Mathematical Modeling of Complement Pathway Dynamics for Target Validation and Selection of Drug Modalities for Complement Therapies.

Motivation: The complement pathway plays a critical role in innate immune defense against infections. Dysregulation between activation and regulation of the complement pathway is widely known to contribute to several diseases. Nevertheless, very few drugs that target complement proteins have made it to the final regulatory approval because of factors such as high concentrations and dosing requirements for complement proteins and serious side effects from complement inhibition. Methods: A quantitative systems pharmacology (QSP) model of the complement pathway has been developed to evaluate potential drug targets to inhibit complement activation in autoimmune diseases. The model describes complement activation via the alternative and terminal pathways as well as the dynamics of several regulatory proteins. The QSP model has been used to evaluate the effect of inhibiting complement targets on reducing pathway activation caused by deficiency in factor H and CD59. The model also informed the feasibility of developing small-molecule or large-molecule antibody drugs by predicting the drug dosing and affinity requirements for potential complement targets. Results: Inhibition of several complement proteins was predicted to lead to a significant reduction in complement activation and cell lysis. The complement proteins that are present in very high concentrations or have high turnover rates (C3, factor B, factor D, and C6) were predicted to be challenging to engage with feasible doses of large-molecule antibody compounds (≤20 mg/kg). Alternatively, complement fragments that have a short half-life (C3b, C3bB, and C3bBb) were predicted to be challenging or infeasible to engage with small-molecule compounds because of high drug affinity requirements (>1 nM) for the inhibition of downstream processes. The drug affinity requirements for disease severity reduction were predicted to differ more than one to two orders of magnitude than affinities needed for the conventional 90% target engagement (TE) for several proteins. Thus, the QSP model analyses indicate the importance for accounting for TE requirements for achieving reduction in disease severity endpoints during the lead optimization stage.

Mathematical Modelling of Alternative Pathway of Complement System.

The complement system (CS) is an integral part of innate immunity and can be activated via three different pathways. The alternative pathway (AP) has a central role in the function of the CS. The AP of complement system is implicated in several human disease pathologies. In the absence of triggers, the AP exists in a time-invariant resting state (physiological steady state). It is capable of rapid, potent and transient activation response upon challenge with a trigger. Previous models of AP have focused on the activation response. In order to understand the molecular machinery necessary for AP activation and regulation of a physiological steady state, we built parsimonious AP models using experimentally supported kinetic parameters. The models further allowed us to test quantitative roles played by negative and positive regulators of the pathway in order to test hypotheses regarding their mechanisms of action, thus providing more insight into the complex regulation of AP.

Discovery of novel irreversible inhibitors of interleukin (IL)-2-inducible tyrosine kinase (Itk) by targeting cysteine 442 in the ATP pocket.

IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both TH1 and TH2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.

Chemokine receptor 4 plays a key role in T cell recruitment into the airways of asthmatic patients.

T lymphocytes of the Th2 type are central orchestrators of airway inflammation in asthma. The mechanisms that regulate their accumulation in the asthmatic airways remains poorly understood. We tested the hypothesis that CCR4, preferentially expressed on T lymphocytes of the Th2 type, plays a critical role in this process. We enumerated by flow cytometry the CCR4-expressing T cells from blood, induced sputum, and biopsy samples of patients with asthma and control subjects. We showed a positive correlation between the numbers of peripheral blood CCR4+ T cells and asthma severity, provided evidence of preferential accumulation of CCR4+ T cells in asthmatic airways, and demonstrated that CCR4+ but not CCR4- cells from patients with asthma produce Th2 cytokines. Explanted airway mucosal biopsy specimens, acquired by bronchoscopy from subjects with asthma, were challenged with allergen and the explant supernatants assayed for T cell chemotactic activity. Allergen-induced ex vivo production of the CCR4 ligand, CCL17 was raised in explants from patients with asthma when compared with healthy controls. Using chemotaxis assays, we showed that the T cell chemotactic activity generated by bronchial explants can be blocked with a selective CCR4 antagonist or by depleting CCR4+ cells from responder cells. These results provide evidence that CCR4 might play a role in allergen-driven Th2 cell accumulation in asthmatic airways. Targeting this chemokine receptor in patients with asthma might reduce Th2 cell-driven airway inflammation; therefore, CCR4 antagonists could be an effective new therapy for asthma. This study also provides wider proof of concept for using tissue explants to study immunomodulatory drugs for asthma.