The mitochondrial permeability transition pore is a recognized drug target for neurodegenerative conditions such as multiple sclerosis and for ischemia-reperfusion injury in the brain and heart. The ...peptidylprolyl isomerase, cyclophilin D (CypD, PPIF), is a positive regulator of the pore, and genetic down-regulation or knock-out improves outcomes in disease models. Current inhibitors of peptidylprolyl isomerases show no selectivity between the tightly conserved cyclophilin paralogs and exhibit significant off-target effects, immunosuppression, and toxicity. We therefore designed and synthesized a new mitochondrially targeted CypD inhibitor, JW47, using a quinolinium cation tethered to cyclosporine. X-ray analysis was used to validate the design concept, and biological evaluation revealed selective cellular inhibition of CypD and the permeability transition pore with reduced cellular toxicity compared with cyclosporine. In an experimental autoimmune encephalomyelitis disease model of neurodegeneration in multiple sclerosis, JW47 demonstrated significant protection of axons and improved motor assessments with minimal immunosuppression. These findings suggest that selective CypD inhibition may represent a viable therapeutic strategy for MS and identify quinolinium as a mitochondrial targeting group for in vivo use.
Counteracting innate immunity is essential for successful viral replication. Host cyclophilins (Cyps) have been implicated in viral evasion of host antiviral responses, although the mechanisms are ...still unclear. Here, we show that hepatitis C virus (HCV) co-opts the host protein CypA to aid evasion of antiviral responses dependent on the effector protein kinase R (PKR). Pharmacological inhibition of CypA rescues PKR from antagonism by HCV NS5A, leading to activation of an interferon regulatory factor-1 (IRF1)-driven cell intrinsic antiviral program that inhibits viral replication. These findings further the understanding of the complexity of Cyp-virus interactions, provide mechanistic insight into the remarkably broad antiviral spectrum of Cyp inhibitors, and uncover novel aspects of PKR activity and regulation. Collectively, our study identifies a novel antiviral mechanism that harnesses cellular antiviral immunity to suppress viral replication.
The inhibition of Aurora kinases in order to arrest mitosis and subsequently inhibit tumor growth via apoptosis of proliferating cells has generated significant discussion within the literature. We ...report a novel class of Aurora kinase inhibitors based upon a phthalazinone pyrazole scaffold. The development of the phthalazinone template resulted in a potent Aurora-A selective series of compounds (typically >1000-fold selectivity over Aurora-B) that display good pharmacological profiles with significantly improved oral bioavailability compared to the well studied Aurora inhibitor VX-680.
CRH and urotensin I (UI) are neuroendocrine peptides that belong to the superfamily of corticotropin-releasing factors. In mammals, these peptides regulate the stress response and other central ...nervous system functions, whereas in fish an involvement for UI in osmoregulation has also been suggested. We have identified, characterized, and localized the genes encoding these peptides in a unique fish neuroendocrine organ, the caudal neurosecretory system (CNSS). The CRH and UI precursors, isolated from a European flounder CNSS library, consist of 168 and 147 amino acid residues, respectively, with an overall homology of approximately 50%. Both precursors contain a signal peptide, a divergent cryptic region and a 41-amino acid mature peptide with cleavage and amidation sites. Genomic organization showed that whole CRH and UI coding sequences are contained in a single exon. Northern blot analysis and quantitative PCR of a range of tissues confirmed the CNSS as a major site of expression of both CRH and UI and thus serves as a likely source of circulating peptides. In situ hybridization demonstrated that CRH and UI colocalize to the same cells of the CNSS. Our findings suggest that, in euryhaline fish, the CNSS is a major site of production of CRH and probably contributes to the high circulating levels observed in response to specific environmental challenges. Furthermore, the localization of CRH and UI within the same cell population suggests an early, possibly shared role for these peptides in controlling stress-mediated adaptive plasticity.
Some of melatonin's (Mel) well-established physiological effects are mediated via high-affinity cell-membrane receptors belonging to the superfamily of G-protein-coupled receptors. Specific binding ...of ligand 2-¹²⁵Iiodomelatonin, using membrane preparations from osmoregulatory tissues of flounder, rainbow trout and sea bream, together with Mel concentrations in the tissues and plasma were studied. The kidney, gill and small intestine samples were collected during the day and at night. The dissociation constants (K d) and maximal binding densities (B max) were calculated for each tissue at 11:00 and 23:00 h. The binding sites with K d values in the tissues in the picomolar range indicated the high affinity. K d and B max values were tissue- and species-dependent. The GTP analogue Guanosine 5'-O-(3-thiotriphosphate) treatment significantly reduced the B max value, indicating that the 2-¹²⁵Iiodomelatonin-binding sites are probably coupled to a G-protein. No daily variations in K d and B max values were observed. These are the first studies of the presence of 2-¹²⁵Iiodomelatonin-binding sites in the small intestine, kidney tubule and gill of fish. The data strongly suggest new potential targets for Mel action and the influence of Mel on water/ion balance in fish. The intestine seems to be a site of Mel synthesis and/or an active accumulation of the hormone.
Chiral complexes of aluminum containing the salcyen ligand framework, (R,R)-salcyenAlX (X = Me, OSiMe2 tBu), catalyze the asymmetric addition of diorgano-H-phosphonates to carbonyls: the ...phospho-aldol reaction. Reaction proceeds smoothly at ambient temperature in various solvents and under aerobic conditions, to afford α-hydroxyphosphonate esters (MeO)2P(O)CHR(OH), with enantiomeric excesses (ee's) <50%. Although catalyst activity is linked to the nature of X, ee's appear to be only slightly affected. Substitution within the chiral salcyen ligand framework seems to affect ee principally where there is a steric or structural change near the metal center. Although catalysis is tolerant of small quantities of water, excess water leads to attenuation of enantioselectivity through decomposition of catalyst to afford hydrated aluminas which competes through achiral phospho-aldol catalysis. Kinetic analyses reveal a second-order polynomial relationship of the form x/(A0 − x)A0 = k 2 t + k 2 t 2, where A0 is the initial concentration of H-phosphonate and carbonyl and x the degree of reaction. This may suggest that the metal complex must first be converted to another, more active precursor prior to catalytic turnover. Hammett analyses suggest that carbonyl binding to the metal center results in enhanced ee's, while single-crystal analyses on four aluminum complexes support the view that twisting of the ligand framework, as measured by the five-coordinate τ parameter, from a purely meridional geometry may be advantageous to stereoselectivity. Strategies for future developments are discussed in light of the results herein.
A novel angiotensin I (ANG I) has been isolated from incubates of plasma and kidney extracts of the flounder,
Platichthys flesus, using ion-exchange, gel-permeation, and reverse-phase high ...performance liquid chromatography (HPLC). Its sequence was determined as H–Asn–Arg–Val–Tyr–Ile–His–Pro–Phe–Thr–Leu–OH by sequence analysis and mass spectrometry. No vasopressor activity was detected at the elution position of Asp
1 ANG I in ion-exchange HPLC. The sequence was confirmed by identity of the elution position with the synthetic peptide in two different HPLC systems. When compared with ANG I isolated from other teleost fish, flounder ANG I uniquely has an isoleucine at position 5 rather than valine. Injection of angiotensin II (ANG II) into chronically cannulated flounder resulted in a dose-dependent pressor response, native
Asn
1,
Ile
5
ANG II, was found to elicit pressor responses comparable with those seen when teleost
Asn
1,
Val
5
ANG II and human
Asp
1,
Ile
5
ANG II were injected into flounder over the dose range 0.02–1.00
nmol/kg
−1. Plasma concentrations of the neurohypophysial peptide AVT were measured in chronically cannulated flounder following the injection of ANG II to examine the effect of ANG II on circulating AVT concentration. The injection of
Asn
1,
Ile
5
ANG II (1
nmol
kg
−1) or
Asp
1,
Ile
5
ANG II (2.5
nmol
kg
−1) resulted in a significant fall in the circulating levels of AVT suggesting that ANG II either directly or indirectly negatively influences AVT secretion.
E3 ligase mediated targeted protein degradation is an ever more important area in drug discovery. In the case of cereblon two avenues exist; either heterobifunctional PROTACs, or discrete cereblon ...ligands that induce a specific neosubstrate degron association to the complex. Here we present a degron blocking approach, whereby structural and in silico understandings of degron association has directed ligand design to block IMiD‐like binding of degron containing neosubstrates, thus enhancing PROTAC selectivity. More information can be found in the Research Article by H. Bouguenina, J. J. Caldwell et al.
Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the ...potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo‐substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo‐substrates to attenuate and indeed remove this monovalent degradation function in well‐known CRL4CRBN molecular glues degraders, namely CC‐885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9‐A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC‐induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.
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