Dissecting Parameters Contributing to the Underprediction of Aldehyde Oxidase-Mediated Metabolic Clearance of Drugs.

We investigated the effect of variability and instability in aldehyde oxidase (AO) content and activity on the scaling of in vitro metabolism data. AO content and activity in human liver cytosol (HLC) and five recombinant human AO preparations (rAO) were determined using targeted proteomics and carbazeran oxidation assay, respectively. AO content was highly variable as indicated by the relative expression factor (REF; i.e., HLC to rAO content) ranging from 0.001 to 1.7 across different in vitro systems. The activity of AO in HLC degrades at a 10-fold higher rate in the presence of the substrate as compared with the activity performed after preincubation without substrate. To scale the metabolic activity from rAO to HLC, a protein-normalized activity factor (pnAF) was proposed wherein the activity was corrected by AO content, which revealed up to sixfold higher AO activity in HLC versus rAO systems. A similar value of pnAF was observed for another substrate, ripasudil. Physiologically based pharmacokinetic (PBPK) modeling revealed a significant additional clearance (CL; 66%), which allowed for the successful prediction of in vivo CL of four other substrates, i.e., O-benzyl guanine, BIBX1382, zaleplon, and zoniporide. For carbazeran, the metabolite identification study showed that the direct glucuronidation may be contributing to around 12% elimination. Taken together, this study identified differential protein content, instability of in vitro activity, role of additional AO clearance, and unaccounted metabolic pathways as plausible reasons for the underprediction of AO-mediated drug metabolism. Consideration of these factors and integration of REF and pnAF in PBPK models will allow better prediction of AO metabolism. SIGNIFICANCE STATEMENT: This study elucidated the plausible reasons for the underprediction of aldehyde oxidase (AO)-mediated drug metabolism and provided recommendations to address them. It demonstrated that integrating protein content and activity differences and accounting for the loss of AO activity, as well as consideration of extrahepatic clearance and additional pathways, would improve the in vitro to in vivo extrapolation of AO-mediated drug metabolism using physiologically based pharmacokinetic modeling.

Optimization of genetic code expansion in the baculovirus expression vector system (BEVS).

The production of recombinant proteins containing unnatural amino acids, commonly known as genetic code expansion (GCE), represents a breakthrough in protein engineering that allows for the creation of proteins having novel designed properties. The naturally occurring orthogonal pyrrolysine tRNA/aminoacyl-tRNApyl synthetase pair (tRNApyl/PylRS) found in Methanosarcinaceae species has provided a rich platform for protein engineers to build a library of amino acid derivatives suitable for the introduction of novel chemical functionalities. While reports of the production of such recombinant proteins utilizing the tRNApyl/PylRS pair, or mutants thereof, is commonplace in Escherichia coli and mammalian cell expression systems, there has only been a single such report of GCE in the other stalwart of recombinant protein production, the baculovirus expression vector system (BEVS). However, that report formulates protein production within the designs of the MultiBac expression system [1]. The current study frames protein production within the strategies of the more commonplace Bac-to-Bac system of recombinant baculovirus production, via the development of novel baculovirus transfer vectors that harbor the tRNApyl/PylRS pair. The production of recombinant proteins harboring an unnatural amino acid(s) was examined using both an in cis and an in trans arrangement of the tRNApyl/PylRS pair relative to the target protein ORF i.e. the latter resides, respectively, on either the same vector as the tRNApyl/PylRS pair, or on a separate vector and deployed in a viral co-infection experiment. Aspects of the transfer vector designs and the viral infection conditions were investigated.

Production of active recombinant human aldehyde oxidase (AOX) in the baculovirus expression vector system (BEVS) and deployment in a pre-clinical fraction-of-control AOX compound exposure assay.

Human aldehyde oxidase (AOX) has emerged as a key enzyme activity for consideration in modern drug discovery. The enzyme catalyzes the oxidation of a wide variety of compounds, most notably azaheterocyclics that often form the building blocks of small molecule therapeutics. Failure to consider and assess AOX drug exposure early in the drug development cycle can have catastrophic consequences for novel compounds entering the clinic. AOX is a complex molybdopterin-containing iron-sulfur flavoprotein comprised of two identical 150 kDa subunits that has proven difficult to produce in recombinant form, and a commercial source of the purified human enzyme is currently unavailable. Thus, the potential exposure of novel drug development candidates to human AOX metabolism is usually assessed by using extracts of pooled human liver cytosol as a source of the enzyme. This can complicate the assignment of AOX-specific compound exposure due to its low activity and the presence of contaminating enzymes that may have overlapping substrate specificities. Herein is described a two-step process for the isolation of recombinant human AOX dimers to near homogeneity following production in the baculovirus expression vector system (BEVS). The deployment of this BEVS-produced recombinant human AOX as a substitute for human liver extracts in a fraction-of-control AOX compound-exposure screening assay is described. The ability to generate this key enzyme activity readily in a purified recombinant form provides for a more accurate and convenient approach to the assessment of new compound exposure to bona fide AOX drug metabolism.

Axitinib effectively inhibits BCR-ABL1(T315I) with a distinct binding conformation.

The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30-50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR-ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR-ABL1-driven leukaemia. Axitinib potently inhibited BCR-ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.

Multiple conformational states of the HPK1 kinase domain in complex with sunitinib reveal the structural changes accompanying HPK1 trans-regulation.

Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 in vitro In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.17-3.00-Å resolutions. The native nonphosphorylated cocrystal structure revealed an inactive dimer in which the activation loop of each monomer partially occupies the ATP- and substrate-binding sites of the partner monomer. In contrast, the structure of the protein with a doubly phosphorylated activation loop exhibited an active kinase conformation with a greatly reduced monomer-monomer interface. Conversely, the phosphomimetic mutant cocrystal structure disclosed an alternative arrangement in which the activation loops are in an extended domain-swapped configuration. These structural results indicate that HPK1 is a highly dynamic kinase that undergoes trans-regulation via dimer formation and extensive intramolecular and intermolecular remodeling of the activation segment.

High-volume shake flask cultures as an alternative to cellbag technology for recombinant protein production in the baculovirus expression vector system (BEVS).

Recombinant protein production in the baculovirus expression vector system (BEVS) has emerged as a system of choice for the production of recombinant human proteins for R&D purposes. Scale-up protein production in insect cells past the one or two liter volume generally utilizes disposable cellbag bioreactors that provide a means to scale to the 5-25L range in a single vessel. However, cellbags can be expensive and their use requires capital investment in dedicated rocker platforms and their associated air pumps and exhaust heaters. Additional equipment, such as tube welders and liquid pumps are often also deployed for the sterile transfer of media outside of a biosafety cabinet. Herein it is reported that Sf9, Sf21 and High Five insect cells demonstrate normal growth characteristics when cultured at the 2.5 L level in 3 L Erlenmeyer flasks, or at the 4.5 L level in 5 L Erlenmeyer flasks in standard laboratory shakers. In addition, a direct comparison of the expression levels of four separate proteins at the 4.5 L scale in 5 L flasks versus those at the 5 L scale in 10 L cellbags demonstrates that protein production is equal to, or slightly better, in the flasks versus the cellbags. The adoption of high-volume shake flasks for routine recombinant protein production in insect cells has a number of advantages over disposable bioreactors in terms of ease of use, and equipment and disposables costs.

Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1.

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.

Design and Synthesis of Functionally Active 5-Amino-6-Aryl Pyrrolopyrimidine Inhibitors of Hematopoietic Progenitor Kinase 1.

Immune activating agents represent a valuable class of therapeutics for the treatment of cancer. An area of active research is expanding the types of these therapeutics that are available to patients via targeting new biological mechanisms. Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of immune signaling and a target of high interest for the treatment of cancer. Herein, we present the discovery and optimization of novel amino-6-aryl pyrrolopyrimidine inhibitors of HPK1 starting from hits identified via virtual screening. Key components of this discovery effort were structure-based drug design aided by analyses of normalized B-factors and optimization of lipophilic efficiency.

Synthesis of Deuterated Heterocycles with Me2NCD(OMe)2 and Evaluation of the Products for Metabolism by Aldehyde Oxidase.

The metabolic oxidation of drug-like small molecules by aldehyde oxidase (AO) has commonly been mitigated through the incorporation of deuterium at the oxidation site. We report that dimethylformamide dimethyl acetal and related compounds undergo rapid CH to CD isotopic exchange upon exposure to methanol-d and similar deuterated alcohols. This isotopic exchange process can be used to synthesize Me2NCD(OMe)2 and has significant implications for the use of Me2NCD(OMe)2 in the synthesis of specifically deuterium-labeled compounds. The application of Me2NCD(OMe)2 to the synthesis of various heterocycles that have been associated with AO metabolism is described, and we report the impact of deuteration on the rate of in vitro AO-mediated metabolism.

Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays.

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.

Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus.

A new protein expression vector design utilizing an N-terminal six-histidine tag and tobacco etch virus protease cleavage site upstream of the hepatitis C virus NS5A sequence has resulted in a more straightforward purification method and improved yields of purified NS5A domain I protein. High-resolution diffracting crystals of NS5A domain I (amino acids 33 to 202) [NS5A(33-202)] were obtained by using detergent additive crystallization screens, leading to the structure of a homodimer which is organized differently from that published previously (T. L. Tellinghuisen, J. Marcotrigiano, and C. M. Rice, Nature 435:374-379, 2005) yet is consistent with a membrane association model for NS5A. The monomer-monomer interface of NS5A(33-202) features an extensive buried surface area involving the most-highly conserved face of each monomer. The two alternate structural forms of domain I now available may be indicative of the multiple roles emerging for NS5A in viral RNA replication and viral particle assembly.

Biochemical characterization of recombinant hepatitis C virus nonstructural protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity.

While nonstructural protein 4B (NS4B) from hepatitis C virus (HCV) is absolutely required for viral propagation, a full understanding of the enzymatic properties of this protein is lacking. Previous studies suggest that NS4B is located at the endoplasmic reticulum and that the protein structure consists of four central transmembrane domains with the N- and C-termini located in the cytoplasm of the host cell. To characterize the enzymatic activity of NS4B, the full-length protein with a C-terminal His tag was expressed in Sf9 insect cells and stabilized with nonionic detergents during purification. Chemical cross-linking experiments using GTP-gamma-azidoanilide and ATP-gamma-azidoanilide and equilibrium binding analyses with GTPgammaS and ATPgammaS show that both GTP and ATP are bound by NS4B, with ATP displaying a higher affinity. Analyses of enzymatic reactions catalyzed by NS4B indicate that the terminal phosphate groups of ATP, GTP, and GDP are removed to produce ADP, GDP, and GMP, respectively. The k(cat) for hydrolysis of GTP by purified NS4B compared favorably with the k(cat) for hydrolysis of GTP by Ras-p21 in the absence of GTPase activating proteins (GAPs). In addition to the hydrolysis of NTP and NDP substrates, adenylate kinase activity was detected in purified preparations of NS4B with the reverse reaction 2ADP --> ATP + ADP, yielding a larger k(cat) compared to that of the forward reaction ATP + AMP --> 2ADP. These studies suggest that HCV NS4B possesses both adenylate kinase activity and nucleotide hydrolase activity. Mutation of amino acids in the Walker A and B motifs of NS4B resulted in decreased affinity for both GTPgammaS and ATPgammaS as well as decreased ATP hydrolysis and AK activity.

Discovery of Allosteric, Potent, Subtype Selective, and Peripherally Restricted TrkA Kinase Inhibitors.

Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are activated by hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). Moreover, the NGF antibody tanezumab has provided clinical proof of concept for inhibition of the TrkA kinase pathway in pain leading to significant interest in the development of small molecule inhibitors of TrkA. However, achieving TrkA subtype selectivity over TrkB and TrkC via a Type I and Type II inhibitor binding mode has proven challenging and Type III or Type IV allosteric inhibitors may present a more promising selectivity design approach. Furthermore, TrkA inhibitors with minimal brain availability are required to deliver an appropriate safety profile. Herein, we describe the discovery of a highly potent, subtype selective, peripherally restricted, efficacious, and well-tolerated series of allosteric TrkA inhibitors that culminated in the delivery of candidate quality compound 23.

Small molecule inhibitors reveal PTK6 kinase is not an oncogenic driver in breast cancers.

Protein tyrosine kinase 6 (PTK6, or BRK) is aberrantly expressed in breast cancers, and emerging as an oncogene that promotes tumor cell proliferation, migration and evasion. Both kinase-dependent and -independent functions of PTK6 in driving tumor growth have been described, therefore targeting PTK6 kinase activity by small molecule inhibitors as a therapeutic approach to treat cancers remains to be validated. In this study, we identified novel, potent and selective PTK6 kinase inhibitors as a means to investigate the role of PTK6 kinase activity in breast tumorigenesis. We report here the crystal structures of apo-PTK6 and inhibitor-bound PTK6 complexes, providing the structural basis for small molecule interaction with PTK6. The kinase inhibitors moderately suppress tumor cell growth in 2D and 3D cell cultures. However, the tumor cell growth inhibition shows neither correlation with the PTK6 kinase activity inhibition, nor the total or activated PTK6 protein levels in tumor cells, suggesting that the tumor cell growth is independent of PTK6 kinase activity. Furthermore, in engineered breast tumor cells overexpressing PTK6, the inhibition of PTK6 kinase activity does not parallel the inhibition of tumor cell growth with a >500-fold shift in compound potencies (IC50 values). Overall, these findings suggest that the kinase activity of PTK6 does not play a significant role in tumorigenesis, thus providing important evidence against PTK6 kinase as a potential therapeutic target for breast cancer treatment.

Discovery of Potent, Selective, and Peripherally Restricted Pan-Trk Kinase Inhibitors for the Treatment of Pain.

Hormones of the neurotrophin family, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), are known to activate the family of Tropomyosin receptor kinases (TrkA, TrkB, and TrkC). Moreover, inhibition of the TrkA kinase pathway in pain has been clinically validated by the NGF antibody tanezumab, leading to significant interest in the development of small molecule inhibitors of TrkA. Furthermore, Trk inhibitors having an acceptable safety profile will require minimal brain availability. Herein, we discuss the discovery of two potent, selective, peripherally restricted, efficacious, and well-tolerated series of pan-Trk inhibitors which successfully delivered three candidate quality compounds 10b, 13b, and 19. All three compounds are predicted to possess low metabolic clearance in human that does not proceed via aldehyde oxidase-catalyzed reactions, thus addressing the potential clearance prediction liability associated with our current pan-Trk development candidate PF-06273340.

Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells.

A protocol is described for the production of both intracellularly expressed and secreted selenomethionyl-derivatized recombinant proteins in baculovirus-infected insect cells. The method results in the production of recombinant soluble proteins with an SeMet occupancy of approximately 75% and with a recovery of approximately 20% that of native protein expression. The method is independent of the percentage methionine content of the protein and is reliable and consistent. Similar results are obtained using either Spodoptera frugiperda Sf9 or Trichoplusia ni High Five insect cells as the expression host, and when cultures are grown in either shake flasks or in Wave BioReactors.

Structures of the cancer-related Aurora-A, FAK, and EphA2 protein kinases from nanovolume crystallography.

Protein kinases are important drug targets in human cancers, inflammation, and metabolic diseases. This report presents the structures of kinase domains for three cancer-associated protein kinases: ephrin receptor A2 (EphA2), focal adhesion kinase (FAK), and Aurora-A. The expression profiles of EphA2, FAK, and Aurora-A in carcinomas suggest that inhibitors of these kinases may have inherent potential as therapeutic agents. The structures were determined from crystals grown in nanovolume droplets, which produced high-resolution diffraction data at 1.7, 1.9, and 2.3 A for FAK, Aurora-A, and EphA2, respectively. The FAK and Aurora-A structures are the first determined within two unique subfamilies of human kinases, and all three structures provide new insights into kinase regulation and the design of selective inhibitors.

Development of a high-throughput crystal structure-determination platform for JAK1 using a novel metal-chelator soaking system.

Crystals of phosphorylated JAK1 kinase domain were initially generated in complex with nucleotide (ADP) and magnesium. The tightly bound Mg2+-ADP at the ATP-binding site proved recalcitrant to ligand displacement. Addition of a molar excess of EDTA helped to dislodge the divalent metal ion, promoting the release of ADP and allowing facile exchange with ATP-competitive small-molecule ligands. Many kinases require the presence of a stabilizing ligand in the ATP site for crystallization. This procedure could be useful for developing co-crystallization systems with an exchangeable ligand to enable structure-based drug design of other protein kinases.

Economical parallel protein expression screening and scale-up in Escherichia coli.

A novel microfermentation and scale-up platform for parallel protein production in Escherichia coli is described. The vertical shaker device Vertiga, which generates low-volume high density (A(600) approximately 20) Escherichia coli cultures in 96-position deep-well plates without auxiliary oxygen supplementation, has been coupled to a new disposable shake flask design, the Ultra Yield flask, that allows for equally high cell culture densities to be obtained. The Ultra Yield flask, which accommodates up to 1 l in culture volume, has a baffled base and a more vertical wall construction compared to traditional shake flask designs. Experimental data is presented demonstrating that the Ultra Yield flask generates, on average, an equivalent amount of recombinant protein per unit cell culture density as do traditional shake flask designs but at a substantially greater amount per unit volume. The combination of Vertiga and the Ultra Yield flask provides a convenient and scalable low-cost solution to parallel protein production in Escherichia coli.