Predicting the relative binding affinity of mineralocorticoid receptor antagonists by density functional methods.

In drug discovery, prediction of binding affinity ahead of synthesis to aid compound prioritization is still hampered by the low throughput of the more accurate methods and the lack of general pertinence of one method that fits all systems. Here we show the applicability of a method based on density functional theory using core fragments and a protein model with only the first shell residues surrounding the core, to predict relative binding affinity of a matched series of mineralocorticoid receptor (MR) antagonists. Antagonists of MR are used for treatment of chronic heart failure and hypertension. Marketed MR antagonists, spironolactone and eplerenone, are also believed to be highly efficacious in treatment of chronic kidney disease in diabetes patients, but is contra-indicated due to the increased risk for hyperkalemia. These findings and a significant unmet medical need among patients with chronic kidney disease continues to stimulate efforts in the discovery of new MR antagonist with maintained efficacy but low or no risk for hyperkalemia. Applied on a matched series of MR antagonists the quantum mechanical based method gave an R(2) = 0.76 for the experimental lipophilic ligand efficiency versus relative predicted binding affinity calculated with the M06-2X functional in gas phase and an R(2) = 0.64 for experimental binding affinity versus relative predicted binding affinity calculated with the M06-2X functional including an implicit solvation model. The quantum mechanical approach using core fragments was compared to free energy perturbation calculations using the full sized compound structures.

Identification of Novel Series of Potent and Selective Relaxin Family Peptide Receptor 1 (RXFP1) Agonists.

Relaxin H2 is a clinically relevant peptide agonist for relaxin family peptide receptor 1 (RXFP1), but a combination of this hormone's short plasma half-life and the need for injectable delivery limits its therapeutic potential. We sought to overcome these limitations through the development of a potent small molecule (SM) RXFP1 agonist. Although two large SM HTS campaigns failed in identifying suitable hit series, we uncovered novel chemical space starting from the only known SM RXFP1 agonist series, represented by ML290. Following a design-make-test-analyze strategy based on improving early dose to man ranking, we discovered compound 42 (AZ7976), a highly selective RXFP1 agonist with sub-nanomolar potency. We used AZ7976, its 10 000-fold less potent enantiomer 43 and recombinant relaxin H2 to evaluate in vivo pharmacology and demonstrate that AZ7976-mediated heart rate increase in rats was a result of RXFP1 agonism. As a result, AZ7976 was selected as lead for continued optimization.

Discovery of Clinical Candidate AZD5462, a Selective Oral Allosteric RXFP1 Agonist for Treatment of Heart Failure.

Optimization of the highly potent and selective, yet metabolically unstable and poorly soluble hRXFP1 agonist AZ7976 led to the identification of the clinical candidate, AZD5462. Assessment of RXFP1-dependent cell signaling demonstrated that AZD5462 activates a highly similar panel of downstream pathways as relaxin H2 but does not modulate relaxin H2-mediated cAMP second messenger responsiveness. The therapeutic potential of AZD5462 was assessed in a translatable cynomolgus monkey heart failure model. Following 8 weeks of treatment with AZD5462, robust improvements in functional cardiac parameters including LVEF were observed at weeks 9, 13, and 17 without changes in heart rate or mean arterial blood pressure. AZD5462 was well tolerated in both rat and cynomolgus monkey and has successfully completed phase I studies in healthy volunteers. In summary, AZD5462 is a small molecule pharmacological mimetic of relaxin H2 signaling at RXFP1 and holds promise as a potential therapeutic approach to treat heart failure patients.

The allosterically modulated FFAR2 is transactivated by signals generated by other neutrophil GPCRs.

Positive allosteric modulators for free fatty acid receptor 2 (FFAR2/GPR43), that affect receptor function through binding to two distinct allosteric binding sites, were used to determine the correlation between the responses induced in neutrophils by two distinct activation modes; FFAR2 was activated either by the orthosteric agonist propionate or by a receptor transactivation mechanism that activated FFAR2 from the cytosolic side of the neutrophil plasma membrane by signals generated by the neutrophil PAFR (receptor for platelet activating factor), P2Y2R (receptor for ATP), FPR1 (receptor for fMLF) and FPR2 (receptor for WKYMVM). We show that the transactivation signals that activate FFAR2 in the absence of any orthosteric agonist were generated downstream of the signaling G protein that couple to PAFR and P2Y2R. This transactivation of allosterically modulated FFAR2s, by signals generated by PAFR/P2Y2R, represents a novel mechanism by which a G protein coupled receptor can be activated. Weak correlations were obtained when the FFAR2 activity was induced by the transactivation signals generated by PAFRs and P2Y2Rs were compared with the FFAR2 activity induced by the orthosteric agonist propionate. Comparison of the responses for each allosteric modulator revealed that the ratio values, calculated from the peak values of the ATP and propionate responses, varied from 0.2 to 1. Depending on the allosteric modulator, the response induced by the two different mechanisms (orthosteric activation and receptor transactivation, respectively), was equal or the propionate response was more pronounced. Importantly, we conclude that FFAR2 activation from outside (orthosteric activation) and inside (receptor cross-talk/transactivation) can be selectively affected by an allosteric FFAR2 modulator.

AZ2158 is a more potent formyl peptide receptor 1 inhibitor than the commonly used peptide antagonists in abolishing neutrophil chemotaxis.

Formyl peptide receptor 1 (FPR1), a G protein-coupled receptor expressed in phagocytes, recognizes short N-formylated peptides originating from proteins synthesized by bacteria and mitochondria. Such FPR1 agonists are important regulators of neutrophil functions and by that, determinants of inflammatory reactions. As FPR1 is implicated in promoting both pro-inflammatory and pro-resolving responses associated with inflammatory diseases, characterization of ligands that potently and selectively modulate FPR1 induced functions might be of high relevance. Accordingly, a number of FPR1 specific antagonists have been identified and shown to inhibit agonist binding or receptor down-stream signaling as well as neutrophil functions such as granule secretion and NADPH oxidase activity. The inhibitory effect on neutrophil chemotaxis induced by FPR1 agonists has generally not been part of basic antagonist characterization. In this study we show that the inhibitory effects on neutrophil chemotaxis of established FPR1 antagonists (i.e., cyclosporin H, BOC1 and BOC2) are limited. Our data demonstrate that the recently described small molecule AZ2158 is a potent and selective FPR1 antagonist in human neutrophils. In contrast to the already established FPR1 antagonists, AZ2158 also potently inhibits chemotaxis. Whereas the cyclosporin H inhibition was agonist selective, AZ2158 inhibited the FPR1 response induced by both a balanced and a biased FPR1 agonist equally well. In accordance with the species specificity described for many FPR1 ligands, AZ2158 was not recognized by the mouse orthologue of FPR1. Our data demonstrate that AZ2158 may serve as an excellent tool compound for further mechanistic studies of human FPR1 mediated activities.

Multiple ligand recognition sites in free fatty acid receptor 2 (FFA2R) direct distinct neutrophil activation patterns.

The allosteric modulating free fatty acid receptor 2 ligands Cmp58 and AZ1729, increased the activity induced by orthosteric receptor agonists mediating a rise in intracellular calcium ions and activation of the neutrophil NADPH-oxidase. Together, the two modulators triggered an orthosteric-agonist-independent activation of the oxidase without any rise in the concentration of intracellular calcium ions. In this study, structurally diverse compounds presumed to be ligands for free fatty acid receptor 2 were used to gain additional insights into receptor-modulation/signaling. We identified two molecules that activate neutrophils on their own and we classified one as allosteric agonist and the other as orthosteric agonist. Ten compounds were classified as allosteric FFA2R modulators. Of these, one activated neutrophils when combined with AZ1729; the nine remaining compounds activated neutrophils solely when combined with Cmp58. The activation signals were primarily biased when stimulated by two allosteric modulators interacting with different binding sites, such that two complementary modulators together triggered an activation of the NADPH-oxidase but no increase in the intracellular concentration of calcium ions. No neutrophil activation was induced when allosteric receptor modulators suggested to be recognized by the same binding site were combined, results in agreement with our proposed model for activation, in which the receptor has two different sites that selectively bind allosteric modulators. The down-stream signaling mediated by cross-sensitizing allosteric receptor modulators, occurring independent of any orthosteric agonist, represent a new mechanism for activation of the neutrophil NADPH oxidase.

Mineralocorticoid Receptor Antagonists.

Two mineralocorticoid receptor antagonists, spironolactone and eplerenone, are currently approved by the FDA. Several non-steroid based ligands are in clinical trials for indications including heart failure, hypertension and diabetic kidney disease, and even more structurally distinct chemical series are reported in the literature with preclinical data from animal models. Design of new ligands that are both selective over the other oxosteroid receptors (GR, PR and AR) and possess properties compatible with oral dosing, despite the overall lipophilic binding pocket of MR, remains a challenge. High-throughput screening has been successfully used to identify novel starting points in several drug discovery programs, and these were optimized using property based drug design, often aided by protein-ligand X-ray complex structures.

Identification of Mineralocorticoid Receptor Modulators with Low Impact on Electrolyte Homeostasis but Maintained Organ Protection.

The mechanism-based risk for hyperkalemia has limited the use of mineralocorticoid receptor antagonists (MRAs) like eplerenone in cardio-renal diseases. Here, we describe the structure and property-driven lead generation and optimization, which resulted in identification of MR modulators ( S)-1 and ( S)-33. Both compounds were partial MRAs but still demonstrated equally efficacious organ protection as eplerenone after 4 weeks of treatment in uni-nephrectomized rats on high-salt diet and aldosterone infusion. Importantly, and in sharp contrast to eplerenone, this was achieved without substantial changes to the urine Na+/K+ ratio after acute treatment in rat, which predicts a reduced risk for hyperkalemia. This work led to selection of ( S)-1 (AZD9977) as the clinical candidate for treating MR-mediated cardio-renal diseases, including chronic kidney disease and heart failure. On the basis of our findings, we propose an empirical model for prediction of compounds with low risk of affecting the urinary Na+/K+ ratio in vivo.

Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion.

Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 in vitro potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR in vitro potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na+/K+ ratio, while eplerenone increased the Na+/K+ ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia.

Structure-Based Drug Design of Mineralocorticoid Receptor Antagonists to Explore Oxosteroid Receptor Selectivity.

The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high-throughput screening (HTS) campaign. The compound bound to MR with pKi =6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X-ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced-fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11- to 79-fold over PR and 23- to 234-fold over GR was obtained. Given the U-shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pKi =7.3. Two protein-ligand X-ray structures were determined, confirming the hypothesized binding mode for the designed compounds.