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.

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 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.

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.

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.

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.

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).

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).

Skeletal muscle: a dual system to measure glucocorticoid-dependent transactivation and transrepression of gene regulation.

The use of chronic glucocorticoid (GC) therapy for the treatment of inflammatory diseases is limited by associated metabolic side effects, including muscle atrophy. Therefore, selective glucocorticoid receptor-(GR)-binding ligands that maintain anti-inflammatory activity and demonstrate diminished side-effect profiles would have great therapeutic utility. In this work, we use Taqman PCR and ELISA methods to show that GCs can inhibit basal, and lipopolysaccharide (LPS)-stimulated levels of cytokines IL-6 and TNFalpha, and also the chemokine MCP-1 in a non-inflammatory system such as primary human skeletal muscle cells. In the murine C2C12 skeletal muscle cell line we observe a similar effect of GCs on IL-6 and MCP-1; however, in contrast to previous reports, we observe a time-dependent repression of TNFalpha. Furthermore, in skeletal muscle cells, concomitant with cytokine repression, GCs transcriptionally induce glutamine synthetase (GS), a marker for muscle wasting, in an LPS independent manner. Similarly, administration of dexamethasone to mice, previously administered LPS, results in an increase in GS and an inhibition of TNFalpha and MCP-1 in skeletal muscle tissue. Thus, skeletal muscle cells and tissues present a novel system for the identification of selective GR-binding ligands, which simultaneously inhibit cytokine expression in the absence of GS induction.

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.

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.

Effects of pH and low density lipoprotein (LDL) on PCSK9-dependent LDL receptor regulation.

Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are associated with dominant forms of familial hyper- and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an approximately 150-fold higher affinity at an acidic endosomal pH (K(D) = 4.19 nm) compared with a neutral pH (K(D) = 628 nm). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.

Novel ketal ligands for the glucocorticoid receptor: in vitro and in vivo activity.

A novel series of selective ligands for the human glucocorticoid receptor is described. Structure-activity studies focused on variation of B-ring size, ketal ring size, and ketal substitution. These analogs were found to be potent and selective ligands for GR and have partial agonist profiles in functional assays for transactivation (TAT, GS) and transrepression (IL-6). Of these compounds, 27, 28, and 35 were evaluated further in a mouse LPS-induced TNF-alpha secretion model. Compound 28 had an ED(50) of 14.1 mg/kg compared with 0.5 mg/kg for prednisolone in the same assay.