CaMKII inhibition has dual effects on spontaneous Ca2+ release and Ca2+ alternans in ventricular cardiomyocytes from mice with a gain-of-function RyR2 mutation

In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca2+/calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca2+ alternans. Aim: To test whether suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca2+ alternans. Methods and results: We studied spontaneous Ca2+-release events and Ca2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca2+-release events in RyR2‑R2474S cardiomyocytes exposed to the β‑adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca2+ alternans and increased Ca2+ alternans ratio compared with both an inactive analog of KN‑93 and with vehicle-treated controls. This increased propensity for Ca2+ alternans was explained by prolongation of Ca2+-release refractoriness. Importantly, the increased propensity for Ca2+ alternans in KN‑93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild-type. Conclusions: Inhibition of CaMKII efficiently reduces spontaneous Ca2+-release, but promotes Ca2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca2+-release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca2+ alternans should include investigation of both phenomena.

Pharmacologic targeting of Cdc42 GTPase by a small molecule Cdc42 activity-specific inhibitor prevents platelet activation and thrombosis

Gene targeting of Cdc42 GTPase has been shown to inhibit platelet activation. In this study, we investigated a hypothesis that inhibition of Cdc42 activity by CASIN, a small molecule Cdc42 Activity-Specific INhibitor, may down regulate platelet activation and thrombus formation. We investigated the effects of CASIN on platelet activation in vitro and thrombosis in vivo. In human platelets, CASIN, but not its inactive analog Pirl7, blocked collagen induced activation of Cdc42 and inhibited phosphorylation of its downstream effector, PAK1/2. Moreover, addition of CASIN to washed human platelets inhibited platelet spreading on immobilized fibrinogen. Treatment of human platelets with CASIN inhibited collagen or thrombin induced: (a) ATP secretion and platelet aggregation; and (b) phosphorylation of Akt, ERK and p38-MAPK. Pre-incubation of platelets with Pirl7, an inactive analog of CASIN, failed to inhibit collagen induced aggregation. Washing of human platelets after incubation with CASIN eliminated its inhibitory effect on collagen induced aggregation. Intraperitoneal administration of CASIN to wild type mice inhibited ex vivo aggregation induced by collagen but did not affect the murine tail bleeding times. CASIN administration, prior to laser-induced injury in murine cremaster muscle arterioles, resulted in formation of smaller and unstable thrombi compared to control mice without CASIN treatment. These data suggest that pharmacologic targeting of Cdc42 by specific and reversible inhibitors may lead to the discovery of novel antithrombotic agents.

Synthesis of the 6-Substituted Imidazo[1,2-a]Pyridine-3-yl-2- Phosphonopropionic Acids as Potential Inhibitors of Rab Geranylgeranyl Transferase

Twelve phosphonopropionates derived from 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC) were synthesized and evaluated for their activity as inhibitors of protein geranylgeranylation. The nature of the substituent in the C6 position of imidazo[1,2-a]pyridine ring was responsible for the compound's activity against Rab geranylgeranyl transferase (RGGT). The most active inhibitors disrupted Rab11A prenylation in the human cervical carcinoma HeLa cell line. The esterification of carboxylic acid in the phosphonopropionate moiety turned the inhibitor into an inactive analog.

Pharmacological Inhibition of the RNA m6a Writer METTL3 As a Novel Therapeutic Strategy for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. New evidence has revealed an important role for RNA modifications in cancer development and maintenance via the catalytic function of RNA-modifying enzymes. We and others have recently shown that METTL3, the RNA methyltransferase responsible for the deposition of N-6-methyl groups on adenosine (m6A) in mRNA, is a promising therapeutic target for AML1,2. Here we present the in vitro and in vivo characterization of novel small molecule inhibitors of METTL3 as an effective therapeutic strategy in AML. Recently, we generated a comprehensive catalogue of RNA-modifying enzymes that are essential for AML cells using CRISPR-Cas9 recessive screens and characterised METTL3 as a novel therapeutic candidate through its effects on mRNA translational efficiency of key leukemia oncogenes1. Using a structure-guided medicinal chemistry platform we developed and optimised small molecule inhibitors of METTL3 from 2 distinct chemical series. Here we demonstrate that compounds 1 and 2 show biochemical inhibition of METTL3 enzyme with single digit nanomolar potency, while direct binding to METTL3 was confirmed by Surface Plasmon Resonance (SPR) analysis with comparable potency between compounds. Additionally, we developed compound 3 as an inactive analog which was confirmed inactive in enzyme assays (>50 µM IC50). Importantly, we verified that compounds 1 and 2 are selective for METTL3 and do not inhibit a panel of other RNA, DNA or protein methyltransferases tested (>10 µM IC50). Cellular target engagement was confirmed by demonstrating that compounds 1 and 2 reduced m6A levels and inhibited the protein expression of METTL3-dependent m6A substrates in mouse and human AML models, including SP1, with nanomolar potency. Furthermore, treatment of MOLM13 cells with compounds 1 and 2 inhibited their proliferation with comparable potency to SP1 inhibition. The same anti-proliferative effect was observed using a large panel of human AML cell lines. In addition, polyribosome profiling in MOLM13 cells treated with compounds 1 and 2 revealed enhanced blocking of mRNA translation, mirroring the effects derived from the genetic inhibition of METTL3. Notably, all of the above effects were not observed when the inactive analog (compound 3) was used, further highlighting the specificity and sensitivity of our active candidates. We subsequently performed in vivo characterisation of compound 1. This compound exhibited excellent bioavailability after oral or intraperitoneal administration with good dose-proportional exposure in mice and a half-life of 3.5 hours. It also appeared to be well-tolerated with no body weight loss or clinical signs of toxicity. We also evaluated its anti-tumor effects in patient derived xenotransplantation experiments (PDX) as well as transplantation experiments using an MLL-AF9 driven primary murine AML model. Daily dosing of 30 mg/kg significantly inhibited AML expansion and reduced spleen weight compared to vehicle control, indicating a pronounced anti-tumor effect in vivo. Target engagement was confirmed in bone marrow and spleen as measured by the reduction of METTL3-dependent m6A targets. Importantly, we went on to demonstrate that, while the pharmacological inhibition of METTL3 is required for AML cell survival, it was dispensable for normal hematopoiesis. Collectively, we describe the detailed characterization of potent and selective inhibitors of the METTL3 RNA methyltransferase, and demonstrate their activity and utility using biochemical, cellular and in vivo systems. We show that inhibition of METTL3 by small molecules in vivo leads to strong anti-tumor effects in physiologically and clinically relevant models of AML. To our knowledge, this is the first study demonstrating in vivo activity of inhibitors of an RNA methyltransferase, hence providing proof of concept that RNA modifying enzymes represent a new target class for anti-cancer therapeutics. References Barbieri, I. et al. Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature552, 126-131, doi:10.1038/nature24678 (2017). Vu, L. P. et al. The N(6)-methyladenosine (m(6)A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med23, 1369-1376, doi:10.1038/nm.4416 (2017). Disclosures Yankova: STORM THERAPEUTICS: Employment. Fosbeary:STORM THERAPEUTICS: Employment. Hendrick:STORM THERAPEUTICS: Employment. Leggate:STORM THERAPEUTICS: Employment. Ofir-Rosenfeld:STORM THERAPEUTICS: Employment. Sapetschnig:STORM THERAPEUTICS: Employment. Albertella:STORM THERAPEUTICS: Employment. Blackaby:STORM THERAPEUTICS: Employment. Rausch:STORM THERAPEUTICS: Employment. Vassiliou:Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding. Kouzarides:STORM THERAPEUTICS: Equity Ownership.

Selective inhibition of EZH2 and EZH1 enzymatic activity by a small molecule suppresses MLL-rearranged leukemia

Key Points We characterize active vs inactive analog compounds suitable for inhibition of both PRC2-EZH2 and PRC2-EZH1 ex vivo and in vivo. This study is the first to show oral delivery of an EZH2 and EZH1 dual inhibitor as promising therapeutics for MLL-rearranged leukemia.

Excretion of NaCl and KCl loads in mosquitoes. 2. Effects of the small molecule Kir channel modulator VU573 and its inactive analog VU342

The effect of two small molecules VU342 and VU573 on renal functions in the yellow fever mosquito Aedes aegypti was investigated in vitro and in vivo. In isolated Malpighian tubules, VU342 (10 μM) had no effect on the transepithelial secretion of Na+, K+, Cl−, and water. In contrast, 10 μM VU573 first stimulated and then inhibited the transepithelial secretion of fluid when the tubules were bathed in Na+-rich or K+-rich Ringer solution. The early stimulation was blocked by bumetanide, suggesting the transient stimulation of Na-K-2Cl cotransport, and the late inhibition of fluid secretion was consistent with the known block of AeKir1, an Aedes inward rectifier K+ channel, by VU573. VU342 and VU573 at a hemolymph concentration of about 11 μM had no effect on the diuresis triggered by hemolymph Na+ or K+ loads. VU342 at a hemolymph concentration of 420 μM had no effect on the diuresis elicited by hemolymph Na+ or K+ loads. In contrast, the same concentration of VU573 significantly diminished the Na+ diuresis by inhibiting the urinary excretion of Na+, Cl−, and water. In K+-loaded mosquitoes, 420 μM VU573 significantly diminished the K+ diuresis by inhibiting the urinary excretion of K+, Na+, Cl−, and water. We conclude that 1) the effects of VU573 observed in isolated Malpighian tubules are overwhelmed in vivo by the diuresis triggered with the coinjection of Na+ and K+ loads, and 2) at a hemolymph concentration of 420 μM VU573 affects Kir channels systemically, including those that might be involved in the release of diuretic hormones.

Suppression of the cough reflex by inhibition of ERK1/2 activation in the caudal nucleus tractus solitarii of the rabbit

The caudal nucleus tractus solitarii (cNTS), the predominant site of termination of cough-related afferents, has been shown to be a site of action of some centrally acting antitussive agents. A role of ERK1/2 has been suggested in acute central processing of nociceptive inputs. Because pain and cough share similar features, we investigated whether ERK1/2 activation could also be involved in the central transduction of tussive inputs. For this purpose, we undertook the present research on pentobarbital sodium-anesthetized, spontaneously breathing rabbits by using microinjections (30–50 nl) of an inhibitor of ERK1/2 activation (U0126) into the cNTS. Bilateral microinjections of 25 mM U0126 caused rapid and reversible reductions in the cough responses induced by both mechanical and chemical (citric acid) stimulation of the tracheobronchial tree. In particular, the cough number and peak abdominal activity decreased. Bilateral microinjections of 50 mM U0126 completely suppressed the cough reflex without affecting the Breuer-Hering inflation reflex, the pulmonary chemoreflex, and the sneeze reflex. These U0126-induced effects were, to a large extent, reversible. Bilateral microinjections of 50 mM U0124, the inactive analog of U0126, at the same cNTS sites had no effect. This is the first study that provides evidence that ERK1/2 activation within the cNTS is required for the mediation of cough reflex responses in the anesthetized rabbit. These results suggest a role for ERK1/2 in the observed effects via nontranscriptional mechanisms, given the short time involved. They also may provide hints for the development of novel antitussive strategies.

Rebound Bursts in GABAergic Neurons of the Thalamic Reticular Nucleus in Postnatal Mice

Whole cell patch-clamp recordings from GABAergic cells of thalamic reticular nucleus (RTN) in thalamocortical slices made from postnatal day 6 (P6) to 10 (P10) were used to investigate the pattern of rebound bursts (RBs) triggered by an injection of hyperpolarizing current into RTN cells. The number of RBs in the RTN and the overlying Na+/K+ spikes changed in an agedependent manner. The generation of RBs depended largely on the amplitude of the after-hyperpolarizations (AHPs). RB patterns in response to hyperpolarizing current injection into relay cells were markedly different from RB patterns in RTN cells with an after-depolarization. GABAA receptor antagonist bicuculline methiodide (BMI) changed burst firing patterns, increasing the duration of RB and decreasing the amplitude of AHP in RTN cells. Furthermore, local puffs of NMDA in the presence of BMI induced RBs. K+ channel blocker 4-aminopyridine partially mimicked the effect of BMI on AHPs. The shapes of RBs were altered by a selective CaMKII inhibitor KN-62, but not by an inactive analog KN-04.