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Download Author Version (PDF) MedChemComm Accepted Manuscript This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/medchemcomm Page 1 of 6 Journal Name Medicinal Chemistry Communications Dynamic Article Links ► Cite this: DOI: 10.1039/c0xx00000x www.rsc.org/xxxxxx ARTICLE TYPE Optimizing Glucokinase Activator Binding Kinetics to Lower in vivo Hypoglycemia Risk Manuscript Kris A. Borzilleri ‡ Jeffrey A. Pfefferkorn ╢, Angel Guzman-Perez ¥, Shenping Liu ‡, Xiayang Qiu ‡, Boris A. Chrunyk ‡, Xi Song ‡ Meihua Tu ╢, Kevin J. Filipski ╢, Robert Aiello ±, David R. Derksen ‡, Francis J. ‡ † ± ┴ ┼ ± 5 Bourbonais , James Landro , Patricia Bourassa , Theresa D’Aquila , Levenia Baker , Nicole Barrucci , John Litchfield ‡, Karen Atkinson ‡ Timothy P. Rolph ╢ and Jane M. Withka ‡* Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X DOI: 10.1039/b000000x Accepted Activation of glucokinase represents a promising strategy for the treatment of type 2 diabetes; however, 10 drug candidates have failed in clinical trials due to narrow therapeutic index between glucose-lowering efficacy and hypoglycemia. Described herein is a novel strategy for the design of next generation glucokinase activators with increased therapeutic index, which involves the optimization of activator- enzyme binding kinetics (k on , k off ). This approach is based on the idea that activator binding kinetics are relevant to pharmacodynamics since the affinity of activator binding to glucokinase is cooperative with 15 glucose such that, the rate at which an activator dissociates may influence the enzyme’s sensitivity to changes in physiological glucose concentrations. This study provides a compelling example of using fast- off binding kinetics for developing safe and effective activator drugs targeting glucokinase. Introduction activators in various patient populations are needed to further investigate the issue. Glucokinase functions as a key regulator of glucose homeostasis 45 20 and small molecule allosteric activators of this enzyme have been Given the potential therapeutic limitations and compliance issues shown to reduce blood glucose levels by enhancing glucose- (e.g. dose titration and/or dosing with meals) imposed by the risk stimulated insulin secretion in the pancreas while also increasing 1,2 of GKA-induced hypoglycemia, there is continued interest in glucose uptake and reducing glucose production in the liver. Communications drug design strategies to develop next generation agents with The development of glucokinase activators (GKAs) has been an 50 reduced risk. Biochemically, glucokinase catalyzes the 25 active area of pharmaceutical research with several candidates conversion of glucose to glucose-6-phosphate, which is the first advancing to early clinical studies. However, while these GKAs and rate limiting step in glycolysis and glycogen synthesis. It has have shown promising early efficacy in T2DM patients, there has a physiologically optimal affinity for glucose (K or S = 8 also been significant attrition driven by narrow therapeutic m 0.5 mM) and has been shown to demonstrate positive cooperativity windows against hypoglycemia as well as concerns about 5 3,4 55 of glucose binding, facilitated by multiple conformations of this 30 durability and adverse effects on plasma and hepatic lipids. For monomeric protein in solution. 6,7 High resolution structures of example, during a Phase 2 study of GKA MK-0941 (10 – 40 mg, glucokinase complexed with several activator chemotypes TID) conducted in T2DM patients on basal insulin therapy, the revealed an allosteric binding site, 20 Å remote from the catalytic incidence of hypoglycemia (glucose < 50 mg/dL) was 14-16% in site. Activators of glucokinase have been well characterized and the active treatment arms versus 8% for the placebo group. Chemistry 60 are known to modulate enzyme activity by two parameters α and 35 Furthermore, four patients on MK-0941 discontinued from the β, which are defined by a decrease in K and an increase in V , study due to hypoglycemia and one subject in the 20 mg group m max respectively. 8,9,10 Previously, we published an experimental experienced an episode of severe hypoglycemia requiring medical assessment of the hypoglycemic risk associated with different α,β assistance. 4a Additionally, during this same study of MK-0941, activation profiles and successfully identified a “partial activator” tachyphylasis was observed after 14 weeks of treatment. 4a A 65 that demonstrated favorable efficacy and minimal hypoglycemia 40 similar loss of efficacy was also recently reported in a Phase 2 risk in preclinical studies. 9 As an extension of this work, we also study of GKA AZD-1656. 4b The explanation for this began to consider how activator-enzyme binding kinetics might tachyphylasis is currently unclear, and studies with additional influence GKA residency time on the enzyme and influence Medicinal This journal is © The Royal Society of Chemistry [year] [journal] , [year], [vol] , 00–00 | 1 Medicinal Chemistry Communications Page 2 of 6 11 hypoglycemia safety. For many enzyme systems, slow off rates 100pM 1nM 10nM 100nM and long dissociative half-lives (a direct measure of residence 7 times) have been shown to improve target selectivity and reduce off-target safety risks. 12,13 However in the case of GKAs there is 6 1uM 5 positive cooperativity between activator and glucose binding to the enzyme making the apparent potency of an activator dependent on glucose concentrations. 14 As a consequence of this 5 10uM profile, the rate at which an activator dissociates from the enzyme may play a critical role in the ability of glucokinase to rapidly Logon-rate 10 respond to changing physiological glucose levels hence 4 influencing hypoglycemic risk. Finally, while durability of GKA efficacy is not the focus of the current work, it should be Manuscript acknowledged that understanding differences in activator binding 3 kinetics could have additional, longer term, benefit for -4 -3 -2 -1 0 Log off-rate 15 understanding durability differences between activators. Preclinical Hypoglycemia TI < 5 Preclinical Hypoglycemia TI > 5 The influence of binding kinetics on hypoglycemic risk was investigated using a series of structurally diverse GKAs span- Figure 1: Kinetic plot showing relationship between off rates (koff ), on ning a range of in vitro potencies (EC 50 = 10 – 391 nM) for rates (kon ) and binding affinities (K D) represented on the diagonal axis for 20 which, a detailed assessment of binding kinetic, enzyme 60 compounds in Table 1, and determined in SPR. Off rates and on rates are activation and pharmacodynamic profiles were plotted as logarithmic values. Only efficacious compounds are represented in vivo on plot. Accepted determined. Specifically, the binding affinities and kinetics of these activators were characterized against human recombinant glucokinase using surface plasmon resonance (SPR) as shown in 65 To obtain a pharmacokinetic/pharmacodynamic (PK/PD) 25 Table 1. To enable this experiment, biotinylated glucokinase was 8,10 assessment of postprandial efficacy and hypoglycemia risk, these expressed in E. Coli , purified as previously described and activators were evaluated in Wistar rats during an oral glucose captured onto a streptavidin sensor chip to levels ranging from tolerance test (OGTT) as described previously. 9 To evaluate 5000-6000 response units (RU). SPR binding experiments were efficacy, an oral glucose challenge (2 g/kg) was administered 60 carried out in duplicate for three activator concentrations with 3 70 min post dose and the effect of the activator at reducing the 30 fold dilution and in the presence of a saturating glucose glucose AUC excursion during a hyperglycemic state was concentration of 50 mM glucose (representative SPR determined. [GKA] efficacy was defined as concentration of sensorgrams shown in supplementary material Figure S1 for unbound (free) activator affording a 20% reduction in glucose compounds 6 and 15 ). The activator concentrations run in SPR AUC during an OGTT. [GKA] hypoglycemia was defined as the were based on the EC 50 with the top concentration chosen to be 75 concentration of unbound (free) activator reducing fasting plasma 35 several fold over the EC 50 to achieve saturation. Activator glucose to <60 mg/dL. The hypoglycemic risk assessment was binding kinetics to glucokinase as a function of glucose characterized by a therapeutic index (TI) < 5 as calculated by concentrations were attempted but could not be rigorously [GKA] hypoglycemia / [GKA] efficacy and listed as “no” or “yes” determined in SPR due
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