Discovery of Lanraplenib (GS-9876): A Once-Daily Spleen Tyrosine Kinase Inhibitor for Autoimmune Diseases.

Spleen tyrosine kinase (SYK) is a critical regulator of signaling in a variety of immune cell types such as B-cells, monocytes, and macrophages. Accordingly, there have been numerous efforts to identify compounds that selectively inhibit SYK as a means to treat autoimmune and inflammatory diseases. We previously disclosed GS-9973 (entospletinib) as a selective SYK inhibitor that is under clinical evaluation in hematological malignancies. However, a BID dosing regimen and drug interaction with proton pump inhibitors (PPI) prevented development of entospletinib in inflammatory diseases. Herein, we report the discovery of a second-generation SYK inhibitor, GS-9876 (lanraplenib), which has human pharmacokinetic properties suitable for once-daily administration and is devoid of any interactions with PPI. Lanraplenib is currently under clinical evaluation in multiple autoimmune indications.

Discovery of Potent and Selective MTH1 Inhibitors for Oncology: Enabling Rapid Target (In)Validation.

We describe the discovery of three structurally differentiated potent and selective MTH1 inhibitors and their subsequent use to investigate MTH1 as an oncology target, culminating in target (in)validation. Tetrahydronaphthyridine 5 was rapidly identified as a highly potent MTH1 inhibitor (IC50 = 0.043 nM). Cocrystallization of 5 with MTH1 revealed the ligand in a Φ-cis-N-(pyridin-2-yl)acetamide conformation enabling a key intramolecular hydrogen bond and polar interactions with residues Gly34 and Asp120. Modification of literature compound TH287 with O- and N-linked aryl and alkyl aryl substituents led to the discovery of potent pyrimidine-2,4,6-triamine 25 (IC50 = 0.49 nM). Triazolopyridine 32 emerged as a highly selective lead compound with a suitable in vitro profile and desirable pharmacokinetic properties in rat. Elucidation of the DNA damage response, cell viability, and intracellular concentrations of oxo-NTPs (oxidized nucleoside triphosphates) as a function of MTH1 knockdown and/or small molecule inhibition was studied. Based on our findings, we were unable to provide evidence to further pursue MTH1 as an oncology target.

Discovery of Potent and Selective Tricyclic Inhibitors of Bruton's Tyrosine Kinase with Improved Druglike Properties.

In our continued effort to discover and develop best-in-class Bruton's tyrosine kinase (Btk) inhibitors for the treatment of B-cell lymphomas, rheumatoid arthritis, and systemic lupus erythematosus, we devised a series of novel tricyclic compounds that improved upon the druglike properties of our previous chemical matter. Compounds exemplified by G-744 are highly potent, selective for Btk, metabolically stable, well tolerated, and efficacious in an animal model of arthritis.

Btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell-associated damage in IFNα-driven lupus nephritis.

Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton's tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE.

Discovery of highly potent and selective Bruton's tyrosine kinase inhibitors: Pyridazinone analogs with improved metabolic stability.

BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in a suspension of clinical trials. The primary route of metabolism was through cleavage of the acyclic amide bond connecting the terminal tetrahydrobenzothiophene with the central linker aryl ring. SAR studies were focused on reducing metabolic cleavage of this amide, and resulted in the identification of several central aryl linker substituents that conferred improved stability. The most promising substituted aryl linkers were then incorporated into an optimized pyridazinone scaffold, resulting in the identification of lead analog 23, possessing improved potency, metabolic stability and preclinical properties.

Potent and selective Bruton's tyrosine kinase inhibitors: discovery of GDC-0834.

SAR studies focused on improving the pharmacokinetic (PK) properties of the previously reported potent and selective Btk inhibitor CGI-1746 (1) resulted in the clinical candidate GDC-0834 (2), which retained the potency and selectivity of CGI-1746, but with much improved PK in preclinical animal models. Structure based design efforts drove this work as modifications to 1 were investigated at both the solvent exposed region as well as 'H3 binding pocket'. However, in vitro metabolic evaluation of 2 revealed a non CYP-mediated metabolic process that was more prevalent in human than preclinical species (mouse, rat, dog, cyno), leading to a high-level of uncertainly in predicting human pharmacokinetics. Due to its promising potency, selectivity, and preclinical efficacy, a single dose IND was filed and 2 was taken in to a single dose phase I trial in healthy volunteers to quickly evaluate the human pharmacokinetics. In human, 2 was found to be highly labile at the exo-cyclic amide bond that links the tetrahydrobenzothiophene moiety to the central aniline ring, resulting in insufficient parent drug exposure. This information informed the back-up program and discovery of improved inhibitors.

Discovery of GS-9973, a selective and orally efficacious inhibitor of spleen tyrosine kinase.

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.

Image processing techniques for assessing contractility in isolated neonatal cardiac myocytes.

We describe a computational framework for the quantitative assessment of contractile responses of isolated neonatal cardiac myocytes. To the best of our knowledge, this is the first report on a practical and accessible method for the assessment of contractility in neonatal cardiocytes. The proposed methodology is comprised of digital video recording of the contracting cell, signal preparation, representation by polar Fourier descriptors, and contractility assessment. The different processing stages are variants of mathematically sound and computationally robust algorithms very well established in the scientific community. The described computational approach provides a comprehensive assessment of the neonatal cardiac myocyte contraction without the need of elaborate instrumentation. The versatility of the methodology allows it to be employed in determining myocyte contractility almost simultaneously with the acquisition of the Ca(2+) transient and other correlates of cell contraction. The proposed methodology can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease models, transgeneity, or other common applications of neonatal cardiocytes.

Introduction of non-linear elasticity models for characterization of shape and deformation statistics: application to contractility assessment of isolated adult cardiocytes.

BACKGROUND: We are exploring the viability of a novel approach to cardiocyte contractility assessment based on biomechanical properties of the cardiac cells, energy conservation principles, and information content measures. We define our measure of cell contraction as being the distance between the shapes of the contracting cell, assessed by the minimum total energy of the domain deformation (warping) of one cell shape into another. To guarantee a meaningful vis-à-vis correspondence between the two shapes, we employ both a data fidelity term and a regularization term. The data fidelity term is based on nonlinear features of the shapes while the regularization term enforces the compatibility between the shape deformations and that of a hyper-elastic material. RESULTS: We tested the proposed approach by assessing the contractile responses in isolated adult rat cardiocytes and contrasted these measurements against two different methods for contractility assessment in the literature. Our results show good qualitative and quantitative agreements with these methods as far as frequency, pacing, and overall behavior of the contractions are concerned. CONCLUSIONS: We hypothesize that the proposed methodology, once appropriately developed and customized, can provide a framework for computational cardiac cell biomechanics that can be used to integrate both theory and experiment. For example, besides giving a good assessment of contractile response of the cardiocyte, since the excitation process of the cell is a closed system, this methodology can be employed in an attempt to infer statistically significant model parameters for the constitutive equations of the cardiocytes.

Imidazo[1,2-a]pyrazine diaryl ureas: inhibitors of the receptor tyrosine kinase EphB4.

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent and Nexavar, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.

Structure enhancement diffusion and contour extraction for electron tomography of mitochondria.

The interpretation and measurement of the architectural organization of mitochondria depend heavily upon the availability of good software tools for filtering, segmenting, extracting, measuring, and classifying the features of interest. Images of mitochondria contain many flow-like patterns and they are usually corrupted by large amounts of noise. Thus, it is necessary to enhance them by denoising and closing interrupted structures. We introduce a new approach based on anisotropic nonlinear diffusion and bilateral filtering for electron tomography of mitochondria. It allows noise removal and structure closure at certain scales, while preserving both the orientation and magnitude of discontinuities without the need for threshold switches. This technique facilitates image enhancement for subsequent segmentation, contour extraction, and improved visualization of the complex and intricate mitochondrial morphology. We perform the extraction of the structure-defining contours by employing a variational level set formulation. The propagating front for this approach is an approximate signed distance function which does not require expensive re-initialization. The behavior of the combined approach is tested for visualizing the structure of a HeLa cell mitochondrion and the results we obtain are very promising.

Image processing techniques for assessing contractility in isolated adult cardiac myocytes.

We describe a computational framework for the comprehensive assessment of contractile responses of enzymatically dissociated adult cardiac myocytes. The proposed methodology comprises the following stages: digital video recording of the contracting cell, edge preserving total variation-based image smoothing, segmentation of the smoothed images, contour extraction from the segmented images, shape representation by Fourier descriptors, and contractility assessment. The different stages are variants of mathematically sound and computationally robust algorithms very well established in the image processing community. The physiologic application of the methodology is evaluated by assessing overall contraction in enzymatically dissociated adult rat cardiocytes. Our results demonstrate the effectiveness of the proposed approach in characterizing the true, two-dimensional, "shortening" in the contraction process of adult cardiocytes. We compare the performance of the proposed method to that of a popular edge detection system in the literature. The proposed method not only provides a more comprehensive assessment of the myocyte contraction process but also can potentially eliminate historical concerns and sources of errors caused by myocyte rotation or translation during contraction. Furthermore, the versatility of the image processing techniques makes the method suitable for determining myocyte shortening in cells that usually bend or move during contraction. The proposed method can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease modeling, transgeneity, or other common applications to mammalian cardiocytes.

Bifurcation analysis of bubble dynamics in fluidized beds.

We use a low-dimensional, agent-based bubble model to study the changes in the global dynamics of fluidized beds in response to changes in the frequency of the rising bubbles. The computationally based bifurcation analysis shows that at low frequencies, the global dynamics is attracted towards a fixed point since the bubbles interact very little with one another. As the frequency of injection increases, however, the global dynamics undergoes a series of bifurcations to new behaviors that include highly periodic orbits, chaotic attractors, and intermittent behavior between periodic orbits and chaotic sets. Using methods from time-series analysis, we are able to approximate nonlinear models that allow for long-term predictions and the possibility of developing control algorithms.

Design, synthesis, and antiviral properties of 4'-substituted ribonucleosides as inhibitors of hepatitis C virus replication: the discovery of R1479.

A series of 4'-substituted ribonucleoside derivatives has been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The most potent and non-cytotoxic derivative was compound 28 (4'-azidocytidine, R1479) with an IC(50) of 1.28 microM in the HCV replicon system. The triphosphate of compound 28 was prepared and shown to be an inhibitor of RNA synthesis mediated by NS5B (IC(50)=320 nM), the RNA polymerase encoded by HCV. Data on related analogues have been used to generate some preliminary requirements for activity within this series of nucleosides.

Noise-induced intermittent cellular patterns on circular domains.

We study the effects of thermal noise in a stochastic Langevin formulation of a typical example of a pattern-forming system with two-dimensional circular domain. A greater tendency towards dynamic cellular states is observed when the pattern-forming system is subjected to noise, which seems to explain the prevailing behavior of related laboratory experiments. We also report on two-dimensional numerical observations of certain dynamic states, homoclinic intermittent states, which until now, had only been observed in laboratory experiments.

Synthesis of RNA using 2'-O-DTM protection.

tert-Butyldithiomethyl (DTM), a novel hydroxyl protecting group, cleavable under reductive conditions, was developed and applied for the protection of 2'-OH during solid-phase RNA synthesis. This function is compatible with all standard protecting groups used in oligonucleotide synthesis, and allows for fast and high-yield synthesis of RNA. Oligonucleotides containing the 2'-O-DTM groups can be easily deprotected under the mildest possible aqueous and homogeneous conditions. The preserved 5'-O-DMTr function can be used for high-throughput cartridge RNA purification.

Stable second-order scheme for integrating the Kuramoto-Sivanshinsky equation in polar coordinates using distributed approximating functionals.

We present an algorithm for the time integration of nonlinear partial differential equations. The algorithm uses distributed approximating functionals, which are based on an analytic approximation method, in order to achieve highly accurate spatial derivatives. The time integration is based on a second-order unconditionally A -stable Crank-Nicolson scheme with a Newton solver. We apply the integration scheme to the Kuramoto-Sivanshinsky equation in polar coordinates, which presents a significant computational challenge due to the stiffness introduced by the estimation of the spatial derivatives at the origin. We present several stationary and nonstationary solutions of the Kuramoto-Sivanshinsky equation and compare with previous numerical results as well as patterns observed in the combustion front of a circular burner. The numerical results of the proposed scheme reproduces several patterns--rotating two-cell, three-cell, hopping three-cell, stationary two-three-four- and five-cell, stationary 5/1,6/1,7/1,8/2 two-ring patterns, etc.--observed in physical experiments. The scheme is extremely robust and can produce long-term simulations consisting of several thousand frames. Although applied to a very specific problem, the approach of combining the framework of distributed approximating functionals with a Crank-Nicolson based time integration is generalizable to a large class of problems.

Hopping behavior in the Kuramoto-Sivashinsky equation.

We report the first observations of numerical "hopping" cellular flame patterns found in computer simulations of the Kuramoto-Sivashinsky equation. Hopping states are characterized by nonuniform rotations of a ring of cells, in which individual cells make abrupt changes in their angular positions while they rotate around the ring. Until now, these states have been observed only in experiments but not in truly two-dimensional computer simulations. A modal decomposition analysis of the simulated patterns, via the proper orthogonal decomposition, reveals spatio-temporal behavior in which the overall temporal dynamics is similar to that of equivalent experimental states but the spatial dynamics exhibits a few more features that are not seen in the experiments. Similarities in the temporal behavior and subtle differences in the spatial dynamics between numerical hopping states and their experimental counterparts are discussed in more detail.

Super-resolution in time-reversal acoustics.

The phenomenon of super-resolution in time-reversal acoustics is analyzed theoretically and with numerical simulations. A signal that is recorded and then retransmitted by an array of transducers, propagates back though the medium, and refocuses approximately on the source that emitted it. In a homogeneous medium, the refocusing resolution of the time-reversed signal is limited by diffraction. When the medium has random inhomogeneities the resolution of the refocused signal can in some circumstances beat the diffraction limit. This is super-resolution. A theoretical treatment of this phenomenon is given, and numerical simulations which confirm the theory are presented.