Tetsuo Uno

Synthesis, structure-activity relationships, and in vivo efficacy of the novel potent and selective anaplastic lymphoma kinase (ALK) inhibitor 5-chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine (LDK378) currently in phase 1 and phase 2 clinical trials.

The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure-activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.

In vivo incorporation of unnatural amino acids to probe structure, dynamics and ligand binding in a large protein by Nuclear Magnetic Resonance spectroscopy

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

Site-specific labeling of proteins with NMR-active unnatural amino acids

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.

Site-specific protein modifications through pyrroline-carboxy-lysine residues

Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol)s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.

D-Ornithine coopts pyrrolysine biosynthesis to make and insert pyrroline-carboxy-lysine

D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl.

Creating Chemical Diversity to Target Protein Kinases

Protein kinases play crucial roles in regulating virtually every cellular process and are currently attracting tremendous interest as drug targets from the pharmaceutical industry. The major challenges facing the development of the potential kinase inhibitor drugs are: selectivity, physical properties (solubility, molecular weight), and pharmacological properties (bioavailability, half life, toxicity, etc.) This review focuses on how selective protein kinase inhibitors that target the ATP and allosteric binding sites are currently being identified and optimized.

Design and synthesis of benzoazepin-2-one analogs as allosteric binders targeting the PIF pocket of PDK1.

A novel series of benzoazepin-2-ones were designed and synthesized targeting the PIF pocket of AGC protein kinases, among which a series of thioether-linked benzoazepin-2-ones were discovered to bind to the PIF pocket of 3-phosphoinositide-dependent kinase-1 (PDK1), and to displace the PIF peptide with an EC(50) values in the lower micromolar range. The structure-activity relationships (SARs) of the linker region, tail region, and distal region were explored to further optimize these novel binders which target the PIF pocket of PDK1. When tested in an in vitro PDK1 enzymatic assay using a peptide substrate, the benzodiazepin-2-ones increased the activity of the enzyme in a concentration-dependent fashion, indicating these compounds act as PDK1 allosteric activators. These new compounds may be further developed as therapeutic agents for the treatment of diseases where the PDK1-mediated AGC protein kinases are dysregulated.

Syk inhibitors with high potency in presence of blood.

We describe two series of Syk inhibitors which potently abrogate Syk kinase function in enzymatic assays, cellular assays and in primary cells in the presence of blood. Introduction of a 7-aminoindole substituent led to derivatives with good kinase selectivity and little or no hERG channel inhibition (3b, 10c).

Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Phosphopantetheinyl Transferases.

Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.

Site-Specific Dual Labeling of Proteins by Using Small Orthogonal Tags at Neutral pH

To expand the utility of proteinaceous FRET biosensors, we have developed a dual‐labeling approach based on two small bio‐orthogonal tags: pyrroline‐carboxy‐lysine (Pcl) and the S6 peptide. The lack of cross‐reactivity between those tags enables site‐specific two‐color protein conjugation in a one‐pot reaction. Moreover, Pcl/S6 dual‐tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE‐Fc, respectively. Both proteins could be efficiently dual‐labeled with FRET‐compatible fluorescent dyes at neutral pH. In the case of IgE‐Fc, the resulting conjugate enabled the monitoring of IgE binding to its high‐affinity receptor FcεRI, which is a key event in allergic disease.