Dual Mechanism of Interleukin-3 Receptor Blockade by an Anti-Cancer Antibody

SummaryInterleukin-3 (IL-3) is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα) in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD), a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-13 receptors, adopting unique “open” and classical “closed” conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas “open-like” IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a “double hit” cytokine receptor blockade

Structural and functional studies on members of the Aldo-Keto reductase family: Identifying molecular determinants responsible for substrate selectivity and inhibitor potency

The aldo-keto reductase (AKR) superfamily encompasses more than 150 enzymes that catalyse the NADP(H)-dependent reduction of several endogenous and xenobiotic aldehydes and ketones. Human AKRs have been implicated in the development of diabetes, cancer chemotherapeutic drug resistance, tobacco carcinogenesis and other hormone-dependent cancers. As such, there is a growing need to understand the molecular basis for their involvement in disease progression and develop selective inhibitors that could be used as therapeutics. The research project presented in this thesis originates from the need to study the tertiary structures of new members of the AKR family and correlate their unique structural features to enzyme function. Three members of the AKR family, namely AKR1C1, AKR1C21 and AKR1B14 were investigated in this thesis. Human AKR1C1 enzyme regulates activity of progesterone receptors by catalysing the reductive inactivation of progesterone into 20α-hydroxyprogesterone. Aberrant AKR1C1 activity has been associated with several types of cancers, end ometriosis and obesity-related metabolic disorders. In section A of this thesis, we present a new class of salicylic acid based inhibitors of AKR1C1 identified from high throughput virtual screening of the NCI database. These compounds were developed further into potent and selective inhibitors of AKR1C1 using a structure - based drug design approach. Section B of this thesis is dedicated to structure- function analysis of mouse AKR1C21 which is the only enzyme known to exhibit 17α-reductase activity. The role of active site residues in dictating 17α- stereospecificity of this enzyme was investigated by determining the crystal structures of AKR1C21 and its mutants. The mode of binding of substrates and inhibitors in the active site of AKR1C21 was explored through molecular modelling and docking analysis. In section C, we present the first crystal structure of rat AKR1B14 which exhibits significant prostaglandin F2α synthase activity and reduces reactive products derived from lipid peroxidation and glycation with lower Km values than other members of the AKR1B subfamily. Residues involved in binding of coenzyme, bile-acid activator and aldose reductase inhibitors were investigated through molecular modelling and docking analysis. The overall aim of this study was to characterise the mode of binding of substrates and inhibitors within the active site of these enzymes in order to ascertain the role of active site residues in dictating substrate selectivity and inhibitor potency. The structural information obtained from solving the crystal structures of these enzymes was used for high throughput database screening to identify new ligands that could be developed into potent drug-like inhibitors with potential for therapeutic use through a structure based drug design process

Structural and functional studies on members of the Aldo-Keto reductase family: Identifying molecular determinants responsible for substrate selectivity and inhibitor potency

The aldo-keto reductase (AKR) superfamily encompasses more than 150 enzymes that catalyse the NADP(H)-dependent reduction of several endogenous and xenobiotic aldehydes and ketones. Human AKRs have been implicated in the development of diabetes, cancer chemotherapeutic drug resistance, tobacco carcinogenesis and other hormone-dependent cancers. As such, there is a growing need to understand the molecular basis for their involvement in disease progression and develop selective inhibitors that could be used as therapeutics. The research project presented in this thesis originates from the need to study the tertiary structures of new members of the AKR family and correlate their unique structural features to enzyme function. Three members of the AKR family, namely AKR1C1, AKR1C21 and AKR1B14 were investigated in this thesis. Human AKR1C1 enzyme regulates activity of progesterone receptors by catalysing the reductive inactivation of progesterone into 20α-hydroxyprogesterone. Aberrant AKR1C1 activity has been associated with several types of cancers, end ometriosis and obesity-related metabolic disorders. In section A of this thesis, we present a new class of salicylic acid based inhibitors of AKR1C1 identified from high throughput virtual screening of the NCI database. These compounds were developed further into potent and selective inhibitors of AKR1C1 using a structure - based drug design approach. Section B of this thesis is dedicated to structure- function analysis of mouse AKR1C21 which is the only enzyme known to exhibit 17α-reductase activity. The role of active site residues in dictating 17α- stereospecificity of this enzyme was investigated by determining the crystal structures of AKR1C21 and its mutants. The mode of binding of substrates and inhibitors in the active site of AKR1C21 was explored through molecular modelling and docking analysis. In section C, we present the first crystal structure of rat AKR1B14 which exhibits significant prostaglandin F2α synthase activity and reduces reactive products derived from lipid peroxidation and glycation with lower Km values than other members of the AKR1B subfamily. Residues involved in binding of coenzyme, bile-acid activator and aldose reductase inhibitors were investigated through molecular modelling and docking analysis. The overall aim of this study was to characterise the mode of binding of substrates and inhibitors within the active site of these enzymes in order to ascertain the role of active site residues in dictating substrate selectivity and inhibitor potency. The structural information obtained from solving the crystal structures of these enzymes was used for high throughput database screening to identify new ligands that could be developed into potent drug-like inhibitors with potential for therapeutic use through a structure based drug design process

Structure of 3(17)α-hydroxysteroid dehydrogenase (AKR1C21) holoenzyme from an orthorhombic crystal form: an insight into the bifunctionality of the enzyme

The structure of AKR1C21 holoenzyme was determined at 1.8 Å resolution. A model describing the interaction between AKR1C21 and steroid substrates is proposed that explains the bifunctionality of the enzyme

EPO does not promote interaction between the erythropoietin and beta-common receptors

A direct interaction between the erythropoietin (EPOR) and the beta-common (βc) receptors to form an Innate Repair Receptor (IRR) is controversial. On one hand, studies have shown a functional link between EPOR and βc receptor in tissue protection while others have shown no involvement of the βc receptor in tissue repair. To date there is no biophysical evidence to confirm a direct association of the two receptors either in vitro or in vivo. We investigated the existence of an interaction between the extracellular regions of EPOR and the βc receptor in silico and in vitro (either in the presence or absence of EPO or EPO-derived peptide ARA290). Although a possible interaction between EPOR and βc was suggested by our computational and genomic studies, our in vitro biophysical analysis demonstrates that the extracellular regions of the two receptors do not specifically associate. We also explored the involvement of the βc receptor gene (Csf2rb) under anaemic stress conditions and found no requirement for the βc receptor in mice. In light of these studies, we conclude that the extracellular regions of the EPOR and the βc receptor do not directly interact and that the IRR is not involved in anaemic stress

Author Correction: EPO does not promote interaction between the erythropoietin and beta-common receptors (Scientific Reports, (2018), 8, 1, (12457), 10.1038/s41598-018-29865-x)

In Figure 1C, Sites 1 and 2 are incorrectly labelled. The correct Figure 1 appears below.(Figure presented.)

Signalling by the βc family of cytokines

The GM-CSF, IL-3 and IL-5 family of cytokines, also known as the βc family due to their receptors sharing the signalling subunit βc, regulates multiple biological processes such as native and adaptive immunity, inflammation, normal and malignant hemopoieis, and autoimmunity. Australian scientists played a major role in the discovery and biological characterisation of the βc cytokines and their recent work is revealing unique features of cytokine receptor assembly and signalling. Furthermore, specific antibodies have been generated to modulate their function. Characterisation of the structural and dynamic requirements for the activation of the βc receptor family and the molecular definition of downstream signalling pathways are providing new insights into cytokine receptor signalling as well as new therapeutic opportunities.

CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins

The calcium-calmodulin–dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca2+, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495. We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and noncanonical (Ser100) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases