Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although ...genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.
Three nitric oxide synthase (NOS) isoforms, eNOS, nNOS and iNOS, generate nitric oxide (NO) crucial to the cardiovascular, nervous and host defense systems, respectively. Development of ...isoform-selective NOS inhibitors is of considerable therapeutic importance. Crystal structures of nNOS-selective dipeptide inhibitors in complex with both nNOS and eNOS were solved and the inhibitors were found to adopt a curled conformation in nNOS but an extended conformation in eNOS. We hypothesized that a single-residue difference in the active site, Asp597 (nNOS) versus Asn368 (eNOS), is responsible for the favored binding in nNOS. In the D597N nNOS mutant crystal structure, a bound inhibitor switches to the extended conformation and its inhibition of nNOS decreases >200-fold. Therefore, a single-residue difference is responsible for more than two orders of magnitude selectivity in inhibition of nNOS over eNOS by L-N(omega)-nitroarginine-containing dipeptide inhibitors.
In a continuing effort to unravel the structural basis for isoform-selective inhibition of nitric oxide synthase (NOS) by various inhibitors, we have determined the crystal structures of the nNOS and ...eNOS heme domain bound with two d-nitroarginine-containing dipeptide inhibitors, d-Lys-d-ArgNO 2 -NH2 and d-Phe-d-ArgNO 2 -NH2. These two dipeptide inhibitors exhibit similar binding modes in the two constitutive NOS isozymes, which is consistent with the similar binding affinities for the two isoforms as determined by K i measurements. The d-nitroarginine-containing dipeptide inhibitors are not distinguished by the amino acid difference between nNOS and eNOS (Asp 597 and Asn 368, respectively) which is key in controlling isoform selection for nNOS over eNOS observed for the l-nitroarginine-containing dipeptide inhibitors reported previously Flinspach, M., et al. (2004) Nat. Struct. Mol. Biol. 11, 54−59. The lack of a free α-amino group on the d-nitroarginine moiety makes the dipeptide inhibitor steer away from the amino acid binding pocket near the active site. This allows the inhibitor to extend into the solvent-accessible channel farther away from the active site, which enables the inhibitors to explore new isoform-specific enzyme−inhibitor interactions. This might be the structural basis for why these d-nitroarginine-containing inhibitors are selective for nNOS (or eNOS) over iNOS.
A series of N-alkyl-N‘-hydroxyguanidine compounds have recently been characterized as non-amino acid substrates for all three nitric oxide synthase (NOS) isoforms which mimic NO formation from N ...ω-hydroxy-l-arginine. Crystal structures of the nNOS heme domain complexed with either N-isopropyl-N‘-hydroxyguanidine or N-butyl-N‘-hydroxyguanidine reveal two different binding modes in the substrate binding pocket. The binding mode of the latter is consistent with that observed for the substrate N ω-hydroxy-l-arginine bound in the nNOS active site. However, the former binds to nNOS in an unexpected fashion, thus providing new insights into the mechanism on how the hydroxyguanidine moiety leads to NO formation. Structural features of substrate binding support the view that the OH-substituted guanidine nitrogen, instead of the hydroxyl oxygen, is the source of hydrogen supplied to the active ferric−superoxy species for the second step of the NOS catalytic reaction.
Selective inhibition of nitric oxide synthase (NOS) isoforms has great therapeutic potential in the treatment of certain disease states arising from the pathological overproduction of nitric oxide. ...In this study three structures of each NOS isoform were employed to examine selective regions in the active site using the GRID/CPCA approach. In the GRID calculations, 10 probes covering hydrophobic, steric, and hydrogen-bond-acceptor and -donor interactions were used to calculate the molecular interaction fields (MIFs) in the active site. The side chain flexibility of the residues and the grid spacings were considered at the same time. Consensus principal component analysis (CPCA) was applied to analyze the MIFs differences in the active site between the NOS isoforms. By combining the cutout tool with GRID/CPCA pseudofield differential plots, several selective regions in the active site were identified. The selectivity analysis showed that the most important determinants for NOS inhibitor selectivity are hydrophobic and charge−charge interactions. Twenty-five inhibitors of NOS were then docked into the active site using the program AutoDock3.0. The regions identified as being important for selectivity by this method are in excellent agreement with inhibitor structure−activity relationships. A rational usage of the selective region described in this work should make it possible to develop NOS isoform-selective inhibitors.
A series of l-nitroarginine-based dipeptide inhibitors are highly selective for neuronal nitric oxide synthase (nNOS) over the endothelial isoform (eNOS). Crystal structures of these dipeptides bound ...to both isoforms revealed two different conformations, curled in nNOS and extended in eNOS, corresponding to higher and lower binding affinity to the two isoforms, respectively. In previous studies we found that the primary reason for selectivity is that Asp597 in nNOS, which is Asn368 in eNOS, provides greater electrostatic stabilization in the inhibitor complex. While this is the case for smaller dipeptide inhibitors, electrostatic stabilization may no longer be the sole determinant for isoform selectivity with bulkier dipeptide inhibitors. Another residue farther away from the active site, Met336 in nNOS (Val106 in eNOS), is in contact with bulkier dipeptide inhibitors. Double mutants were made to exchange the D597/M336 pair in nNOS with N368/V106 in eNOS. Here we report crystal structures and inhibition constants for bulkier dipeptide inhibitors bound to nNOS and eNOS that illustrate the important role played by residues near the entry to the active site in isoform selective inhibition.
The OX
orexin receptor (OX
R) is a highly expressed G protein-coupled receptor (GPCR) in the brain that regulates wakefulness and circadian rhythms in humans. Antagonism of OX
R is a proven ...therapeutic strategy for insomnia drugs, and agonism of OX
R is a potentially powerful approach for narcolepsy type 1, which is characterized by the death of orexinergic neurons. Until recently, agonism of OX
R had been considered 'undruggable.' We harness cryo-electron microscopy of OX
R-G protein complexes to determine how the first clinically tested OX
R agonist TAK-925 can activate OX
R in a highly selective manner. Two structures of TAK-925-bound OX
R with either a G
mimetic or G
reveal that TAK-925 binds at the same site occupied by antagonists, yet interacts with the transmembrane helices to trigger activating microswitches. Our structural and mutagenesis data show that TAK-925's selectivity is mediated by subtle differences between OX
and OX
receptor subtypes at the orthosteric pocket. Finally, differences in the polarity of interactions at the G protein binding interfaces help to rationalize OX
R's coupling selectivity for G
signaling. The mechanisms of TAK-925's binding, activation, and selectivity presented herein will aid in understanding the efficacy of small molecule OX
R agonists for narcolepsy and other circadian disorders.
Purpose
Over the course of COVID-19 pandemic, evidence has accumulated that SARS-CoV-2 infections may affect multiple organs and have serious clinical sequelae, but on-site clinical examinations with ...non-hospitalized samples are rare. We, therefore, aimed to systematically assess the long-term health status of samples of hospitalized and non-hospitalized SARS-CoV-2 infected individuals from three regions in Germany.
Methods
The present paper describes the COVIDOM-study within the population-based cohort platform (POP) which has been established under the auspices of the NAPKON infrastructure (German National Pandemic Cohort Network) of the national Network University Medicine (NUM). Comprehensive health assessments among SARS-CoV-2 infected individuals are conducted at least 6 months after the acute infection at the study sites Kiel, Würzburg and Berlin. Potential participants were identified and contacted via the local public health authorities, irrespective of the severity of the initial infection. A harmonized examination protocol has been implemented, consisting of detailed assessments of medical history, physical examinations, and the collection of multiple biosamples (e.g., serum, plasma, saliva, urine) for future analyses. In addition, patient-reported perception of the impact of local pandemic-related measures and infection on quality-of-life are obtained.
Results
As of July 2021, in total 6813 individuals infected in 2020 have been invited into the COVIDOM-study. Of these, about 36% wished to participate and 1295 have already been examined at least once.
Conclusion
NAPKON-POP COVIDOM-study complements other Long COVID studies assessing the long-term consequences of an infection with SARS-CoV-2 by providing detailed health data of population-based samples, including individuals with various degrees of disease severity.
Trial registration
Registered at the German registry for clinical studies (DRKS00023742).
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