The structures of salt-inducible kinase 3 in complex with inhibitors reveal determinants for binding and selectivity.

The salt-inducible kinases (SIKs) 1 to 3, belonging to the AMPK-related kinase family, serve as master regulators orchestrating a diverse set of physiological processes such as metabolism, bone formation, immune response, oncogenesis, and cardiac rhythm. Owing to its key regulatory role, the SIK kinases have emerged as compelling targets for pharmacological intervention across a diverse set of indications. Therefore, there is interest in developing SIK inhibitors with defined selectivity profiles both to further dissect the downstream biology and for treating disease. However, despite a large pharmaceutical interest in the SIKs, experimental structures of SIK kinases are scarce. This is likely due to the challenges associated with the generation of proteins suitable for structural studies. By adopting a rational approach to construct design and protein purification, we successfully crystallized and subsequently solved the structure of SIK3 in complex with HG-9-91-01, a potent SIK inhibitor. To enable further SIK3-inhibitor complex structures we identified an antibody fragment that facilitated crystallization and enabled a robust protocol suitable for structure-based drug design. The structures reveal SIK3 in an active conformation, where the ubiquitin-associated domain is shown to provide further stabilization to this active conformation. We present four pharmacologically relevant and distinct SIK3-inhibitor complexes. These detail the key interaction for each ligand and reveal how different regions of the ATP site are engaged by the different inhibitors to achieve high affinity. Notably, the structure of SIK3 in complex with a SIK3 specific inhibitor offers insights into isoform selectivity.

Controlling Liquid Crystal Configuration and Phase Using Multiple Molecular Triggers.

Liquid crystals are able to transform a local molecular interaction into a macroscopic change of state, making them a valuable "smart" material. Here, we investigate a novel polymeric amphiphile as a candidate for molecular triggering of liquid crystal droplets in aqueous background. Using microscopy equipped with crossed polarizers and optical tweezers, we find that the monomeric amphiphile is able to trigger both a fast phase change and then a subsequent transition from nematic to isotropic. We next include sodium dodecyl sulfate (SDS), a standard surfactant, with the novel amphiphilic molecules to test phase transitioning when both were present. As seen previously, we find that the activity of SDS at the surface can result in configuration changes with hysteresis. We find that the presence of the polymeric amphiphile reverses the hysteresis previously observed during such transitions. This work demonstrates a variety of phase and configuration changes of liquid crystals that can be controlled by multiple exogenous chemical triggers.

A class of highly selective inhibitors bind to an active state of PI3Kγ.

We have discovered a class of PI3Kγ inhibitors exhibiting over 1,000-fold selectivity over PI3Kα and PI3Kß. On the basis of X-ray crystallography, hydrogen-deuterium exchange-mass spectrometry and surface plasmon resonance experiments we propose that the cyclopropylethyl moiety displaces the DFG motif of the enzyme away from the adenosine tri-phosphate binding site, inducing a large conformational change in both the kinase- and helical domains of PI3Kγ. Site directed mutagenesis explained how the conformational changes occur. Our results suggest that these cyclopropylethyl substituted compounds selectively inhibit the active state of PI3Kγ, which is unique to these compounds and to the PI3Kγ isoform, explaining their excellent potency and unmatched isoform selectivity that were confirmed in cellular systems. This is the first example of a Class I PI3K inhibitor achieving its selectivity by affecting the DFG motif in a manner that bears similarity to DFG in/out for type II protein kinase inhibitors.

Direct Observation of Liquid Crystal Droplet Configurational Transitions using Optical Tweezers.

Liquid crystals (LCs) are easily influenced by external interactions, particularly at interfaces. When rod-like LC molecules are confined to spherical droplets, they experience a competition between interfacial tension and elastic deformations. The configuration of LCs inside a droplet can be controlled using surfactants that influence the interfacial orientation of the LC molecules in the oil-phase of an oil in water emulsion. Here, we used the surfactant sodium dodecyl sulfate (SDS) to manipulate the orientation of 5CB molecules in a polydisperse emulsion and examined the configuration of the droplets as a function of SDS concentration. We triggered pronounced morphological transitions by altering the SDS concentration while observing an individual LC droplet held in place using an optical tweezer. We compared the experimental configuration changes to predictions from simulations. We observed a hysteresis in the SDS concentration that induced the morphological transition from radial to bipolar and back as well as a fluctuations in the configuration during the transition.

Structure and biophysical characterization of the human full-length neurturin-GFRa2 complex: A role for heparan sulfate in signaling.

Neurturin (NRTN) provides trophic support to neurons and is considered a therapeutic agent for neurodegenerative diseases, such as Parkinson's disease. It binds to its co-receptor GFRa2, and the resulting NRTN-GFRa2 complex activates the transmembrane receptors rearranged during transfection (RET) or the neural cell adhesion molecule (NCAM). We report the crystal structure of NRTN, alone and in complex with GFRa2. This is the first crystal structure of a GFRa with all three domains and shows that domain 1 does not interact directly with NRTN, but it may support an interaction with RET and/or NCAM, via a highly conserved surface. In addition, biophysical results show that the relative concentration of GFRa2 on cell surfaces can affect the functional affinity of NRTN through avidity effects. We have identified a heparan sulfate-binding site on NRTN and a putative binding site in GFRa2, suggesting that heparan sulfate has a role in the assembly of the signaling complex. We further show that mutant NRTN with reduced affinity for heparan sulfate may provide a route forward for delivery of NRTN with increased exposure in preclinical in vivo models and ultimately to Parkinson's patients.

Structural and functional characterization of a specific antidote for ticagrelor.

Ticagrelor is a direct-acting reversibly binding P2Y12 antagonist and is widely used as an antiplatelet therapy for the prevention of cardiovascular events in acute coronary syndrome patients. However, antiplatelet therapy can be associated with an increased risk of bleeding. Here, we present data on the identification and the in vitro and in vivo pharmacology of an antigen-binding fragment (Fab) antidote for ticagrelor. The Fab has a 20 pM affinity for ticagrelor, which is 100 times stronger than ticagrelor's affinity for its target, P2Y12. Despite ticagrelor's structural similarities to adenosine, the Fab is highly specific and does not bind to adenosine, adenosine triphosphate, adenosine 5'-diphosphate, or structurally related drugs. The antidote concentration-dependently neutralized the free fraction of ticagrelor and reversed its antiplatelet activity both in vitro in human platelet-rich plasma and in vivo in mice. Lastly, the antidote proved effective in normalizing ticagrelor-dependent bleeding in a mouse model of acute surgery. This specific antidote for ticagrelor may prove valuable as an agent for patients who require emergency procedures.

Discovery of triazole aminopyrazines as a highly potent and selective series of PI3Kδ inhibitors.

A novel class of potent PI3Kδ inhibitors with >1000-fold selectivity against other class I PI3K isoforms is described. Optimization of the substituents on a triazole aminopyrazine scaffold, emerging from an in-house PI3Kα program, turned moderately selective PI3Kδ compounds into highly potent and selective PI3Kδ inhibitors. These efforts resulted in a series of aminopyrazines with PI3Kδ IC50⩽1nM in the enzyme assay, some of the most selective PI3Kδ inhibitors published to date, with a cell potency in a JeKo-cell assay of 20-120nM.

Discovery of a novel pyrazole series of group X secreted phospholipase A2 inhibitor (sPLA2X) via fragment based virtual screening.

The discovery of potent novel pyrazole containing group X secreted phospholipase A2 inhibitors via structure based virtual screening is reported. Docking was applied on a large set of in-house fragment collection and pharmacophore feature matching was used to filter docking poses. The selected virtual screening hits was run in NMR screening, a potent pyrazole containing fragment hit was identified and confirmed by its complex X-ray structure and the following biochemical assay result. Expansion on the fragment hit has led to further improvement of potency while maintaining high ligand efficiency, thus supporting the further development of this chemical series.

Vestibular Preservation in Pediatric Cochlear Implantation.

OBJECTIVES: Evaluate the rate of preserved vestibular function in pediatric cochlear implant surgery. STUDY DESIGN: Retrospective case review. METHODS: Pre- and post-operative vestibular tests were compared in children who underwent cochlear implantation at a tertiary level pediatric hospital over a 4-year period. RESULTS: Data from 59 implanted ears in 44 children was included. Median age was 2.8 years at initial testing (range 7 months - 21 years) with 1:1 male/female ratio. Implant surgeries were 26 unilateral, 13 bilateral simultaneous, and 5 bilateral sequential. The majority were implanted with slim, non-styletted electrodes (86.4%) via a round window approach (91.5%). Normal pre-operative results were preserved post-operatively on rotary chair testing in 75% (21/28) of patients, cervical vestibular evoked myogenic potential testing in (75%) 30/40 of ears tested, ocular vestibular evoked myogenic potential testing in 85.7% (6/7) of ears tested, video head impulse testing in 100% (9/9) of ears tested, and computerized dynamic posturography in 100% (5/5) of patients tested. Overall, 62.5% of patients had no new deficits on any vestibular test performed post-operatively. CONCLUSIONS: Preservation rates of vestibular function following cochlear implant surgery were higher in this cohort than what has been reported in many earlier studies. Contemporary, less traumatic electrodes and insertion techniques may be a significant factor. The risk of causing a new, severe bilateral vestibular loss with long-term functional impacts appears to be low. Further study is warranted on the impacts of different cochlear implant electrode designs and insertion approaches on post-operative vestibular preservation. LEVEL OF EVIDENCE: 4, Case Series Laryngoscope, 134:1913-1918, 2024.

Discovery and Characterization of a Bicyclic Peptide (Bicycle) Binder to Thymic Stromal Lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pro-inflammatory cytokine involved in the development of asthma and other atopic diseases. We used Bicycle Therapeutics' proprietary phage display platform to identify bicyclic peptides (Bicycles) with high affinity for TSLP, a target that is difficult to drug with conventional small molecules due to the extended protein-protein interactions it forms with both receptors. The hit series was shown to bind to TSLP in a hotspot, that is also used by IL-7Rα. Guided by the first X-ray crystal structure of a small peptide binding to TSLP and the identification of key metabolites, we were able to improve the proteolytic stability of this series in lung S9 fractions without sacrificing binding affinity. This resulted in the potent Bicycle 46 with nanomolar affinity to TSLP (KD = 13 nM), low plasma clearance of 6.4 mL/min/kg, and an effective half-life of 46 min after intravenous dosing to rats.

Solutions for the storage and handling of SPINE standard pucks.

Currently there is no rack system for the long-term storage of SPINE pucks in spite of their commercial availability and heavy usage at the ESRF. The only way to store pucks is in transport dewar canisters which presents a number of limitations and drawbacks. Here a simple affordable rack for storing SPINE pucks is described, which we believe is accessible to not only synchrotrons but also both academic and industrial research laboratories.

Balance and Vestibular Deficits in Pediatric Patients with Autism Spectrum Disorder: An Underappreciated Clinical Aspect.

Children with autism spectrum disorder (ASD) not only have communication and social difficulties, but also exhibit poor balance and motor control ability, which frequently affect daily activities. Effective balance and motor control rely on the integration of somatosensory, visual, and vestibular inputs. Although reports of balance dysfunction in ASD have been documented, comprehensive studies of balance and vestibular function in children with ASD are scarce. In this study, we retrospectively reviewed 36 pediatric patients diagnosed with ASD who underwent balance/vestibular laboratory testing in our speciality clinic. Results from sensory organization test (SOT) or modified clinical test for sensory integration of balance (mCTSIB) found that out of 15 patients, 80% had abnormal findings. Of the children who successfully completed each vestibular test, abnormal responses were observed in 12 (80%) sensory organization tests, 5 (24%) vestibular evoked myogenic potential (VEMP), 22 (66%) videonystagmography (VNG), and 11 (32%) sinusoidal rotary chair tests. These results indicate that balance and vestibular testing may be of diagnostic value for clinicians and providers as an aid in early detection, intervention, and the development of appropriate management and therapies for this patient population. Increased awareness of this topic is warranted to promote better clinical management of this special group of patients and improve their quality of life.

Discovery and optimization of cyclohexane-1,4-diamines as allosteric MALT1 inhibitors.

Inhibition of mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) is a promising strategy to modulate NF-κB signaling, with the potential to treat B-cell lymphoma and autoimmune diseases. We describe the discovery and optimization of (1s,4s)-N,N'-diaryl cyclohexane-1,4-diamines, a novel series of allosteric MALT1 inhibitors, resulting in compound 8 with single digit micromolar cell potency. X-ray analysis confirms that this compound binds to an induced allosteric site in MALT1. Compound 8 is highly selective and has an excellent in vivo rat PK profile with low clearance and high oral bioavailability, making it a promising lead for further optimization.

Design of small molecule inhibitors of acetyl-CoA carboxylase 1 and 2 showing reduction of hepatic malonyl-CoA levels in vivo in obese Zucker rats.

Inhibition of acetyl-CoA carboxylases has the potential for modulating long chain fatty acid biosynthesis and mitochondrial fatty acid oxidation. Hybridization of weak inhibitors of ACC2 provided a novel, moderately potent but lipophilic series. Optimization led to compounds 33 and 37, which exhibit potent inhibition of human ACC2, 10-fold selectivity over inhibition of human ACC1, good physical and in vitro ADME properties and good bioavailability. X-ray crystallography has shown this series binding in the CT-domain of ACC2 and revealed two key hydrogen bonding interactions. Both 33 and 37 lower levels of hepatic malonyl-CoA in vivo in obese Zucker rats.

Fragment-Based Discovery of Novel Allosteric MEK1 Binders.

The MEK1 kinase plays a critical role in key cellular processes, and as such, its dysfunction is strongly linked to several human diseases, particularly cancer. MEK1 has consequently received considerable attention as a drug target, and a significant number of small-molecule inhibitors of this kinase have been reported. The majority of these inhibitors target an allosteric pocket proximal to the ATP binding site which has proven to be highly druggable, with four allosteric MEK1 inhibitors approved to date. Despite the significant attention that the MEK1 allosteric site has received, chemotypes which have been shown structurally to bind to this site are limited. With the aim of discovering novel allosteric MEK1 inhibitors using a fragment-based approach, we report here a screening method which resulted in the discovery of multiple allosteric MEK1 binders, one series of which was optimized to sub-µM affinity for MEK1 with promising physicochemical and ADMET properties.

Discovery of AZD8154, a Dual PI3Kγδ Inhibitor for the Treatment of Asthma.

Starting from our previously described PI3Kγ inhibitors, we describe the exploration of structure-activity relationships that led to the discovery of highly potent dual PI3Kγδ inhibitors. We explored changes in two positions of the molecules, including macrocyclization, but ultimately identified a simpler series with the desired potency profile that had suitable physicochemical properties for inhalation. We were able to demonstrate efficacy in a rat ovalbumin challenge model of allergic asthma and in cells derived from asthmatic patients. The optimized compound, AZD8154, has a long duration of action in the lung and low systemic exposure coupled with high selectivity against off-targets.

The structural basis of cephalosporin formation in a mononuclear ferrous enzyme.

Deacetoxycephalosporin-C synthase (DAOCS) is a mononuclear ferrous enzyme that transforms penicillins into cephalosporins by inserting a carbon atom into the penicillin nucleus. In the first half-reaction, dioxygen and 2-oxoglutarate produce a reactive iron-oxygen species, succinate and CO2. The oxidizing iron species subsequently reacts with penicillin to give cephalosporin and water. Here we describe high-resolution structures for ferrous DAOCS in complex with penicillins, the cephalosporin product, the cosubstrate and the coproduct. Steady-state kinetic data, quantum-chemical calculations and the new structures indicate a reaction sequence in which a 'booby-trapped' oxidizing species is formed. This species is stabilized by the negative charge of succinate on the iron. The binding sites of succinate and penicillin overlap, and when penicillin replaces succinate, it removes the stabilizing charge, eliciting oxidative attack on itself. Requisite groups of penicillin are within 1 A of the expected position of a ferryl oxygen in the enzyme-penicillin complex.

Clavatadine A, a natural product with selective recognition and irreversible inhibition of factor XIa.

Bioassay-guided fractionation of a CH2Cl2/MeOH extract of the sponge Suberea clavata using the serine protease factor XIa to detect antithrombotic activity led to the isolation of the new marine natural products, clavatadines A and B. Clavatadines A and B inhibited factor XIa with IC50's of 1.3 and 27 microM, respectively. A crystal structure of protein-inhibitor (clavatadine A) complex was obtained and revealed interesting selective binding and irreversible inhibition of factor XIa. The cocrystal structure provides guidance for the design and synthesis of future factor XIa inhibitors as antithrombotic agents.

Discovery of Highly Isoform Selective Orally Bioavailable Phosphoinositide 3-Kinase (PI3K)-γ Inhibitors.

In this paper, we describe the discovery and optimization of a new chemotype of isoform selective PI3Kγ inhibitors. Starting from an HTS hit, potency and physicochemical properties could be improved to give compounds such as 15, which is a potent and remarkably selective PI3Kγ inhibitor with ADME properties suitable for oral administration. Compound 15 was advanced into in vivo studies showing dose-dependent inhibition of LPS-induced airway neutrophilia in rats when administered orally.

Insights into cephamycin biosynthesis: the crystal structure of CmcI from Streptomyces clavuligerus.

Cephamycin C-producing microorganisms use two enzymes to convert cephalosporins to their 7alpha-methoxy derivatives. Here we report the X-ray structure of one of these enzymes, CmcI, from Streptomyces clavuligerus. The polypeptide chain of the enzyme folds into a C-terminal Rossmann domain and a smaller N-terminal domain, and the molecule packs as a hexamer in the crystal. The Rossmann domain binds S-adenosyl-L-methionine (SAM) and the demethylated product, S-adenosyl-L-homocysteine, in a fashion similar to the common binding mode of this cofactor in SAM-dependent methyltransferases. There is a magnesium-binding site in the vicinity of the SAM site with a bound magnesium ion ligated by residues Asp160, Glu186 and Asp187. The expected cephalosporin binding site near the magnesium ion is occupied by polyethyleneglycol (PEG) from the crystallisation medium. The geometry of the SAM and the magnesium binding sites is similar to that found in cathechol O-methyltransferase. The results suggest CmcI is a methyltransferase, and its most likely function is to catalyse the transfer of a methyl group from SAM to the 7alpha-hydroxy cephalosporin in the second catalytic reaction of cephamycin formation. Based on the docking of the putative substrate, 7alpha-hydroxy-O-carbamoyldeacetylcephalosporin C, to the structure of the ternary CmcI-Mg2+-SAM complex, we propose a model for substrate binding and catalysis. In this model, the 7-hydroxy group of the beta-lactam ring ligates the Mg2+ with its alpha-side facing the methyl group of SAM at a distance that would allow methylation of the hydroxyl-group.