Fabien Vincent

Developing predictive assays: The phenotypic screening “rule of 3”

Not all phenotypic assays are created equal; critically evaluating the disease relevance of the assay system, stimulus, and readout can help design the most predictive ones. Phenotypic drug discovery approaches can positively affect the translation of preclinical findings to patients. However, not all phenotypic assays are created equal. A critical question then follows: What are the characteristics of the optimal assays? We analyze this question and propose three specific criteria related to the disease relevance of the assay—system, stimulus, and end point—to help design the most predictive phenotypic assays.

Small Molecule Screening in Human Induced Pluripotent Stem Cell-derived Terminal Cell Types

A need for better clinical outcomes has heightened interest in the use of physiologically relevant human cells in the drug discovery process. Patient-specific human induced pluripotent stem cells may offer a relevant, robust, scalable, and cost-effective model of human disease physiology. Small molecule high throughput screening in human induced pluripotent stem cell-derived cells with the intent of identifying novel therapeutic compounds is starting to influence the drug discovery process; however, the use of these cells presents many high throughput screening development challenges. This technology has the potential to transform the way drug discovery is performed.

Discovery of Clinical Candidate 1-{[(2S,3S,4S)-3-Ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoline-6-carboxamide (PF-06650833), a Potent, Selective Inhibitor of Interleukin-1 Receptor Associated Kinase 4 (IRAK4), by Fragment-Based Drug Design

Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1-((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1-yl)prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans

Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of 11 (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor 11 led to its evaluation in several human clinical studies.

Binding site elucidation and structure guided design of macrocyclic IL-17A antagonists.

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

The catalytic mechanism of cyclic gmp-amp synthase (cGAS) and implications for innate immunity and inhibition

Cyclic GMP‐AMP synthase (cGAS) is activated by ds‐DNA binding to produce the secondary messenger 2′,3′‐cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds‐DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2′,3′‐cGAMP formation, and interestingly, describe a catalytic mechanism where 2′,3′‐cGAMP may be a minor product of cGAS compared with linear nucleotides.

Class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion

Despite the important role of apolipoprotein E (apoE) secretion from astrocytes in brain lipid metabolism and the strong association of apoE4, one of the human apoE isoforms, with sporadic and late onset forms of Alzheimer’s disease (AD) little is known about the regulation of astrocytic apoE. Utilizing annotated chemical libraries and a phenotypic screening strategy that measured apoE secretion from a human astrocytoma cell line, inhibition of pan class I histone deacetylases (HDACs) was identified as a mechanism to increase apoE secretion. Knocking down select HDAC family members alone or in combination revealed that inhibition of the class I HDAC family was responsible for enhancing apoE secretion. Knocking down LXRα and LXRβ genes revealed that the increase in astrocytic apoE in response to HDAC inhibition occurred via an LXR-independent pathway. Collectively, these data suggest that pan class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.

Rational approach to highly potent and selective apoptosis signal-regulating kinase 1 (ASK1) inhibitors.

Many diseases are believed to be driven by pathological levels of reactive oxygen species (ROS) and oxidative stress has long been recognized as a driver for inflammatory disorders. Apoptosis signal-regulating kinase 1 (ASK1) has been reported to be activated by intracellular ROS and its inhibition leads to a down regulation of p38-and JNK-dependent signaling. Consequently, ASK1 inhibitors may have the potential to treat clinically important inflammatory pathologies including renal, pulmonary and liver diseases. Analysis of the ASK1 ATP-binding site suggested that Gln756, an amino acid that rarely occurs at the GK+2 position, offered opportunities for achieving kinase selectivity for ASK1 which was applied to the design of a parallel medicinal chemistry library that afforded inhibitors of ASK1 with nanomolar potency and excellent kinome selectivity. A focused optimization strategy utilizing structure-based design resulted in the identification of ASK1 inhibitors with low nanomolar potency in a cellular assay, high selectivity when tested against kinase and broad pharmacology screening panels, and attractive physicochemical properties. The compounds we describe are attractive tool compounds to inform the therapeutic potential of ASK1 inhibition.

OP0155 Development of A JAK3 Specific Inhibitor Clinical Candidate: Functional Differentiation of JAK3 Selective Inhibition over PAN-JAK or JAK1 Selective Inhibition

Background Janus kinase (JAK) inhibitors targeting multiple JAK isoforms, JAK1 and 2, or selective against JAK1 are currently utilized in clinical practice or being developed for the treatment of various inflammatory and oncological diseases. No truly JAK3-selective inhibitor has reached the clinic. JAK3 signal in pairs with JAK1 to transduce signal elicited from six known cytokines (IL-2, IL-4, IL-7, IL-9, IL-15 & IL-21) binding to the gamma-common (g-c) chain cytokine receptors. JAK1 in addition to be required for g-c chain cytokine receptors signaling can also signal in pair with JAK2 or TYK2 and is required for other cytokine receptors signaling such as type I and II interferons and IL-6 and IL-10 families of cytokines. Other cytokines such as IL-12 and IL-23 are signaling via JAK2 and TYK2. Additionally, hematopoietic cytokines such as EPO and TPO as well as cytokines such as IL-3 and IL-5 signal via homodimers of JAK2. Objectives Develop a JAK3 selective inhibitor that will inhibit signaling from the g-c chain cytokine receptors at therapeutic dosing without inhibiting signaling by other cytokines such as hematopoietic cytokines, type I and II interferons and IL-6, IL-12 and IL-10 families of cytokines. Methods A JAK3-specific covalent ATP competitive inhibitor was assessed functionally in vitro and in vivo. Functional differentiation of JAK3 selective inhibition as compare to pan-JAK inhibition (tofacitinib) or JAK1 selective inhibition was also assessed. Results The inhibitor showed JAK3 selective inhibition in biochemical and cellular assays. It inhibits Th1 and Th17 cell differentiation and function. Importantly, sparing JAK1 inhibition, through the selectivity of this inhibitor, preserved anti-inflammatory functions such as the differentiation of alternatively activated M2a macrophages. Unlike pan-JAK or JAK1-selective inhibitors, JAK3 selective inhibition also preserved IL-10-dependent suppression of the production of pro-inflammatory cytokines following LPS treatment in macrophages while maintaining the suppression of TNF and IL-1 responses in IL-27-primed macrophages. Further characterization of this compound in the rat adjuvant-induced arthritis (AIA) demonstrated efficacy at reducing disease pathology. Conclusions We have identified the first trully selective JAK3 inhibitor with potency and properties that make it suitable for further clinical development. Disclosure of Interest J.-B. Telliez Employee of: Pfizer, L. Wang Employee of: Pfizer, J. Jussif Employee of: Pfizer, T. Lin Employee of: Pfizer, L. Li Employee of: Pfizer, E. Moy Employee of: Pfizer, W. Li Employee of: Pfizer, Y. Zhao Employee of: Pfizer, K. Crouse Employee of: Pfizer, P. Symanowicz Employee of: Pfizer, M. Hegen Employee of: Pfizer, M. E. Banker Employee of: Pfizer, F. Vincent Employee of: Pfizer, J. Clark Employee of: Pfizer, A. Thorarensen Employee of: Pfizer