Expression, purification, and characterization of TYK-2 kinase domain, a member of the Janus kinase family.

The Janus kinase family consists of four members: JAK-1, -2, -3 and TYK-2. While JAK-2 and JAK-3 have been well characterized biochemically, there is little data on TYK-2. Recent work suggests that TYK-2 may play a critical role in the development of a number of inflammatory processes. We have carried out a series of biochemical studies to better understand TYK-2 enzymology and its inhibition profile, in particular how the TYK-2 phosphorylated forms differ from each other and from the other JAK family members. We have expressed and purified milligram quantities of the TYK-2 kinase domain (KD) to high purity and developed a method to separate the non-, mono- (pY(1054)) and di-phosphorylated forms of the enzyme. Kinetic studies (k(cat(app))/K(m(app))) indicated that phosphorylation of the TYK-2-KD (pY(1054)) increased the catalytic efficiency 4.4-fold compared to its non-phosphorylated form, while further phosphorylation to generate the di-phosphorylated enzyme imparted no further increase in activity. These results are in contrast to those obtained with the JAK-2-KD and JAK-3-KD, where little or no increase in activity occurred upon mono-phosphorylation, while di-phosphorylation resulted in a 5.1-fold increase in activity for the JAK-2-KD. Moreover, ATP-competitive inhibitors demonstrated 10-30-fold shifts in potency (K(i(app))) as a result of the TYK-2-KD phosphorylation state, while the shifts for JAK-3-KD were only 2-3-fold and showed little or no change for JAK-2-KD. Thus, the phosphorlyation state imparted differential effects on both activity and inhibition within the JAK family of kinases.

Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain.

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K(m) and k(cat)) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus.

Compounds capable of stimulating soluble guanylate cyclase (sGC) activity might become important new tools to treat hypertension. While rational design of these drugs would be aided by elucidation of the sGC three-dimensional structure and molecular mechanism of activation, such efforts also require quantities of high quality enzyme that are challenging to produce. We implemented the titerless infected-cells preservation and scale-up (TIPS) methodology to express the heterodimeric sGC. In the TIPS method, small-scale insect cell cultures were first incubated with a recombinant baculovirus which replicated in the cells. The baculovirus-infected insect cells (BIIC) were harvested and frozen prior to cell lysis and the subsequent escape of the newly replicated virus into the culture supernatant. Thawed BIIC stocks were ultimately used for subsequent scale up. As little as 1 mL of BIIC was needed to infect a 100-L insect cell culture, in contrast to the usual 1L of high-titer, virus stock supernatants. The TIPS method eliminates the need and protracted time for titering virus supernatants, and provides stable, concentrated storage of recombinant baculovirus in the form of infected cells. The latter is particularly advantageous for virus stocks which are unstable, such as those for sGC, and provides a highly efficient alternative for baculovirus storage and expression. The TIPS process enabled efficient scale up to 100-L batches, each producing about 200mg of active sGC. Careful adjustment of expression culture conditions over the course of several 100-L runs provided uniform starting titers, specific activity, and composition of contaminating proteins that facilitated development of a process that reproducibly yielded highly active, purified sGC.

Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells.

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M(r)=144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS-PAGE corresponding to the alpha (M(r) approximately 77,000) and beta (M(r) approximately 70,000) sGC subunits. It showed an A(430)/A(280)=1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k(cat)/K(m)=1.7 x 10(-3)s(-1)microM(-1) that increased to 5.8 x 10(-1)s(-1)microM(-1) upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure-function relationships.

High yield purification of soluble guanylate cyclase from bovine lung.

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), is a cytosolic, heme-containing, heterodimeric enzyme that catalyzes the conversion of guanosine 5'-triphosphate (GTP) to 3,5'-cyclic guanosine monophosphate (cGMP) and pyrophosphate (PPi) in the presence of Mg2+. Cyclic GMP is then involved in transmitting the NO activating signals to a variety of downstream effectors such as cyclic-nucleotide-gated channels, protein kinases, and phosphodiesterases. In this work, sGC has been purified from bovine lung. The lungs were subjected to grinding and extraction with buffer at physiological pH followed by centrifugation. The resulting solution was subjected to successive column chromatography on DEAE- and Q-Sepharose, Ceramic Hydroxyapatite, Resource Q, and GTP-agarose. The purified enzyme migrated as a two-band protein on SDS-PAGE corresponding to sGC subunits alpha (M(r)=77,532) and beta (M(r)=70,500) and had an A(280 nm)/A(430 nm) of approximately 1 indicating one heme per heterodimer. The yield of enzyme was 8-10mg from 4 to 5 kg bovine lungs. V(max) and K(m) of non-stimulated sGC were 22 nmol/mg/min and 180 microM, respectively. Upon stimulation with the NO donor 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, the V(max) increased to 1330 nmol/mg/min while the K(m) dropped to 43 microM. The quality and quantity of enzyme make it suitable for studies to probe the structure and catalytic mechanism of this enzyme and for research related to drug discovery.

Design, synthesis, and crystal structure of hydroxyethyl secondary amine-based peptidomimetic inhibitors of human beta-secretase.

The design and synthesis of a novel series of potent and cell permeable peptidomimetic inhibitors of the human beta-secretase (BACE) are described. These inhibitors feature a hydroxyethyl secondary amine isostere and a novel aromatic ring replacement for the C-terminus. The crystal structure of BACE in complex with this hydroxyethyl secondary amine isostere inhibitor is also presented.

Differential kinetics and inhibition of purified recombinant tyrosine kinase 2 (TYK-2) and its catalytic domain JH-1.

The Janus kinase (JAK) family consists of four members: JAK-1, -2, -3 and tyrosine kinase 2 (TYK-2). Recent work suggests that cytokine signaling through TYK-2 may play a critical role in a number of inflammatory processes. We recently described the purification and characterization of phosphorylated isoforms of the TYK-2 kinase domain (TYK-2 KD) and its high resolution 3D structure in the presence of inhibitors. We now report the expression and a two-step purification procedure for the doubly tagged full-length construct, H6-FL-TYK-2-FLAG, and examine its properties compared to those of TYK-2 KD. In the presence of ATP and a peptide substrate, H6-FL-TYK-2-FLAG showed a marked lag in phosphopeptide product formation, while TYK-2 KD showed no such lag. This lag could be eliminated by ATP pretreatment, suggesting that the H6-FL-TYK-2-FLAG enzyme was activated by phosphorylation. The potencies of several nanomolar inhibitors were similar for TYK-2 KD and H6-FL-TYK-2-FLAG. However, these same inhibitors were about 1000 times less potent inhibiting the autophosphorylation of H6-FL-TYK-2-FLAG than they were inhibiting the phosphorylation of a peptide substrate modeled after the activation loop sequence of TYK-2. This intriguing result suggests that autophosphorylation and, thus, activation of H6-FL-TYK-2-FLAG is relatively insensitive to inhibition and that present inhibitors act to inhibit TYK-2 subsequent to activation. Inhibition of TYK-2 autophosphorylation may represent a new area of investigation for the JAK family.

Structural and thermodynamic characterization of the TYK2 and JAK3 kinase domains in complex with CP-690550 and CMP-6.

Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.

Potent and selective isophthalamide S2 hydroxyethylamine inhibitors of BACE1.

The design and synthesis of a novel series of potent BACE1 hydroxyethylamine inhibitors. These inhibitors feature hydrogen bonding substituents at the C-5 position of the isophthalamide ring with improved selectivity over cathepsin D.

Design of potent inhibitors of human beta-secretase. Part 2.

We describe an optimized series of acyclic hydroxyethylamine transition state isosteres of beta-secretase that incorporates a variety of P(2) side chains that yield potent inhibitors with excellent cellular activity. A 2.2A crystal structure of compound 13 is shown.

Design of potent inhibitors of human beta-secretase. Part 1.

We describe a novel series of potent inhibitors of human beta-secretase. These compounds possess the hydroxyethyl amine transition state isostere. A 2.5A crystal structure of inhibitor 32 bound to BACE is provided.

Electrophoretic mobility of Alzheimer's amyloid-beta peptides in urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The 43-amino acid Alzheimer's amyloid-beta peptide (Abeta peptide) retains a predominantly alpha-helix and beta-strand structure in sodium dodecyl sulfate (SDS) solution. This conformer has a high tendency to aggregate during conventional SDS-polyacrylamide gel electrophoresis (PAGE). Both the secondary structure and the proclivity for aggregation are obviated by the use of urea-SDS-PAGE: In 8M urea-with or without SDS-the Abeta peptide becomes 100% random coil and remains monomeric. However, during electrophoresis in this medium, the peptide and its truncated variants do not obey the law of mass/mobility relationship that most proteins-including Abeta peptides-follow in conventional SDS-PAGE. Rather, the smaller carboxy-terminally truncated peptides migrate slower than the larger full-length peptide, while the amino terminally truncated peptide does migrate faster than the full-length Abeta peptide. Thus, despite their small size (2-4kDa) and minor differences between their lengths, the Abeta peptides display a wide separation in this low-porosity (12% acrylamide) gel. We found that this unusual electrophoretic mobility in 8M urea is due to the fact that the quantity of [35S]SDS bound to the Abeta peptides, instead of being proportional to the total number of amino acids, is rather proportional to the sum of the hydrophobicity consensus indices of the constituent amino acids. It is then their hydrophobicity and, hence, the net negative charges contributed by the peptide-bound SDS that plays a major role in determining the mobility of Abeta peptides in 8M urea-SDS-PAGE. The high selectivity of the 8M urea-SDS-PAGE method allowed us to detect the presence of hitherto unknown Abeta peptide variants that were secreted in the conditioned medium by cultured HeLa cells.

Employing a superior BACE1 cleavage sequence to probe cellular APP processing.

The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.