Daniel F. Ortwine

Discovery and characterization of a novel inhibitor of matrix metalloprotease-13 that reduces cartilage damage in vivo without joint fibroplasia side effects.

Matrix metalloproteinase-13 (MMP13) is a Zn2+-dependent protease that catalyzes the cleavage of type II collagen, the main structural protein in articular cartilage. Excess MMP13 activity causes cartilage degradation in osteoarthritis, making this protease an attractive therapeutic target. However, clinically tested MMP inhibitors have been associated with a painful, joint-stiffening musculoskeletal side effect that may be due to their lack of selectivity. In our efforts to develop a disease-modifying osteoarthritis drug, we have discovered MMP13 inhibitors that differ greatly from previous MMP inhibitors; they do not bind to the catalytic zinc ion, they are noncompetitive with respect to substrate binding, and they show extreme selectivity for inhibiting MMP13. By structure-based drug design, we generated an orally active MMP13 inhibitor that effectively reduces cartilage damage in vivo and does not induce joint fibroplasias in a rat model of musculoskeletal syndrome side effects. Thus, highly selective inhibition of MMP13 in patients may overcome the major safety and efficacy challenges that have limited previously tested non-selective MMP inhibitors. MMP13 inhibitors such as the ones described here will help further define the role of this protease in arthritis and other diseases and may soon lead to drugs that safely halt cartilage damage in patients.

Histone Deacetylase (HDAC) Inhibitor Kinetic Rate Constants Correlate with Cellular Histone Acetylation but Not Transcription and Cell Viability

Background: The effect of HDAC inhibitor kinetic properties on biological function is currently unknown. Results: The kinetic rate constants of HDAC inhibitors differentially affect histone acetylation, cell viability, and gene expression. Conclusion: Evaluating HDAC inhibitor properties using histone acetylation is not predictive of their function on cellular activity. Significance: Characterizing the biological effect of different HDAC inhibitors will help to evaluate their clinical utility. Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.

Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist.

A channel involved in pain perception Voltage-gated sodium (Nav) channels propagate electrical signals in muscle cells and neurons. In humans, Nav1.7 plays a key role in pain perception. It is challenging to target a particular Nav isoform; however, arylsulfonamide antagonists selective for Nav1.7 have been reported recently. Ahuja et al. characterized the binding of these small molecules to human Nav channels. To further investigate the mechanism, they engineered a bacterial Nav channel to contain features of the Nav1.7 voltage-sensing domain that is targeted by the antagonist and determined the crystal structure of the chimera bound to an inhibitor. The structure gives insight into the mechanism of voltage sensing and will enable the design of more-selective Nav channel antagonists. Science, this issue p. 10.1126/science.aac5464 Structural studies give insight into how a human sodium channel involved in pain perception can be selectively inhibited. INTRODUCTION Voltage-gated sodium (Nav) channels open and close ion-selective pores in response to changes in membrane potential, and this gating underlies the generation of action potentials. Nav channels are large membrane proteins that contain four peripheral voltage-sensor domains (VSD1–4) that influence the functional state of the central ion-conducting pore. Mutations within the nine human Nav channel isoforms are associated with migraine (Nav1.1), epilepsy (Nav1.1–Nav1.3, Nav1.6), pain (Nav1.7–Nav1.9), cardiac (Nav1.5), and muscle paralysis (Nav1.4) syndromes. Accordingly, Nav channel blockers are used for the treatment of many neurological and cardiovascular disorders. These drugs bind within the central pore domain and generally lack isoform selectivity owing to the high sequence conservation found among Nav channels, limiting their therapeutic utility. In this study, we focused on a recently identified class of isoform-selective small-molecule antagonists that target a unique binding site on the fourth voltage-sensor domain, VSD4. Here we report the structural determination of such small-molecule aryl sulfonamide antagonists in complex with human Nav1.7 VSD4. Our studies demonstrate how this important new class of gating modifier engages VSD4 to inhibit Nav channel activity through a “voltage-sensor trapping” mechanism. RATIONALE For structural studies, we devised a novel protein-engineering strategy that overcomes the technical complexities of producing full-length human Nav channels. Exploiting the evolutionary relationship between human and bacterial Nav channels, we fused portions of Nav1.7 VSD4 onto the bacterial channel NavAb. Using ligand-binding assays and alanine-scanning mutagenesis, we demonstrated that the antagonist binding site present in the human Nav1.7 channel is preserved within this human VSD4-NavAb chimeric channel. This chimeric construct allowed purification, crystallization, and structure determination of potent aryl sulfonamide antagonists in complex with the human Nav1.7 VSD4 binding site. RESULTS Functional studies using patch-clamp electrophysiology revealed that aryl sulfonamide inhibitors bind with high affinity to an isoform-selective and extracellularly accessible site on VSD4. These inhibitors show a high level of state dependence, potently blocking human Nav1.7 only when VSD4 is in its activated conformation. Our crystallographic studies revealed that the anionic warhead from the aryl sulfonamide inhibitors directly engages the fourth gating charge residue (R4) on the voltage-sensing S4 helix, effectively trapping VSD4 in its activated state. Isoform selectivity is achieved by inhibitor interactions with nonconserved residues found on the S2 and S3 transmembrane helices. The drug receptor site is partially submerged within the membrane bilayer, and a peripherally bound phospholipid was observed to form a tripartite complex with the antagonist and channel. CONCLUSION A new crystallization strategy has enabled the structural determination of VSD4 from human Nav1.7 in complex with potent, state-dependent, isoform-selective small-molecule antagonists. Mechanistically, inhibitor binding traps VSD4 in an activated conformation, which stabilizes a nonconductive state of the channel, and likely prevents recovery from inactivation. Unique phospholipid interactions and an exposed inhibitor binding site expand the importance of the membrane bilayer in ion channel biology. We anticipate that these structures will enable drug design efforts aimed at other voltage-gated ion channels and may accelerate the development of new treatments for pain that selectively target Nav1.7. Drug binding sites in sodium channels. (Left) Top-view model of human Nav1.7. When open, sodium passes through the channel. Blocking drugs lacking isoform selectivity bind to a conserved site within the central pore. Isoform-selective inhibitors bind to a distinct site on VSD4. (Right) Strategy for Nav1.7 crystallography. Portions of Nav1.7 VSD4 were grafted onto a tetrameric channel (NavAb) and crystallized. (Inset) Side view of aryl sulfonamide binding site with the S4 helix and arginine gating charges highlighted pink. Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.

The evaluation of 25 chiral stationary phases and the utilization of sub-2.0μm coated polysaccharide chiral stationary phases via supercritical fluid chromatography.

A rapid screening method to identify the best conditions for chiral separations is described. We analyzed a representative set of 80 racemic compounds against 25 different chiral stationary phases with three different mobile phases to identify the combination of columns and mobile phases that will separate the most compounds on the initial screen. While the OD separated the largest number of compounds, we found the best combination of six columns to be the AD, AS, AY, CC4, ID and Whelk-O1. The second team included the CCC, Cellulose-1, Cellulose-3 or OJ, IA, IE and IF. All 80 compounds were separated with a resolution range of 0.65-15.36. Screening the covalently bonded phases provided separation for 79 of the 80 compounds. We also found ethanol (0.1% NH4OH) separated more compounds than methanol (0.1% NH4OH) or isopropanol (0.1% NH4OH). As part of this study, we also compared the effectiveness of stationary phases that have the same chiral selector. Finally, we demonstrated the effectiveness of using a fast, 1.5-min screening method that utilizes a 1.7μm coated polysaccharide chiral stationary phase.

Identification, Characterization, and Implications of Species-Dependent Plasma Protein Binding for the Oral Hedgehog Pathway Inhibitor Vismodegib (GDC-0449)

Vismodegib (GDC-0449) is is an orally available selective Hedgehog pathway inhibitor in development for cancer treatment. The drug is ≥95% protein bound in plasma at clinically relevant concentrations and has an approximately 200-fold longer single dose half-life in humans than rats. We have identified a strong linear relationship between plasma drug concentrations and α-1-acid glycoprotein (AAG) in a phase I study. Biophysical and cellular techniques have been used to reveal that vismodegib strongly binds to human AAG (K(D) = 13 μM) and binds albumin with lower affinity (K(D) = 120 μM). Additionally, binding to rat AAG is reduced ∼20-fold relative to human, whereas the binding affinity to rat and human albumin was similar. Molecular docking studies reveal the reason for the signficiant species dependence on binding. These data highlight the utility of biophysical techniques in creating a comprehensive picture of protein binding across species.

Structure based design of an in vivo active hydroxamic acid inhibitor of P. aeruginosa LpxC

Lipid A is an essential component of the Gram negative outer membrane, which protects the bacterium from attack of many antibiotics. The Lipid A biosynthesis pathway is essential for Gram negative bacterial growth and is unique to these bacteria. The first committed step in Lipid A biosynthesis is catalysis by LpxC, a zinc dependent deacetylase. We show the design of an LpxC inhibitor utilizing a robust model which directed efficient design of picomolar inhibitors. Analysis of physiochemical properties drove design to focus on an optimal lipophilicity profile. Further structure based design took advantage of a conserved water network over the active site, and with the optimal lipophilicity profile, led to an improved LpxC inhibitor with in vivo activity against wild type Pseudomonas aeruginosa.

Pyrimidoaminotropanes as Potent, Selective, and Efficacious Small Molecule Kinase Inhibitors of the Mammalian Target of Rapamycin (mTOR)

We have recently reported a series of tetrahydroquinazoline (THQ) mTOR inhibitors that produced a clinical candidate 1 (GDC-0349). Through insightful design, we hoped to discover and synthesize a new series of small molecule inhibitors that could attenuate CYP3A4 time-dependent inhibition commonly observed with the THQ scaffold, maintain or improve aqueous solubility and oral absorption, reduce free drug clearance, and selectively increase mTOR potency. Through key in vitro and in vivo studies, we demonstrate that a pyrimidoaminotropane based core was able to address each of these goals. This effort culminated in the discovery of 20 (GNE-555), a highly potent, selective, metabolically stable, and efficacious mTOR inhibitor.

Potent, Selective, and Orally Bioavailable Inhibitors of the Mammalian Target of Rapamycin Kinase Domain Exhibiting Single Agent Antiproliferative Activity

Selective inhibitors of mammalian target of rapamycin (mTOR) kinase based upon saturated heterocycles fused to a pyrimidine core were designed and synthesized. Each series produced compounds with K(i) < 10 nM for the mTOR kinase and >500-fold selectivity over closely related PI3 kinases. This potency translated into strong pathway inhibition, as measured by phosphorylation of mTOR substrate proteins and antiproliferative activity in cell lines with a constitutively active PI3K pathway. Two compounds exhibiting suitable mouse PK were profiled in in vivo tumor models and were shown to suppress mTORC1 and mTORC2 signaling for over 12 h when dosed orally. Both compounds were additionally shown to suppress tumor growth in vivo in a PC3 prostate cancer model over a 14 day study.

Potent, selective, and orally bioavailable inhibitors of mammalian target of rapamycin (mTOR) kinase based on a quaternary substituted dihydrofuropyrimidine.

A series of inhibitors of mTOR kinase based on a quaternary-substituted dihydrofuropyrimidine was designed and synthesized. The most potent compounds in this series inhibited mTOR kinase with K(i) < 1.0 nM and were highly (>100×) selective for mTOR over the closely related PI3 kinases. Compounds in this series showed inhibition of the pathway and antiproliferative activity in cell-based assays. Furthermore, these compounds had excellent mouse PK, and showed a robust PK-PD relationship in a mouse model of cancer.

Property- and structure-guided discovery of a tetrahydroindazole series of interleukin-2 inducible T-cell kinase inhibitors.

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays a major role in T-cell signaling downstream of the T-cell receptor (TCR), and considerable efforts have been directed toward discovery of ITK-selective inhibitors as potential treatments of inflammatory disorders such as asthma. Using a previously disclosed indazole series of inhibitors as a starting point, and using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.