Low-molecular weight inhibitors of the alternative complement pathway.

Dysregulation of the alternative complement pathway predisposes individuals to a number of diseases. It can either be evoked by genetic alterations in or by stabilizing antibodies to important pathway components and typically leads to severe diseases such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, C3 glomerulopathy, and age-related macular degeneration. In addition, the alternative pathway may also be involved in many other diseases where its amplifying function for all complement pathways might play a role. To identify specific alternative pathway inhibitors that qualify as therapeutics for these diseases, drug discovery efforts have focused on the two central proteases of the pathway, factor B and factor D. Although drug discovery has been challenging for a number of reasons, potent and selective low-molecular weight (LMW) oral inhibitors have now been discovered for both proteases and several molecules are in clinical development for multiple complement-mediated diseases. While the clinical development of these inhibitors initially focuses on diseases with systemic and/or peripheral tissue complement activation, the availability of LMW inhibitors may also open up the prospect of inhibiting complement in the central nervous system where its activation may also play an important role in several neurodegenerative diseases.

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.

Design, Synthesis, and Preclinical Characterization of Selective Factor D Inhibitors Targeting the Alternative Complement Pathway.

Complement factor D (FD), a highly specific S1 serine protease, plays a central role in the amplification of the alternative complement pathway (AP) of the innate immune system. Dysregulation of AP activity predisposes individuals to diverse disorders such as age-related macular degeneration, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis type II, and paroxysmal nocturnal hemoglobinuria. Previously, we have reported the screening efforts and identification of reversible benzylamine-based FD inhibitors (1 and 2) binding to the open active conformation of FD. In continuation of our drug discovery program, we designed compounds applying structure-based approaches to improve interactions with FD and gain selectivity against S1 serine proteases. We report herein the design, synthesis, and medicinal chemistry optimization of the benzylamine series culminating in the discovery of 12, an orally bioavailable and selective FD inhibitor. 12 demonstrated systemic suppression of AP activation in a lipopolysaccharide-induced AP activation model as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD.

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.

Discovery of Highly Potent and Selective Small-Molecule Reversible Factor D Inhibitors Demonstrating Alternative Complement Pathway Inhibition in Vivo.

The highly specific S1 serine protease factor D (FD) plays a central role in the amplification of the complement alternative pathway (AP) of the innate immune system. Genetic associations in humans have implicated AP activation in age-related macular degeneration (AMD), and AP dysfunction predisposes individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The combination of structure-based hit identification and subsequent optimization of the center (S)-proline-based lead 7 has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound 2 exerted excellent potency in 50% human whole blood in vitro and blocked AP activity ex vivo after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor 2 demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD.

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.

Plasma contact system activation drives anaphylaxis in severe mast cell-mediated allergic reactions.

BACKGROUND: Anaphylaxis is an acute, potentially lethal, multisystem syndrome resulting from the sudden release of mast cell-derived mediators into the circulation. OBJECTIVES AND METHODS: We report here that a plasma protease cascade, the factor XII-driven contact system, critically contributes to the pathogenesis of anaphylaxis in both murine models and human subjects. RESULTS: Deficiency in or pharmacologic inhibition of factor XII, plasma kallikrein, high-molecular-weight kininogen, or the bradykinin B2 receptor, but not the B1 receptor, largely attenuated allergen/IgE-mediated mast cell hyperresponsiveness in mice. Reconstitutions of factor XII null mice with human factor XII restored susceptibility for allergen/IgE-mediated hypotension. Activated mast cells systemically released heparin, which provided a negatively charged surface for factor XII autoactivation. Activated factor XII generates plasma kallikrein, which proteolyzes kininogen, leading to the liberation of bradykinin. We evaluated the contact system in patients with anaphylaxis. In all 10 plasma samples immunoblotting revealed activation of factor XII, plasma kallikrein, and kininogen during the acute phase of anaphylaxis but not at basal conditions or in healthy control subjects. The severity of anaphylaxis was associated with mast cell degranulation, increased plasma heparin levels, the intensity of contact system activation, and bradykinin formation. CONCLUSIONS: In summary, the data collectively show a role of the contact system in patients with anaphylaxis and support the hypothesis that targeting bradykinin generation and signaling provides a novel and alternative treatment strategy for anaphylactic attacks.

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.

Functional and binding characterizations of urotensin II-related peptides in human and rat urotensin II-receptor assay.

Urotensin II (U-II; cyclo5-10[H-Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH]) is a potent vasoconstrictor in mammals, and it is postulated that it plays a central role in cardiovascular homeostasis. Thus, we initiated a structure-to-function analysis of this peptide characterized by a N-terminal tail and a cyclic core formed through a disulfide bridging. A total of 41 analogs focusing on these characteristics were developed and evaluated using a binding assay on membranes from a stable HEK-293 cell line containing the human or rat U-II receptor, a functional assay for Ca2+ mobilization on transiently transfected CHO-K1 cells with the human or rat U-II receptor, and a rat thoracic aorta bioassay. At first, the focus was applied on peptide compounds containing exocyclic modifications. From this series, it appeared that only valine-11 played a significant role although it is not an essential amino acid. Similarly, endocyclic and ring transformations of hU-II were also studied. In most cases, a detrimental effect on affinity and biological activity was observed. However, two compounds, [Tyr6]hU-II and [Phe9]hU-II, retained affinity and activity. So far, our binding, functional, and pharmacological data clearly demonstrated the minor contribution of the N-terminal segment and the essential role of the cyclic structure. More particularly, three residues within the loop, i.e., Trp-7, Lys-8, and Tyr-9, are required for receptor recognition and activation. This three-pole feature, kept by the disulfide bond in a correct spatial arrangement, appears as the key pharmacophore for the U-II receptor.

Identification of nonpeptidic urotensin II receptor antagonists by virtual screening based on a pharmacophore model derived from structure-activity relationships and nuclear magnetic resonance studies on urotensin II.

The vasoactive cyclic 11-amino acid peptide urotensin II (U-II) has recently been discovered as the endogenous ligand of the orphan G-protein-coupled receptor GPR14. As U-II might be involved in the regulation of cardiovascular homeostasis and pathology, a nonpeptidic GPR14/U-II antagonist is of considerable basic and therapeutic interest. We have performed structure-activity relationship studies on U-II by investigating 25 peptide analogues to mobilize intracellular calcium in GPR14-transfected CHO cells, demonstrating that only the side chains of the residues Trp-7, Lys-8, and Tyr-9 are required for receptor recognition and activation. The solution structure of U-II derived by nuclear magnetic resonance has served as a structural template for a three-dimensional three point pharmacophore query for the virtual screening of the Aventis compound repository for nonpeptidic U-II receptor antagonists. Highly active lead compounds of six different scaffold classes could be identified, antagonizing the biological activity of U-II in vitro. The most potent compound identified by the virtual screening approach, 1-(3-carbamimidoyl-benzyl)-4-methyl-1H-indole-2-carboxylic acid (naphthalen-1-ylmethyl)amide, reveals an IC(50) of 400 nM in a functional fluorometric imaging plate reader assay and constitutes a promising lead.