and 48 hours post dose (Table S8). Error bars denote standard error. Asterisks denote significant change compared to vehicle treatment within each time point (students t-test p value < 0.05*, p<0.01**, p<0.001***). Arrows on far right denote effects of PEG-FGF21 Q108 at 2.5 mg/kg at the RNA level after 24 hours of treatment (2 day time point) in WT chow-fed mice (IWAT).</p

High Resolution Discovery Proteomics Reveals Candidate Disease Progression Markers of Alzheimer’s Disease in Human Cerebrospinal Fluid

<div><p>Disease modifying treatments for Alzheimer’s disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p<0.0001) and SME-2 (p = 0.0004) for evaluation in a second independent longitudinal cohort of human CSF from post-mortem diagnosed AD patients and age-matched and case-matched control patients. In cohort-2, SME-1, identified as neuronal secretory protein VGF, and SME-2, identified as neuronal pentraxin receptor-1 (NPTXR), in AD were 21% (p = 0.039) and 17% (p = 0.026) lower, at baseline, respectively, than in controls. Linear mixed model analysis in the longitudinal cohort estimate a decrease in the levels of VGF and NPTXR at the rate of 10.9% and 6.9% per year in the AD patients, whereas both markers increased in controls. Because these markers are detected by mass spectrometry without the need for antibody reagents, targeted MS based assays provide a clear translation path for evaluating selected AD disease-progression markers with high analytical precision in the clinic.</p></div