Discovery and Preclinical Characterization of BIIB129, a Covalent, Selective, and Brain-Penetrant BTK Inhibitor for the Treatment of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disease with an underlying pathology characterized by inflammation-driven neuronal loss, axonal injury, and demyelination. Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase and member of the TEC family of kinases, is involved in the regulation, migration, and functional activation of B cells and myeloid cells in the periphery and the central nervous system (CNS), cell types which are deemed central to the pathology contributing to disease progression in MS patients. Herein, we describe the discovery of BIIB129 (25), a structurally distinct and brain-penetrant targeted covalent inhibitor (TCI) of BTK with an unprecedented binding mode responsible for its high kinome selectivity. BIIB129 (25) demonstrated efficacy in disease-relevant preclinical in vivo models of B cell proliferation in the CNS, exhibits a favorable safety profile suitable for clinical development as an immunomodulating therapy for MS, and has a low projected total human daily dose.

Optimization of a novel piperazinone series as potent selective peripheral covalent BTK inhibitors.

BTK is a tyrosine kinase playing an important role in B cell and myeloid cell functions through B cell receptor (BCR) signaling and Fc receptor (FcR) signaling. Selective inhibition of BTK has the potential to provide therapeutical benefits to patients suffering from autoimmune diseases. Here we report the design, optimization, and characterization of novel potent and highly selective covalent BTK inhibitors. Starting from a piperazinone hit derived from a selective reversible inhibitor, we solved the whole blood cellular potency issue by introducing an electrophilic warhead to reach Cys481. This design led to a covalent irreversible BTK inhibitor series with excellent kinase selectivity as well as good whole blood CD69 cellular potency. Optimization of metabolic stability led to representative compounds like 42, which demonstrated strong cellular target occupancy and inhibition of B-cell proliferation measured by proximal and distal functional activity.

Discovery and Preclinical Characterization of BIIB091, a Reversible, Selective BTK Inhibitor for the Treatment of Multiple Sclerosis.

Multiple Sclerosis is a chronic autoimmune neurodegenerative disorder of the central nervous system (CNS) that is characterized by inflammation, demyelination, and axonal injury leading to permeant disability. In the early stage of MS, inflammation is the primary driver of the disease progression. There remains an unmet need to develop high efficacy therapies with superior safety profiles to prevent the inflammation processes leading to disability. Herein, we describe the discovery of BIIB091, a structurally distinct orthosteric ATP competitive, reversible inhibitor that binds the BTK protein in a DFG-in confirmation designed to sequester Tyr-551, an important phosphorylation site on BTK, into an inactive conformation with excellent affinity. Preclinical studies demonstrated BIB091 to be a high potency molecule with good drug-like properties and a safety/tolerability profile suitable for clinical development as a highly selective, reversible BTKi for treating autoimmune diseases such as MS.

Discovery of Potent, Selective, and Brain-Penetrant Apoptosis Signal-Regulating Kinase 1 (ASK1) Inhibitors that Modulate Brain Inflammation In Vivo.

Apoptosis signal-regulating kinase 1 (ASK1) is one of the key mediators of the cellular stress response that regulates inflammation and apoptosis. To probe the therapeutic value of modulating this pathway in preclinical models of neurological disease, we further optimized the profile of our previously reported inhibitor 3. This effort led to the discovery of 32, a potent (cell IC50 = 25 nM) and selective ASK1 inhibitor with suitable pharmacokinetic and brain penetration (rat Cl/Clu = 1.6/56 L/h/kg and Kp,uu = 0.46) for proof-of-pharmacology studies. Specifically, the ability of 32 to inhibit ASK1 in the central nervous system (CNS) was evaluated in a human tau transgenic (Tg4510) mouse model exhibiting elevated brain inflammation. In this study, transgenic animals treated with 32 (at 3, 10, and 30 mg/kg, BID/PO for 4 days) showed a robust reduction of inflammatory markers (e.g., IL-1ß) in the cortex, thus confirming inhibition of ASK1 in the CNS.

Next-generation Bruton's tyrosine kinase inhibitor BIIB091 selectively and potently inhibits B cell and Fc receptor signaling and downstream functions in B cells and myeloid cells.

OBJECTIVES: Bruton's tyrosine kinase (BTK) plays a non-redundant signaling role downstream of the B-cell receptor (BCR) in B cells and the receptors for the Fc region of immunoglobulins (FcR) in myeloid cells. Here, we characterise BIIB091, a novel, potent, selective and reversible small-molecule inhibitor of BTK. METHODS: BIIB091 was evaluated in vitro and in vivo in preclinical models and in phase 1 clinical trial. RESULTS: In vitro, BIIB091 potently inhibited BTK-dependent proximal signaling and distal functional responses in both B cells and myeloid cells with IC50s ranging from 3 to 106 nm, including antigen presentation to T cells, a key mechanism of action thought to be underlying the efficacy of B cell-targeted therapeutics in multiple sclerosis. BIIB091 effectively sequestered tyrosine 551 in the kinase pocket by forming long-lived complexes with BTK with t 1/2 of more than 40 min, thereby preventing its phosphorylation by upstream kinases. As a key differentiating feature of BIIB091, this property explains the very potent whole blood IC50s of 87 and 106 nm observed with stimulated B cells and myeloid cells, respectively. In vivo, BIIB091 blocked B-cell activation, antibody production and germinal center differentiation. In phase 1 healthy volunteer trial, BIIB091 inhibited naïve and unswitched memory B-cell activation, with an in vivo IC50 of 55 nm and without significant impact on lymphoid or myeloid cell survival after 14 days of dosing. CONCLUSION: Pharmacodynamic results obtained in preclinical and early clinical settings support the advancement of BIIB091 in phase 2 clinical trials.

Discovery of CNS-Penetrant Apoptosis Signal-Regulating Kinase 1 (ASK1) Inhibitors.

Apoptosis signal-regulating kinase 1 (ASK1) is a key mediator in the apoptotic and inflammatory cellular stress response. To investigate the therapeutic value of modulating this pathway in neurological disease, we have completed medicinal chemistry studies to identify novel CNS-penetrant ASK1 inhibitors starting from peripherally restricted compounds reported in the literature. This effort led to the discovery of 21, a novel ASK1 inhibitor with good potency (cell IC50 = 138 nM), low clearance (rat Cl/Clu = 0.36/6.7 L h-1 kg-1) and good CNS penetration (rat K p,uu = 0.38).

Human PLD structures enable drug design and characterization of isoenzyme selectivity.

Phospholipase D enzymes (PLDs) are ubiquitous phosphodiesterases that produce phosphatidic acid (PA), a key second messenger and biosynthetic building block. Although an orthologous bacterial Streptomyces sp. strain PMF PLD structure was solved two decades ago, the molecular basis underlying the functions of the human PLD enzymes (hPLD) remained unclear based on this structure due to the low homology between these sequences. Here, we describe the first crystal structures of hPLD1 and hPLD2 catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes. Furthermore, structure-based mutation studies and structures of inhibitor-hPLD complexes allowed us to elucidate the binding modes of dual and isoform-selective inhibitors, highlight key determinants of isoenzyme selectivity and provide a basis for further structure-based drug discovery and functional characterization of this therapeutically important superfamily of enzymes.

Rational Design and Optimization of a Novel Class of Macrocyclic Apoptosis Signal-Regulating Kinase 1 Inhibitors.

Structural analysis of a known apoptosis signal-regulating kinase 1 (ASK1) inhibitor bound to its kinase domain led to the design and synthesis of the novel macrocyclic inhibitor 8 (cell IC50 = 1.2 µM). The profile of this compound was optimized for CNS penetration following two independent strategies: a rational design approach leading to 19 and a parallel synthesis approach leading to 26. Both analogs are potent ASK1 inhibitors in biochemical and cellular assays (19, cell IC50 = 95 nM; 26, cell IC50 = 123 nM) and have moderate to low efflux ratio (ER) in an MDR1-MDCK assay (19, ER = 5.2; 26, ER = 1.5). In vivo PK studies revealed that inhibitor 19 had moderate CNS penetration (Kpuu = 0.17) and analog 26 had high CNS penetration (Kpuu = 1.0).

Rapid Resolution of Polyhydramnios Foretells Circulatory Collapse for the Donor Twin in Feto-Fetal Transfusion Syndrome.

Feto-fetal transfusion syndrome is a pathological process unique to diamniotic monochorionic pregnancies. It is the consequence of an unbalanced fetal blood flow through communicating vessels within a shared placenta. When it occurs, a polyuric, hypervolemic recipient twin co-exists with a hypovolemic oliguric donor. The presence of polyhydramnios or oligohydramnios is considered a poor prognostic indicator, whereas normal amniotic fluid volumes indicate a lack of clinically significant twintwin transfusion. In addition, the spontaneous normalization of amniotic fluid volume is usually seen as a favorable prognostic sign. Here, however, we present a case of feto-fetal transfusion in a 31 year-old primigravida at 19 week, in which the spontaneous normalization of amniotic fluid volume in the recipient twin preceded the death of the donor.

Quantitative determination of human interleukin 22 (IL-22) in serum using Singulex-Erenna® technology.

Interleukin-22 (IL-22) is a key mediator of inflammatory processes associated with diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis. The measurement of this cytokine in human plasma may provide insight into safety, pharmacodynamics and efficacy of drugs targeting inflammatory pathways. However, commonly used immunoassays are not sufficiently sensitive to measure baseline concentrations of IL-22. Here we describe the analytical validation of an ultrasensitive assay for the measurement of IL-22 in human serum using the Erenna® system by Singulex (Alameda, CA). The lower limit of quantification (LLOQ) of the Erenna assay estimated at 0.2pg/mL was sensitive enough to measure IL-22 in all human serum samples tested. The assay ranged from 0.2 to 100.0pg/mL and showed good dilution linearity. The inter-assay and intra-assay imprecision were <9% and <7% CV respectively. The accuracy determined by spiked recovery in serum samples was >86%. In addition, the results using Erenna assay correlated well with those using the IL-22 Quantikine immunoassay (R&D Systems, Minneapolis, MN) with a coefficient R(2) of 0.9285. However the Erenna assay showed an improved sensitivity by approximately 2 logs. These results show that this novel assay offers a significant improvement over previous methods for high-sensitive quantitative measurement of IL-22 in human serum samples.

The importance of family management, closeness with father and family structure in early adolescent alcohol use.

AIMS: To examine the importance of family management, family structure and father-adolescent relationships on early adolescent alcohol use. DESIGN: Cross-sectional data was collected across 30 randomly selected Australian communities stratified to represent a range of socio-economic and regional variation. SETTING: Data were collected during school time from adolescents attending a broad range of schools. PARTICIPANTS: The sample consisted of a combined 8256 students (aged 10-14 years). MEASUREMENTS: Students completed a web-based survey as part of the Healthy Neighbourhoods project. FINDINGS: Family management-which included practices such as parental monitoring and family rules about alcohol use-had the strongest and most consistent relationship with alcohol use in early adolescence. Adolescents reporting higher family management were less likely to have drunk alcohol in their life-time, less likely to drink alcohol in the preceding 30 days and less likely to have had an alcohol binge. Adolescents reporting emotionally close relationships with their fathers were less likely to have drunk alcohol in their life-time and less likely to have had an alcohol binge in the preceding fortnight. CONCLUSIONS: Findings indicate that family management practices may contribute to alcohol abstinence in adolescents. Furthermore, emotionally close father-adolescent relationships may also foster abstinence; however, fathers' drinking behaviours need to be considered.

Evaluation of respiration-correlated digital tomosynthesis in lung.

Digital tomosynthesis (DTS) with a linear accelerator-mounted imaging system provides a means of reconstructing tomographic images from radiographic projections over a limited gantry arc, thus requiring only a few seconds to acquire. Its application in the thorax, however, often results in blurred images from respiration-induced motion. This work evaluates the feasibility of respiration-correlated (RC) DTS for soft-tissue visualization and patient positioning. Image data acquired with a gantry-mounted kilovoltage imaging system while recording respiration were retrospectively analyzed from patients receiving radiotherapy for non-small-cell lung carcinoma. Projection images spanning an approximately 30 degrees gantry arc were sorted into four respiration phase bins prior to DTS reconstruction, which uses a backprojection, followed by a procedure to suppress structures above and below the reconstruction plane of interest. The DTS images were reconstructed in planes at different depths through the patient and normal to a user-selected angle close to the center of the arc. The localization accuracy of RC-DTS was assessed via a comparison with CBCT. Evaluation of RC-DTS in eight tumors shows visible reduction in image blur caused by the respiratory motion. It also allows the visualization of tumor motion extent. The best image quality is achieved at the end-exhalation phase of the respiratory motion. Comparison of RC-DTS with respiration-correlated cone-beam CT in determining tumor position, motion extent and displacement between treatment sessions shows agreement in most cases within 2-3 mm, comparable in magnitude to the intraobserver repeatability of the measurement. These results suggest the method's applicability for soft-tissue image guidance in lung, but must be confirmed with further studies in larger numbers of patients.

Use of corticocancellous allogeneic bone blocks for augmentation of alveolar bone defects.

PURPOSE: The use of autogenous block bone grafts in bone regeneration procedures for alveolar ridge augmentation can be limited by donor site morbidity and complications. The purpose of the present study was to evaluate the efficacy of allogeneic corticocancellous iliac block grafts used for ridge augmentation prior to implant placement. MATERIALS AND METHODS: Forty-one patients with severe ridge volume deficiency underwent augmentation using allogeneic corticocancellous iliac block bone grafts. After rigid fixation of the graft, the site was covered with a freeze-dried allogeneic dura mater membrane, and the wound was closed with tension-free suturing. Implants were placed 3 to 4 months after surgery. Three to 6 months after implant placement, panoramic radiographs were taken and implants were uncovered for prosthetic restoration. RESULTS: Of the 57 grafts placed, one showed 2.5 mm of resorption at the superior buccal aspect of the graft. No other clinical problems were observed. The block grafts were clinically well integrated into the recipient sites and the augmented bone remained stable throughout the implant placement procedures. Of the 84 implants placed, only one failed to integrate. CONCLUSION: These results demonstrate that the use of allogeneic corticocancellous iliac block bone grafts in conjunction with guided bone regeneration principles is a viable alternative to autogenous grafts in selected patients with alveolar ridge deficiencies.

A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.

PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

From phase-based to displacement-based gating: a software tool to facilitate respiration-gated radiation treatment.

The Varian Real-time Position Management (RPM) system allows respiratory gating based on either the phase or displacement (amplitude) of the breathing waveform. A problem in clinical application is that phase-based gating, required for respiration-correlated (4D-CT) simulation, is not robust to irregular breathing patterns during treatment, and a widely used system version (1.6) does not provide an easy means to change from a phase-based gate into an equivalent displacement-based one. We report on the development and evaluation of a robust method to convert phase-gate thresholds, set by the physician, into equivalent displacement-gate thresholds to facilitate its clinical application to treatment. The software tool analyzes the respiration trace recorded during the 4D-CT simulation, and determines a relationship between displacement and phase through a functional fit. The displacement gate thresholds are determined from an average of two values of this function, corresponding to the start and end thresholds of the original phase gate. The software tool was evaluated in two ways: first, whether in-gate residual target motion and predicted treatment beam duty cycle are equivalent between displacement gating and phase gating during 4D-CT simulation (using retrospective phase recalculation); second, whether residual motion is improved with displacement gating during treatment relative to phase gating (using real-time phase calculation). Residual target motion was inferred from the respiration traces and quantified in terms of mean and standard deviation in-gate displacement measured relative to the value at the start of the recorded trace. For retrospectively-calculated breathing traces compared with real-time calculated breathing traces, we evaluate the inaccuracies of real-time phase calculation by measuring the phase gate position in each trace as well as the mean in-gate displacement and standard deviation of the displacement. Retrospectively-calculated data from ten patients were analyzed. The patient averaged in-gate mean +/- standard deviation displacement (representing residual motion) was reduced from 0.16 +/- 0.14 cm for phase gating under simulation conditions to 0.12 +/- 0.08 cm for displacement gating. Evaluation of respiration traces under treatment conditions (real-time phase calculation) showed that the average displacement gate threshold results in a lower in-gate mean and residual motion (variance) for all patients studied. The patient-averaged in-gate mean +/- standard deviation displacement was reduced from 0.26 +/- 0.18 cm for phase gating (under treatment conditions) to 0.15 +/- 0.09 cm for displacement gating. Real-time phase gating sometimes leads to gating on incorrect portions of the breathing cycle when the breathing trace is irregular. Displacement gating is less prone to such errors, as evidenced by the lower in-gate residual motion in a large majority of cases.

Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants.

PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9DeltaC-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9DeltaC bound to EGF(AB)(H306Y), a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB)(H306Y)-PCSK9 interaction is pH-independent, LDLR(H306Y) binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9DeltaC containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.

Discovery of betamethasone 17alpha-carbamates as dissociated glucocorticoid receptor modulators in the rat.

A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.

Functional analysis of sites within PCSK9 responsible for hypercholesterolemia.

Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. PCSK9 binds the LDL receptor (LDLR), and addition of PCSK9 to cells promotes degradation of LDLR. PCSK9 mutant proteins associated with hypercholesterolemia (S127R and D374Y) are more potent in decreasing LDL uptake than is wild-type PCSK9. To better understand the mechanism by which mutations at the Ser127 and Asp374 residues of PCSK9 influence PCSK9 function, a limited vertical scanning mutagenesis was performed at both sites. S127R and S127K proteins were more potent in decreasing LDL uptake than was wild-type PCSK9, and each D374 mutant tested was more potent in reducing LDL uptake when the proteins were added exogenously to cells. The potencies of D374 mutants in lowering LDL uptake correlated with their ability to interact with LDLR in vitro. Combining S127R and D374Y was also found to have an additive effect in enhancing PCSK9's ability to reduce LDL uptake. Modeling of PCSK9 S127 and D374 mutations indicates that mutations that enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at Ser127 and Asp374 residues modulate PCSK9's ability to regulate LDLR function through distinct mechanisms.