SLC6A19 inhibition facilitates urinary neutral amino acid excretion and lowers plasma phenylalanine.

BACKGROUNDThe toxic accumulation of phenylalanine (Phe) in the brain underlies the neurological presentation of phenylketonuria (PKU). Solute carrier family 6 member 19 (SLC6A19) is the major transporter responsible for the (re)absorption of Phe in the kidney and intestine. Here, we describe the characterization of the first small molecule SLC6A19 inhibitor to enter clinical development for the treatment of PKU.

Novel Corrector for Variants of SLC6A8: A Therapeutic Opportunity for Creatine Transporter Deficiency.

Mutations in creatine transporter SLC6A8 cause creatine transporter deficiency (CTD), which is responsible for 2% of all cases of X-linked intellectual disability. CTD has no current treatments and has a high unmet medical need. Inspired by the transformational therapeutic impact of small molecule "correctors" for the treatment of cystic fibrosis, which bind to mutated versions of the CFTR ion channel to promote its trafficking to the cell surface, we sought to identify small molecules that could stabilize SLC6A8 as a potential treatment for CTD.

A Probe-Free Occupancy Assay to Assess a Targeted Covalent Inhibitor of Receptor Tyrosine-Protein Kinase erbB-2.

Establishing target engagement is fundamental to effective target-based drug development. It paves the way for efficient medicinal chemistry design and definitive answers about target validation in the clinic. For irreversible targeted covalent inhibitor (TCI) drugs, there is a unique opportunity to establish and quantify the target engagement or occupancy.

SMaSh: A Streptavidin Mass Shift Assay for Rapidly Quantifying Target Occupancy by Irreversible Inhibitors.

The streptavidin mass shift (SMaSh) assay is a robust and fast approach for quantifying target protein occupancy by a covalent inhibitor or ligand. It exploits the biotin-streptavidin bond using the Simple Western platform. One measurement on a single sample determines both total and occupied target protein simultaneously and is, therefore, self-normalizing.

A Chemoproteomics Approach to Determine the Mechanism of Testicular Toxicity for the Bruton's Tyrosine Kinase Inhibitor CC-292.

During drug development, potential safety issues can occur at any time. Understanding the cause of a toxicity can help with deciding on how to advance the drug development program. Chemoproteomics provides a way to help understand the cause of a toxicity wherein the affected tissue is accessible and can be probed with a covalently binding compound that is analogous to the offending drug.

Labeling Preferences of Diazirines with Protein Biomolecules.

Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome.

Efficacy of a Covalent ERK1/2 Inhibitor, CC-90003, in KRAS-Mutant Cancer Models Reveals Novel Mechanisms of Response and Resistance.

As a critical signaling node, ERK1/2 are attractive drug targets, particularly in tumors driven by activation of the MAPK pathway. Utility of targeting the MAPK pathway has been demonstrated by clinical responses to inhibitors of MEK1/2 or RAF kinases in some mutant BRAF-activated malignancies. Unlike tumors with mutations in BRAF, those with mutations in KRAS (>30% of all cancers and >90% of certain cancer types) are generally not responsive to inhibitors of MEK1/2 or RAF.

Transcriptional and post-translational modifications of B-Raf in quinol-thioether induced tuberous sclerosis renal cell carcinoma.

Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2(EK/+) ), bearing a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) gene, with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. These tumors have increased protein expression of B-Raf, C-Raf (Raf-1), and increased expression and activity of ERK kinase.

S-adenosyl-l-methionine protection of acetaminophen mediated oxidative stress and identification of hepatic 4-hydroxynonenal protein adducts by mass spectrometry.

Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-l-methionine (SAMe) treatment 1hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins.

In vitro and in vivo characterization of irreversible mutant-selective EGFR inhibitors that are wild-type sparing.

Patients with non-small cell lung carcinoma (NSCLC) with activating mutations in epidermal growth factor receptor (EGFR) initially respond well to the EGFR inhibitors erlotinib and gefitinib. However, all patients relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR (EGFR(WT)).

Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC.

Unlabelled: Patients with non-small cell lung cancer (NSCLC) with activating EGF receptor (EGFR) mutations initially respond to first-generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, frequently driven by the EGFR(T790M) mutation. CO-1686 is a novel, irreversible, and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR, including T790M, while exhibiting minimal activity toward the wild-type (WT) receptor.

Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans.

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed.

Orally active fumagillin analogues: transformations of a reactive warhead in the gastric environment.

Semisynthetic analogues of fumagillin, 1, inhibit methionine aminopeptidase-2 (MetAP2) and have entered the clinic for the treatment of cancer. An optimized fumagillin analogue, 3 (PPI-2458), was found to be orally active, despite containing a spiroepoxide function that formed a covalent linkage to the target protein. In aqueous acid, 3 underwent ring-opening addition of water and HCl, leading to four products, 4-7, which were characterized in detail.

Discovery of a potent and isoform-selective targeted covalent inhibitor of the lipid kinase PI3Kα.

PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallography, that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified.

Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and in vivo activity.

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, [(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability.

The frequency of 1,4-benzoquinone-lysine adducts in cytochrome c correlate with defects in apoptosome activation.

Electrophile-mediated post-translational modifications (PTMs) are known to cause tissue toxicities and disease progression. These effects are mediated via site-specific modifications and structural disruptions associated with such modifications. 1,4-Benzoquinone (BQ) and its quinone-thioether metabolites are electrophiles that elicit their toxicity via protein arylation and the generation of reactive oxygen species.

Selective irreversible inhibition of a protease by targeting a noncatalytic cysteine.

Designing selective inhibitors of proteases has proven problematic, in part because pharmacophores that confer potency exploit the conserved catalytic apparatus. We developed a fundamentally different approach by designing irreversible inhibitors that target noncatalytic cysteines that are structurally unique to a target in a protein family. We have successfully applied this approach to the important therapeutic target HCV protease, which has broad implications for the design of other selective protease inhibitors.

Identification of chemical-adducted proteins in urine by multi-dimensional protein identification technology (LC/LC-MS/MS).

Recent technological advancements in mass spectrometry facilitate the detection of chemical-induced posttranslational modifications (PTMs) that may alter cell signaling pathways or alter the structure and function of the modified proteins. To identify such protein adducts (Kleiner et al., Chem Res Toxicol 11:1283-1290, 1998), multi-dimensional protein identification technology (MuDPIT) has been utilized.

One-dimensional western blotting coupled to LC-MS/MS analysis to identify chemical-adducted proteins in rat urine.

The environmental toxicant hydroquinone (HQ) and its glutathione conjugates (GSHQs) cause renal cell necrosis via a combination of redox cycling and the covalent adduction of proteins within the S₃ segment of the renal proximal tubules in the outer stripe of the outer medulla (OSOM). Following administration of 2-(glutathion-S-yl)HQ (MGHQ) (400 μmol/kg, i.v.

Utilization of LC-MS/MS analyses to identify site-specific chemical protein adducts in vitro.

Biologically reactive intermediates are formed following metabolism of xenobiotics, and during normal oxidative metabolism. These reactive species are electrophilic in nature and are capable of forming stable adducts with target proteins. These covalent protein modifications can initiate processes that lead to acute tissue injury or chronic disease.

Utilization of MALDI-TOF to determine chemical-protein adduct formation in vitro.

Biological reactive intermediates can be created via metabolism of xenobiotics during the process of chemical elimination. They can also be formed as by-products of cellular metabolism, which produces reactive oxygen and nitrogen species. These reactive intermediates tend to be electrophilic in nature, which enables them to interact with tissue macromolecules, disrupting cellular signaling processes and often producing acute and chronic toxicities.

Carbamate analogues of fumagillin as potent, targeted inhibitors of methionine aminopeptidase-2.

Inhibition of methionine aminopeptidase-2 (MetAP2) represents a novel approach to antiangiogenic therapy. We describe the synthesis and activity of fumagillin analogues that address the pharmacokinetic and safety liabilities of earlier candidates in this compound class. Two-step elaboration of fumagillol with amines yielded a diverse series of carbamates at C6 of the cyclohexane spiroepoxide.

An inhibitor of methionine aminopeptidase type-2, PPI-2458, ameliorates the pathophysiological disease processes of rheumatoid arthritis.

Objective: To elucidate the role of methionine aminopeptidase type-2 (MetAP-2) in the clinical pathology of rheumatoid arthritis, arthritis was induced in rats by administration of peptidoglycan-polysaccharide (PG-PS).

Design: The inhibitor of MetAP-2, PPI-2458, was administered orally at 5 mg/kg every other day during 3 distinct phases of the disease. In vitro studies were performed to clarify in vivo findings.

Quinone electrophiles selectively adduct "electrophile binding motifs" within cytochrome c.

Electrophiles generated endogenously, or via the metabolic bioactivation of drugs and other environmental chemicals, are capable of binding to a variety of nucleophilic sites within proteins. Factors that determine site selective susceptibility to electrophile-mediated post-translational modifications, and the consequences of such alterations, remain largely unknown. To identify and characterize chemical-mediated protein adducts, electrophiles with known toxicity were utilized.