Discovery of 4-((2S,4S)-4-Ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic Acid (LNP023), a Factor B Inhibitor Specifically Designed To Be Applicable to Treating a Diverse Array of Complement Mediated Diseases.

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.

Small-molecule factor B inhibitor for the treatment of complement-mediated diseases.

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.

Structure-Based Library Design and Fragment Screening for the Identification of Reversible Complement Factor D Protease Inhibitors.

Chronic dysregulation of alternative complement pathway activation has been associated with diverse clinical disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinurea. Factor D is a trypsin-like serine protease with a narrow specificity for arginine in the P1 position, which catalyzes the first enzymatic reaction of the amplification loop of the alternative pathway. In this article, we describe two hit finding approaches leading to the discovery of new chemical matter for this pivotal protease of the complement system: in silico active site mapping for hot spot identification to guide rational structure-based design and NMR screening of focused and diverse fragment libraries. The wealth of information gathered by these complementary approaches enabled the identification of ligands binding to different subpockets of the latent Factor D conformation and was instrumental for understanding the binding requirements for the generation of the first known potent noncovalent reversible Factor D inhibitors.

Small-molecule factor D inhibitors targeting the alternative complement pathway.

Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

Gift from Nature: Cyclomarin†A Kills Mycobacteria and Malaria Parasites by Distinct Modes of Action.

Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarin A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarin A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarin A binds a dimeric PfAp3Aase and prevents the formation of the enzyme⋅substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarin A in Mycobacterium tuberculosis. Thus, cyclomarin A is a rare example of a natural product with two distinct and specific modes of action.

Exploring T cell reactivity to gliadin in young children with newly diagnosed celiac disease.

Class II major histocompatibility molecules confer disease risk in Celiac disease (CD) by presenting gliadin peptides to CD4 T cells in the small intestine. Deamidation of gliadin peptides by tissue transglutaminase creates immunogenic peptides presented by HLA-DQ2 and DQ8 molecules to activate proinflammatory CD4 T cells. Detecting gliadin specific T cell responses from the peripheral blood has been challenging due to low circulating frequencies and heterogeneity in response to gliadin epitopes. We investigated the peripheral T cell responses to alpha and gamma gliadin epitopes in young children with newly diagnosed and untreated CD. Using peptide/MHC recombinant protein constructs, we are able to robustly stimulate CD4 T cell clones previously derived from intestinal biopsies of CD patients. These recombinant proteins and a panel of α- and γ-gliadin peptides were used to assess T cell responses from the peripheral blood. Proliferation assays using peripheral blood mononuclear cells revealed more CD4 T cell responses to α-gliadin than γ-gliadin peptides with a single deamidated α-gliadin peptide able to identify 60% of CD children. We conclude that it is possible to detect T cell responses without a gluten challenge or in vitro stimulus other than antigen, when measuring proliferative responses.

Discovery of C-(1-aryl-cyclohexyl)-methylamines as selective, orally available inhibitors of dipeptidyl peptidase IV.

The successful launches of dipeptidyl peptidase IV (DPP IV) inhibitors as oral anti-diabetics warrant and spur the further quest for additional chemical entities in this promising class of therapeutics. Numerous pharmaceutical companies have pursued their proprietary candidates towards the clinic, resulting in a large body of published chemical structures associated with DPP IV. Herein, we report the discovery of a novel chemotype for DPP IV inhibition based on the C-(1-aryl-cyclohexyl)-methylamine scaffold and its optimization to compounds which selectively inhibit DPP IV at low-nM potency and exhibit an excellent oral pharmacokinetic profile in the rat.

A quantitative homogeneous assay for fragile X mental retardation 1 protein.

BACKGROUND: Hypermethylation of the fragile X mental retardation 1 gene FMR1 results in decreased expression of FMR1 protein FMRP, which is the underlying cause of Fragile X syndrome - an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism. Disease-modifying therapies for Fragile X syndrome are thus aimed at treatments that increase the FMRP expression levels in the brain. We describe the development and characterization of two assays for simple and quantitative detection of FMRP protein. METHOD: Antibodies coupled to fluorophores that can be employed for time-resolved Förster's resonance energy transfer were used for the development of homogeneous, one-step immunodetection. Purified recombinant human FMRP and patient cells were used as control samples for assay development. RESULTS: The assays require small sample amounts, display high stability and reproducibility and can be used to quantify endogenous FMRP in human fibroblasts and peripheral blood mononuclear cells. Application of the assays to FXS patient cells showed that the methods can be used both for the characterization of clinical FXS patient samples as well as primary readouts in drug-discovery screens aimed at increasing endogenous FMRP levels in human cells. CONCLUSION: This study provides novel quantitative detection methods for FMRP in FXS patient cells. Importantly, due to the simplicity of the assay protocol, the method is suited to be used in screening applications to identify compounds or genetic interventions that result in increased FMRP levels in human cells.

A novel class of oral direct renin inhibitors: highly potent 3,5-disubstituted piperidines bearing a tricyclic p3-p1 pharmacophore.

A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S3-S1 pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay. X-ray crystallography demonstrated the eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the rh-renin prime site. The design of the potent and selective (3S,5R)-12 bearing a P3(sp)-tethered tricyclic P3-P1 pharmacophore derived from 3 is described. (3S,5R)-12 showed oral bioavailability in rats and demonstrated blood pressure lowering activity in the double-transgenic rat model.

Novel heterocyclic DPP-4 inhibitors for the treatment of type 2 diabetes.

Novel deazaxanthine-based DPP-4 inhibitors have been identified that are potent (IC(50) <10nM) and highly selective versus other dipeptidyl peptidases. Their synthesis and SAR are reported, along with initial efforts to improve the PK profile through decoration of the deazaxanthine core. Optimisation of compound 3a resulted in the identification of compound (S)-4i, which displayed an improved in vitro and ADME profile. Further enhancements to the PK profile were possible by changing from the deazahypoxanthine to the deazaxanthine template, culminating in compound 12g, which displayed good ex vivo DPP-4 inhibition and a superior PK profile in rat, suggestive of once daily dosing in man.

Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization.

Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core, N- and C-terminal domains required for protein-protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post-translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N- and C-terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C-terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N-terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzyme's first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor-like region (amino acids 70-205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.

Aspartic proteases in drug discovery.

Aspartic proteases are the smallest class of human proteases with only 15 members. Over the past years, they have received considerable attention as potential targets for pharmaceutical intervention since many have been shown to play important roles in physiological and pathological processes. Despite numerous efforts, however, the only inhibitors for aspartic proteases currently on the market are directed against the HIV protease, an aspartic protease of viral origin. Nevertheless, several inhibitors including those targeting renin, BACE1 and gamma-secretase are in clinical or preclinical development, and some other aspartic proteases are discussed as potential drug target. The crystal structures of seven human aspartic proteases have now been solved and, together with a detailed kinetic understanding of their catalytic mechanism, this has greatly contributed to the design and discovery of novel inhibitors for this protease class. This review describes current aspartic protease drug targets and summarizes the drug discovery efforts in this field. In addition, it highlights recent developments which may lead to a new generation of aspartic protease inhibitors.

Backbone 1H, 13C, and 15N resonance assignments for the 26-kD human de-ubiquitinating enzyme UCH-L3.

To facilitate NMR spectroscopy studies of interactions with various ligands and potential inhibitors, we report the NMR backbone resonance assignments for the 26 kD human enzyme UCH-L3, a member of the ubiquitin C-hydrolase family of ubiquitin-specific cysteine proteases.

Structural basis of ubiquitin recognition by the deubiquitinating protease USP2.

Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs.

Crystal structure of human BACE2 in complex with a hydroxyethylamine transition-state inhibitor.

BACE2 is a membrane-bound aspartic protease of the A1 family with a high level of sequence homology to BACE1. While BACE1 is involved in the generation of amyloid plaques in Alzheimer's disease by cleaving Abeta-peptides from the amyloid precursor protein, the physiological function of BACE2 is not well understood. BACE2 appears to be associated with the early onset of dementia in patients with Down's syndrome, and it has been shown to be highly expressed in breast cancers. Further, it may participate in the function of normal and abnormal processes of human muscle biology. Similar to other aspartic proteases, BACE2 is expressed as an inactive zymogen requiring the cleavage of its pro-sequence during the maturation process. We have produced mature BACE2 by expression of pro-BACE2 in Escherichia coli as inclusion bodies, followed by refolding and autocatalytic activation at pH 3.4. Using a C and N-terminally truncated BACE2 variant, we were able to crystallize and determine the crystal structure of mature BACE2 in complex with a hydroxyethylamine transition-state mimetic inhibitor at 3.1 angstroms resolution. The structure of BACE2 follows the general fold of A1 aspartic proteases. However, similar to BACE1, its C-terminal domain is significantly larger than that of the other family members. Furthermore, the structure of BACE2 reveals differences in the S3, S2, S1' and S2' active site substrate pockets as compared to BACE1, and allows, therefore, for a deeper understanding of the structural features that may facilitate the design of selective BACE1 or BACE2 inhibitors.

Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion.

For a long time, prolyl endopeptidase (PEP) was believed to inactivate neuropeptides in the extracellular space. However, reports on the intracellular activity of PEP suggest additional, as yet unidentified, physiological functions for this enzyme. Here, we demonstrate using biochemical methods of subcellular fractionation, immunocytochemical double-labelling procedures and localization of PEP-enhanced green fluorescent protein fusion proteins that PEP is mainly localized to the perinuclear space, and is associated with the microtubulin cytoskeleton in human neuroblastoma and glioma cell lines. Disassembly of the microtubules by nocodazole treatment disrupts both the fibrillar tubulin and PEP labelling. Furthermore, in a two-hybrid screen, PEP was identified as binding partner of tubulin. These findings indicate novel functions for PEP in axonal transport and/or protein secretion. Indeed, a metabolic labelling approach revealed that both PEP inhibition and PEP antisense mRNA expression result in enhanced peptide/protein secretion from human U-343 glioma cells. Because disturbances in intracellular transport and protein secretion mechanisms are associated with a number of ageing-associated neurodegenerative diseases, cell-permeable PEP inhibitors may be useful for the application in a variety of related clinical conditions.

Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2.

Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay.

Crystal structure of an activation intermediate of cathepsin E.

Cathepsin E is an intracellular, non-lysosomal aspartic protease expressed in a variety of cells and tissues. The protease has proposed physiological roles in antigen presentation by the MHC class II system, in the biogenesis of the vasoconstrictor peptide endothelin, and in neurodegeneration associated with brain ischemia and aging. Cathepsin E is the only A1 aspartic protease that exists as a homodimer with a disulfide bridge linking the two monomers. Like many other aspartic proteases, it is synthesized as a zymogen which is catalytically inactive towards its natural substrates at neutral pH and which auto-activates in an acidic environment. Here we report the crystal structure of an activation intermediate of human cathepsin E at 2.35A resolution. The overall structure follows the general fold of aspartic proteases of the A1 family, and the intermediate shares many features with the intermediate 2 on the proposed activation pathway of aspartic proteases like pepsin C and cathepsin D. The pro-sequence is cleaved from the protease and remains stably associated with the mature enzyme by forming the outermost sixth strand of the interdomain beta-sheet. However, different from these other aspartic proteases the pro-sequence of cathepsin E remains intact after cleavage from the mature enzyme. In addition, the active site of cathepsin E in the crystal is occupied by N-terminal amino acid residues of the mature protease in the non-primed binding site and by an artificial N-terminal extension of the pro-sequence from a neighboring molecule in the primed site. The crystal structure of the cathepsin E/pro-sequence complex, therefore, provides further insight into the activation mechanism of aspartic proteases.

Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid with 2,4-dinitrophenyl groups at various positions.

The development of renin inhibitors for the treatment of hypertension requires highly sensitive substrates to evaluate potency and to characterize the mechanism of tight-binding inhibitors. A series of intramolecularly quenched fluorogenic renin substrates, based on the N-terminal tetradecapeptide sequence of human angiotensinogen (hTDP), was synthesized using a solid-phase technique. Incorporation of the fluorescent amino acid L-Amp [L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid] and the DNP (2,4-dinitrophenyl) group at various positions resulted in >90% quenching efficiency and strong product fluorescence. Shortening the hTDP sequence to an octapeptide from histidine in P5 to histidine in P3' (substrate 3) resulted in an acceptable k(cat)/K(m) (41000 M(-1).s(-1)) and further systematic variation gave substrate 9, DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-L-Amp, with a k(cat)/K(m) value of 350000 M(-1).s(-1) and 94% quenching efficiency. The free side chain of lysine, replacing the isoleucine residue at P6 position in the angiotensinogen sequence, contributed to the increased value for k(cat). The pH dependence of k(cat)/K(m) for renin and substrate 9 showed that the optimal pH is at pH 6-7. It also showed two titrating groups on the acidic side of the pH optimum, and one titrating group with a pK(a) of 7.8 on the alkaline side. The combination of good kinetic and spectroscopic properties resulted in a >20-fold improvement in the sensitivity of renin assay, compared with the commercial substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg [where EDANS is 5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid and DABCYL is 4-(4-dimethylaminophenylazo)benzoic acid] (k(cat)/K(m)=268000 M(-1) x s(-1), quenching efficiency <80%). The detection limit in a microplate renin assay was 60 pM, making substrate 9 well suited for the evaluation of inhibitors at picomolar concentrations.