Functional characterization of inactivating ABCC8 variants causing congenital hyperinsulinism.

Congenital hyperinsulinism (CHI; OMIM: 256450) is characterized by persistent insulin secretion despite severe hypoglycemia. The most common causes are variants in the ATP-binding cassette subfamily C member 8(ABCC8) and potassium inwardly-rectifying channel subfamily J member 11(KCNJ11) genes. These encode ATP-sensitive potassium (KATP) channel subunit sulfonylurea receptor 1 (SUR1) and inwardly rectifying potassium channel (Kir6.2) proteins. A 7-day-old male infant presented with frequent hypoglycemic episodes and was clinically diagnosed with CHI, underwent trio-whole-exome sequencing, revealing compound heterozygous ABCC8 variants (c.307C>T, p.His103Tyr; and c.3313_3315del, p.Ile1105del) were identified. In human embryonic kidney 293 (HEK293) and rat insulinoma cells (INS-1) transfected with wild-type and variant plasmids, KATP channels formed by p.His103Tyr were delivered to the plasma membrane, whereas p.Ile1105del or double variants (p.His103Tyr coupled with p.Ile1105del) failed to be transported to the plasma membrane. Compared to wild-type channels, the channels formed by the variants (p.His103Tyr; p.Ile1105del) had elevated basal [Ca2+]i, but did not respond to stimulation by glucose. Our results provide evidence that the two ABCC8 variants may be related to CHI owing to defective trafficking and dysfunction of KATP channels.

Structural Insights into ATP-Sensitive Potassium Channel Mechanics: A Role of Intrinsically Disordered Regions.

Commonly used techniques, such as CryoEM or X-ray, are not able to capture the structural reorganizations of disordered regions of proteins (IDR); therefore, it is difficult to assess their functions in proteins based exclusively on experiments. To fill this gap, we used computational molecular dynamics (MD) simulation methods to capture IDR dynamics and trace biological function-related interactions in the Kir6.2/SUR1 potassium channel. This ATP-sensitive octameric complex, one of the critical elements in the insulin secretion process in human pancreatic ß-cells, has four to five large, disordered fragments. Using unique MD simulations of the full Kir6.2/SUR1 channel complex, we present an in-depth analysis of the dynamics of the disordered regions and discuss the possible functions they could have in this system. Our MD results confirmed the crucial role of the N-terminus of the Kir6.2 fragment and the L0-loop of the SUR1 protein in the transfer of mechanical signals between domains that trigger insulin release. Moreover, we show that the presence of IDRs affects natural ligand binding. Our research takes us one step further toward understanding the action of this vital complex.

The Emerging Structural Pharmacology of ATP-Sensitive Potassium Channels.

ATP-sensitive potassium channels (KATP) are energy sensors that participate in a range of physiologic processes. These channels are also clinically validated drug targets. For decades, KATP inhibitors have been prescribed for diabetes and KATP activators have been used for the treatment of hypoglycemia, hypertension, and hair loss. In this Emerging Concepts article, we highlight our current knowledge about the drug binding modes observed using cryogenic electron microscopy techniques. The inhibitors and activators bind to two distinct sites in the transmembrane domain of the sulfonylurea receptor (SUR) subunit. We also discuss the possible mechanism of how these drugs allosterically modulate the dimerization of SUR nucleotide-binding domains (NBDs) and thus KATP channel activity. SIGNIFICANCE STATEMENT: ATP-sensitive potassium channels (KATP) are fundamental to energy homeostasis, and they participate in many vital physiological processes. KATP channels are important drug targets. Both KATP inhibitors (insulin secretagogues) and KATP activators are broadly used clinically for the treatment of related diseases. Recent cryogenic electron microscopy studies allow us to understand the emerging concept of KATP structural pharmacology.

A high-affinity cocaine binding site associated with the brain acid soluble protein 1.

Cocaine exerts its stimulant effect by inhibiting dopamine (DA) reuptake, leading to increased dopamine signaling. This action is thought to reflect the binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share cocaine's behavioral actions. Further, recent reports show more potent actions of the drug, implying the existence of a high-affinity receptor for cocaine. We now report high-affinity binding of cocaine associated with the brain acid soluble protein 1 (BASP1) with a dissociation constant (Kd) of 7 nM. Knocking down BASP1 in the striatum inhibits [3H]cocaine binding to striatal synaptosomes. Depleting BASP1 in the nucleus accumbens but not the dorsal striatum diminishes locomotor stimulation in mice. Our findings imply that BASP1 is a pharmacologically relevant receptor for cocaine.

Fluorescence Imaging of Neural Activity, Neurochemical Dynamics, and Drug-Specific Receptor Conformation with Genetically Encoded Sensors.

Recent advances in fluorescence imaging permit large-scale recording of neural activity and dynamics of neurochemical release with unprecedented resolution in behaving animals. Calcium imaging with highly optimized genetically encoded indicators provides a mesoscopic view of neural activity from genetically defined populations at cellular and subcellular resolutions. Rigorously improved voltage sensors and microscopy allow for robust spike imaging of populational neurons in various brain regions. In addition, recent protein engineering efforts in the past few years have led to the development of sensors for neurotransmitters and neuromodulators. Here, we discuss the development and applications of these genetically encoded fluorescent indicators in reporting neural activity in response to various behaviors in different biological systems as well as in drug discovery. We also report a simple model to guide sensor selection and optimization.

A rational blueprint for the design of chemically-controlled protein switches.

Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches.

The Effect of DREADD Activation of Leptin Receptor Positive Neurons in the Nucleus of the Solitary Tract on Sleep Disordered Breathing.

Obstructive sleep apnea (OSA) is recurrent obstruction of the upper airway due to the loss of upper airway muscle tone during sleep. OSA is highly prevalent, especially in obesity. There is no pharmacotherapy for OSA. Previous studies have demonstrated the role of leptin, an adipose-tissue-produced hormone, as a potent respiratory stimulant. Leptin signaling via a long functional isoform of leptin receptor, LEPRb, in the nucleus of the solitary tract (NTS), has been implicated in control of breathing. We hypothesized that leptin acts on LEPRb positive neurons in the NTS to increase ventilation and maintain upper airway patency during sleep in obese mice. We expressed designer receptors exclusively activated by designer drugs (DREADD) selectively in the LEPRb positive neurons of the NTS of Leprb-Cre-GFP mice with diet-induced obesity (DIO) and examined the effect of DREADD ligand, J60, on tongue muscle activity and breathing during sleep. J60 was a potent activator of LEPRb positive NTS neurons, but did not stimulate breathing or upper airway muscles during NREM and REM sleep. We conclude that, in DIO mice, the stimulating effects of leptin on breathing during sleep are independent of LEPRb signaling in the NTS.

Receptor-Based Pharmacophore Modeling in the Search for Natural Products for COVID-19 Mpro.

Considering the urgency of the COVID-19 pandemic, we developed a receptor-based pharmacophore model for identifying FDA-approved drugs and hits from natural products. The COVID-19 main protease (Mpro) was selected for the development of the pharmacophore model. The model consisted of a hydrogen bond acceptor, donor, and hydrophobic features. These features demonstrated good corroboration with a previously reported model that was used to validate the present model, showing an RMSD value of 0.32. The virtual screening was carried out using the ZINC database. A set of 208,000 hits was extracted and filtered using the ligand pharmacophore mapping, applying the lead-like properties. Lipinski's filter and the fit value filter were used to minimize hits to the top 2000. Simultaneous docking was carried out for 200 hits for natural drugs belonging to the FDA-approved drug database. The top 28 hits from these experiments, with promising predicted pharmacodynamic and pharmacokinetic properties, are reported here. To optimize these hits as Mpro inhibitors and potential treatment options for COVID-19, bench work investigations are needed.

Protocol for behavioral tests using chemogenetically manipulated mice.

Behavioral analyses using mice chemogenetically manipulated by designer receptors exclusively activated by designer drugs (DREADDs) are powerful tools to elucidate neural functions. Here, we describe the detailed protocols for stereotaxic surgery, adeno-associated virus (AAV)-mediated introduction to Gq-DREADDs in mice, and for behavioral testing and analyses related to anxiety, risk assessment, and burying behaviors. A series of these tests are useful in evaluating animal anxiety and their defensive response patterns to potential threats. For complete details on the use and execution of this protocol, please refer to Horii-Hayashi et al. (2021).

Solid-lipid nanoparticle formulation improves antiseizure action of cryptolepine.

Following the high treatment gap and massive impact of epilepsy on global health particularly in low- and middle-income countries, our study aims to investigate cryptolepine, the major alkaloid of Cryptolepis sanguinolenta as well as its solid-lipid nanoparticle formulation for potential antiseizure activity. Cryptolepine was isolated and a solid-lipid formulation was prepared. Antiseizure activity of Solid-Lipid Nanoparticle formulation of cryptolepine (SLN-CRYP) was investigated using Pentylenetetrazole (PTZ)-induced model of seizure-like behaviors in Zebrafish with 2.5 and 5 mg/kg each of cryptolepine and SLN-CRYP. Drug receptor binding and permeability of the compound across the Blood Brain Barrier (BBB) were also assessed. SLN formulation of cryptolepine increased its permeability to the BBB from 0.32 × 10-6 cm/s to 10.81 × 10-6 cm/s. 2.5 and 5 mg/kg of SLN-CRYP significantly reduced mean seizure score (P = 0.0018; F(6, 63) = 23.52) and significantly increased (P < 0.0001; F(6, 63) = 65.41) latency to onset of seizures. The total distance swam by fish administered with 2.5 and 5 mg/kg of SLN-CRYP was significantly (P < 0.000; F(6, 63) = 161.9) decreased. 5 mg/kg of cryptolepine also significantly decreased swimming distance. Cryptolepine exhibited inhibitory modulation of human voltage-gated calcium channels (Cav1.2), H1-receptor, Peripheral Benzodiazepine Receptor and Sigma 2 receptor with a high Ki values of 6133.38 nM and 2945.0 nM, indicating less potent antagonism on Cav1.2 and Sigma 2 receptors compared to Nifedipine and Haloperidol respectively. This study reveals that the solid-lipid nanoparticle formulation of cryptolepine improves its BBB permeability and hence antiseizure activity.

Estimating drug potency in the competitive target mediated drug disposition (TMDD) system when the endogenous ligand is included.

Predictions for target engagement are often used to guide drug development. In particular, when selecting the recommended phase 2 dose of a drug that is very safe, and where good biomarkers for response may not exist (e.g. in immuno-oncology), a receptor occupancy prediction could even be the main determinant in justifying the approved dose, as was the case for atezolizumab. The underlying assumption in these models is that when the drug binds its target, it disrupts the interaction between the target and its endogenous ligand, thereby disrupting downstream signaling. However, the interaction between the target and its endogenous binding partner is almost never included in the model. In this work, we take a deeper look at the in vivo system where a drug binds to its target and disrupts the target's interaction with an endogenous ligand. We derive two simple steady state inhibition metrics (SSIMs) for the system, which provides intuition for when the competition between drug and endogenous ligand should be taken into account for guiding drug development.

WNK4 kinase: from structure to physiology.

With no lysine kinase-4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that causes familial hyperkalemic hypertension. This disease is mainly driven by increased downstream activation of the Ste20/SPS1-related proline-alanine-rich kinase/oxidative stress responsive kinase-1-NCC pathway, which increases salt reabsorption in the distal convoluted tubule and indirectly impairs renal K+ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

SAMPL7 blind predictions using nonequilibrium alchemical approaches.

In the context of the SAMPL7 challenge, we computed, employing a non-equilibrium (NE) alchemical technique, the standard binding free energy of two series of host-guest systems, involving as a host the Isaac's TrimerTrip, a Cucurbituril-like open cavitand, and the Gilson's Cyclodextrin derivatives. The adopted NE alchemy combines enhanced sampling molecular dynamics simulations with driven fast out-of-equilibrium alchemical trajectories to recover the free energy via the Jarzynski and Crooks NE theorems. The GAFF2 non-polarizable force field was used for the parametrization. Performances were acceptable and similar in accuracy to those we submitted for Gibb's Deep Cavity Cavitands in the previous SAMPL6 host-guest challenge, confirming the reliability of the computational approach and exposing, in some cases, some important deficiencies of the GAFF2 non-polarizable force field.

Cocaine vaccine dAd5GNE protects against moderate daily and high-dose "binge" cocaine use.

The cocaine vaccine dAd5GNE is comprised of a disrupted serotype 5 adenovirus gene therapy vector covalently conjugated to the cocaine analog GNE. The vaccine evokes a high titer of circulating anti-cocaine antibodies that prevent cocaine from reaching its cognate receptors in the central nervous system. Prior studies have demonstrated the efficacy of dAd5GNE in models of occasional, moderate cocaine use. However, previous studies have not sufficiently evaluated the efficacy of dAd5GNE in models of the repetitive and high-dose "binge" use patterns common in human addicts. In the present study, we evaluated the capacity of dAd5GNE vaccination to protect against "binge" cocaine use and circumstances where vaccinated addicts attempt to override the vaccine. We modeled repetitive daily cocaine use in vaccinated Balb/c mice and African green monkeys, and evaluated high-dose "binge" scenarios in Balb/c mice. In each model of daily use the dAd5GNE vaccine prevented cocaine from reaching the central nervous system. In the high-dose "binge" model, vaccination decreased cocaine-induced hyperactivity and reduced the number of cocaine-induced seizures. Based on this data and our prior data in rodents and nonhuman primates, we have initiated a clinical trial evaluating the dAd5GNE anti-cocaine vaccine as a potential therapy for cocaine addicts who wish to stop cocaine use. If dAd5GNE vaccination is safe and produces high anti-cocaine antibody titers in the clinic, we hypothesize that the vaccine will restrict the access of cocaine to the central nervous system and inhibit cocaine-induced "highs" even in the context of moderate daily and high-dose "binge" use that might otherwise cause a drug-induced overdose.

A single unified model for fitting simple to complex receptor response data.

The fitting of complex receptor-response data where fractional response and occupancy do not match is challenging. They encompass important cases including (a) the presence of "receptor reserve" and/or partial agonism, (b) multiple responses assessed at different vantage points along a pathway, (c) responses that are different along diverging downstream pathways (biased agonism), and (d) constitutive activity. For these, simple models such as the well-known Clark or Hill equations cannot be used. Those that can, such as the operational (Black&Leff) model, do not provide a unified approach, have multiple nonintuitive parameters that are challenging to fit in well-defined manner, have difficulties incorporating binding data, and cannot be reduced or connected to simpler forms. We have recently introduced a quantitative receptor model (SABRE) that includes parameters for Signal Amplification (γ), Binding affinity (Kd), Receptor activation Efficacy (ε), and constitutive activity (εR0). It provides a single equation to fit complex cases within a full two-state framework with the possibility of incorporating receptor occupancy data (i.e., experimental Kds). Simpler cases can be fit by using consecutively reduced forms obtained by constraining parameters to specific values, e.g., εR0 = 0: no constitutive activity, γ = 1: no amplification (Emax-type fitting), and ε = 1: no partial agonism (Clark equation). Here, a Hill-type extension is introduced (n ≠ 1), and simulated and experimental receptor-response data from simple to increasingly complex cases are fitted within the unified framework of SABRE with differently constrained parameters.

PGF2α stimulates the 10-pS Cl- channel and thiazide-sensitive Na+-Cl- cotransporter in the distal convoluted tubule.

We used patch-clamp and Western blot analysis to test whether PGF2α stimulates the basolateral 10-pS Cl- channel and thiazide-sensitive Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) via a prostaglandin F receptor (FP-R). Single channel and whole cell recordings demonstrated that PGF2α stimulated the 10-pS Cl- channel in the DCT. The stimulatory effect of PGF2α on the Cl- channel was mimicked by a FP-R agonist, latanoprost, but was abrogated by blocking FP-R with AL8810. Also, the effect of PGF2α on the Cl- channel in the DCT was recapitulated by stimulating PKC but was blocked by inhibiting PKC. Furthermore, inhibition of p38 MAPK but not ERK blocked the effect of PGF2α on the 10-pS Cl- channel. Inhibition of NADPH oxidase also abrogated the stimulatory effect of PGF2α on the 10-pS Cl- channel, while the addition of 10 µM H2O2 mimicked the stimulatory effect of PGF2α on the 10-pS Cl- channel. Moreover, superoxide-related species may mediate the stimulatory effect of PGF2α on the 10-pS Cl- channel because the stimulatory effect of PGF2α and H2O2 was not additive. Western blot analysis showed that infusion of PGF2α in vivo not only increased the expression of FP-R but also increased the expression of total NCC and phosphorylated NCC. We conclude that PGF2α stimulates the basolateral 10-pS Cl- channel in the DCT by activating FP-R through PKC/p38 MAPK and NADPH oxidase-dependent pathways. The stimulatory effects of PGF2α on the Cl- channel and NCC may contribute to PGF2α-induced increases in NaCl reabsorption in the DCT.

Does Pharmacodynamics of Drugs Change After Presenting them as Nanoparticles Like their Pharmacokinetics?

Nowadays, the breakthrough in different medical branches makes it feasible to designate new methods of drug delivery to achieve the most cost-effective and the least unpleasant consequenceimposing solutions to overcome a wide range of diseases. Nanoparticle (NP) drugs entered the therapeutic system, especially in cancer chemotherapy. These drugs are quite well-known for two traits of being long-acting and less toxic. For a long time, it has been investigated how NPs will change the kinetics of drugs. However, there are a few studies that inclined their attention to how NPs affect the dynamics of drugs. In this review, the latter point will mainly be discussed in an example-based manner. Besides, other particular features of NPs will be briefly noted. NPs are capable of affecting the biologic system as much as a drug. Moreover, NPs could arise a wide variety of effects by triggering their own receptors. NPs are able to change a receptor function and manipulate its downstream signaling cascade.

Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development.

Electron cryo-microscopy (cryo-EM) has revolutionized structure determination of membrane proteins and holds great potential for structure-based drug discovery. Here we discuss the potential of cryo-EM in the rational design of therapeutics for membrane proteins compared to X-ray crystallography. We also detail recent progress in the field of drug receptors, focusing on cryo-EM of two protein families with established therapeutic value, the γ-aminobutyric acid A receptors (GABAARs) and G protein-coupled receptors (GPCRs). GABAARs are pentameric ion channels, and cryo-EM structures of physiological heteromeric receptors in a lipid environment have uncovered the molecular basis of receptor modulation by drugs such as diazepam. The structures of ten GPCR-G protein complexes from three different classes of GPCRs have now been determined by cryo-EM. These structures give detailed insights into molecular interactions with drugs, GPCR-G protein selectivity, how accessory membrane proteins alter receptor-ligand pharmacology, and the mechanism by which HIV uses GPCRs to enter host cells.

Binding energies of the drugs capreomycin and streptomycin in complex with tuberculosis bacterial ribosome subunits.

Despite advances, tuberculosis remains a significant infectious disease, whose mortality presents alarming numbers. Although it can be cured, the number of cases of antimicrobial resistant strains is increasing, requiring the use of less efficient second-line drugs. Capreomycin and streptomycin are part of this group, being antibiotics whose mechanism of action is the inhibition of protein synthesis when interacting with the tuberculosis bacterial ribosome. Their binding mechanisms are distinct: capreomycin is able to bind to both ribosomal (30S and 50S) subunits, whereas streptomycin binds only to the smaller one (30S). In this context, the biochemical characterization of these binding sites for a proper understanding of their complex interactions is of crucial importance to increase their efficacy. Through crystallographic data and computer simulations, in this work we calculated the interaction binding energies of capreomycin and streptomycin in complex with the tuberculosis bacterial ribosome subunits, by using density functional theory (DFT) within the molecular fractionation with conjugated caps (MFCC) approach. For capreomycin in the 30S (50S) subunit, we investigated the binding energies of 44 (30) residues presented within a pocket radius of 14 Å (30 Å). Regarding streptomycin, 60 nucleotide (25 amino acid) residues distributed up to 12.5 Å (15 Å) away from the drug in the 30S subunit (S12 protein) were taken into account. We also identify the contributions of hydrogen bonds and hydrophobic interactions in the drug-receptor complex, and the regions of the drugs that most contributed to the anchorages of them in their binding sites, as well as identify residues that are most associated with mutations.

Determination of Real Time in Vivo Drug Receptor Occupancy for a Covalent Binding Drug as a Clinical Pharmacodynamic Biomarker by Immunocapture-LC-MS/MS.

We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug. The real time RO was calculated based on the concentrations of DB-R and the free receptor (which is now QB-R) that were obtained from each sample. This strategy has been successfully applied to the measurement of the RO for Bruton's tyrosine kinase (BTK) in the blood lysate of monkeys after dosing with branebrutinib (BMS-986195), a covalent BTK inhibitor being evaluated to treat rheumatoid arthritis. A custom-made quencher, which is more reactive to BTK than branebrutinib, was added in excess amount to bind with all available free BTK to form quencher-bound BTK (QB-BTK) during blood sample collection. To measure a wide range of % BTK RO, including those of <5% or >95%, the required LLOQ at 0.125 nM for QB-BTK and 0.250 nM for drug-bound BTK (DB-BTK) in blood lysate were successfully achieved by using this IC-LC-MS/MS strategy. This proof-of-concept assay demonstrated its suitability with high throughput for real time in vivo BTK RO measurement as a pharmacodynamic (PD) biomarker for clinical drug development.