Discovery and Design of First Benzylamine-Based Ligands Binding to an Unlocked Conformation of the Complement Factor D

Complement Factor D, a serine protease of the S1 family and key component of the alternative pathway amplification loop, represents a promising target for the treatment of several prevalent and rare diseases linked to the innate immune system. Previously reported FD inhibitors have been shown to bind to the FD active site in its self-inhibited conformation characterized by the presence of a salt bridge at the bottom of the S1 pocket between Asp189 and Arg218. We report herein a new set of small-molecule FD ligands that harbor a basic S1 binding moiety directly binding to the carboxylate of Asp189, thereby displacing the Asp189-Arg218 ionic interaction and significantly changing the conformation of the self-inhibitory loop

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: <i>in silico</i> 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

The Discovery of Novel Potent <i>trans</i>-3,4-Disubstituted Pyrrolidine Inhibitors of the Human Aspartic Protease Renin from in Silico Three-Dimensional (3D) Pharmacophore Searches

The small-molecule <i>trans</i>-3,4-disubstituted pyrrolidine <b>6</b> was identified from in silico three-dimensional (3D) pharmacophore searches based on known X-ray structures of renin–inhibitor complexes and demonstrated to be a weakly active inhibitor of the human enzyme. The unexpected binding mode of the more potent enantiomer (3<i>S</i>,4<i>S</i>)-<b>6a</b> in an extended conformation spanning the nonprime and S1′ pockets of the recombinant human (rh)-renin active site was elucidated by X-ray crystallography. Initial structure–activity relationship work focused on modifications of the hydrophobic diphenylamine portion positioned in S1 and extending toward the S2 pocket. Replacement with an optimized P3–P1 pharmacophore interacting to the nonsubstrate S3^sp cavity eventually resulted in significantly improved in vitro potency and selectivity. The prototype analogue (3<i>S</i>,4<i>S</i>)-<b>12a</b> of this new class of direct renin inhibitors exerted blood pressure lowering effects in a hypertensive double-transgenic rat model after oral administration

Structure-Based Design of Substituted Piperidines as a New Class of Highly Efficacious Oral Direct Renin Inhibitors

A <i>cis-</i>configured 3,5-disubstituted piperidine direct renin inhibitor, (<i>syn</i>,<i>rac</i>)-<b>1</b>, was discovered as a high-throughput screening hit from a target-family tailored library. Optimization of both the prime and the nonprime site residues flanking the central piperidine transition-state surrogate resulted in analogues with improved potency and pharmacokinetic (PK) properties, culminating in the identification of the 4-hydroxy-3,5-substituted piperidine <b>31</b>. This compound showed high <i>in vitro</i> potency toward human renin with excellent off-target selectivity, 60% oral bioavailability in rat, and dose-dependent blood pressure lowering effects in the double-transgenic rat model

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

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 (<i>S</i>)-proline-based lead <b>7</b> has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound <b>2</b> exerted excellent potency in 50% human whole blood <i>in vitro</i> and blocked AP activity <i>ex vivo</i> after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor <b>2</b> demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD

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

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 (<i>S</i>)-proline-based lead <b>7</b> has led to the discovery of noncovalent reversible and selective human factor D (FD) inhibitors with drug-like properties. The orally bioavailable compound <b>2</b> exerted excellent potency in 50% human whole blood <i>in vitro</i> and blocked AP activity <i>ex vivo</i> after oral administration to monkeys as demonstrated by inhibition of membrane attack complex (MAC) formation. Inhibitor <b>2</b> demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide (LPS)-induced systemic AP activation in mice expressing human FD

Design, synthesis and pre-clinical 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 (AMD), atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis type II (MPGNII) and paroxysmal nocturnal hemoglobinuria (PNH). 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 (LPS)-induced AP activation model, as well as local ocular suppression in intravitreal injection-induced AP activation model in mice expressing human FD