Extrahelical Binding Site for a 1H-Imidazo[4,5-c]quinolin-4-amine A3 Adenosine Receptor Positive Allosteric Modulator on Helix 8 and Distal Portions of Transmembrane Domains 1 and 7.

This study describes the localization and computational prediction of a binding site for the A3 adenosine receptor (A3AR) positive allosteric modulator 2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-(3,4-dichlorophenyl)amine (LUF6000). The work reveals an extrahelical lipid-facing binding pocket disparate from the orthosteric binding site that encompasses transmembrane domain (TMD) 1, TMD7, and Helix (H) 8, which was predicted by molecular modeling and validated by mutagenesis. According to the model, the nearly planar 1H-imidazo[4,5-c]quinolinamine ring system lies parallel to the transmembrane segments, inserted into an aromatic cage formed by π-π stacking interactions with the side chains of Y2847.55 in TMD7 and Y2938.54 in H8 and by π-NH bonding between Y2847.55 and the exocyclic amine. The 2-cyclohexyl group is positioned "upward" within a small hydrophobic subpocket created by residues in TMDs 1 and 7, while the 3,4-dichlorophenyl group extends toward the lipid interface. An H-bond between the N-1 amine of the heterocycle and the carbonyl of G291.49 further stabilizes the interaction. Molecular dynamics simulations predicted two metastable intermediates, one resembling a pose determined by molecular docking and a second involving transient interactions with Y2938.54; in simulations, each of these intermediates converges into the final bound state. Structure-activity-relationships for replacement of either of the identified exocyclic or endocyclic amines with heteroatoms lacking H-bond donating ability were consistent with the hypothetical pose. Thus, we characterized an allosteric pocket for 1H-imidazo[4,5-c]quinolin-4-amines that is consistent with data generated by orthogonal methods, which will aid in the rational design of improved A3AR positive allosteric modulators. SIGNIFICANCE STATEMENT: Orthosteric A3AR agonists have advanced in clinical trials for inflammatory conditions, liver diseases, and cancer. Thus, the clinical appeal of selective receptor activation could extend to allosteric enhancers, which would induce site- and time-specific activation in the affected tissue. By identifying the allosteric site for known positive allosteric modulators, structure-based drug discovery modalities can be enabled to enhance the pharmacological properties of the 1H-imidazo[4,5-c]quinolin-4-amine class of A3AR positive allosteric modulators.

Structural Manipulations of Marine Natural Products Inspire a New Library of 3-Amino-1,2,4-Triazine PDK Inhibitors Endowed with Antitumor Activity in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the main aggressive types of cancer, characterized by late prognosis and drug resistance. Among the main factors sustaining PDAC progression, the alteration of cell metabolism has emerged to have a key role in PDAC cell proliferation, invasion, and resistance to standard chemotherapeutic agents. Taking into account all these factors and the urgency in evaluating novel options to treat PDAC, in the present work we reported the synthesis of a new series of indolyl-7-azaindolyl triazine compounds inspired by marine bis-indolyl alkaloids. We first assessed the ability of the new triazine compounds to inhibit the enzymatic activity of pyruvate dehydrogenase kinases (PDKs). The results showed that most of derivatives totally inhibit PDK1 and PDK4. Molecular docking analysis was executed to predict the possible binding mode of these derivatives using ligand-based homology modeling technique. Evaluation of the capability of new triazines to inhibit the cell growth in 2D and 3D KRAS-wild-type (BxPC-3) and KRAS-mutant (PSN-1) PDAC cell line, was carried out. The results showed the capacity of the new derivatives to reduce cell growth with a major selectivity against KRAS-mutant PDAC PSN-1 on both cell models. These data demonstrated that the new triazine derivatives target PDK1 enzymatic activity and exhibit cytotoxic effects on 2D and 3D PDAC cell models, thus encouraging further structure manipulation for analogs development against PDAC.

Lessons Learnt from COVID-19: Computational Strategies for Facing Present and Future Pandemics.

Since its outbreak in December 2019, the COVID-19 pandemic has caused the death of more than 6.5 million people around the world. The high transmissibility of its causative agent, the SARS-CoV-2 virus, coupled with its potentially lethal outcome, provoked a profound global economic and social crisis. The urgency of finding suitable pharmacological tools to tame the pandemic shed light on the ever-increasing importance of computer simulations in rationalizing and speeding up the design of new drugs, further stressing the need for developing quick and reliable methods to identify novel active molecules and characterize their mechanism of action. In the present work, we aim at providing the reader with a general overview of the COVID-19 pandemic, discussing the hallmarks in its management, from the initial attempts at drug repurposing to the commercialization of Paxlovid, the first orally available COVID-19 drug. Furthermore, we analyze and discuss the role of computer-aided drug discovery (CADD) techniques, especially those that fall in the structure-based drug design (SBDD) category, in facing present and future pandemics, by showcasing several successful examples of drug discovery campaigns where commonly used methods such as docking and molecular dynamics have been employed in the rational design of effective therapeutic entities against COVID-19.

Targeting the I7L Protease: A Rational Design for Anti-Monkeypox Drugs?

The latest monkeypox virus outbreak in 2022 showcased the potential threat of this viral zoonosis to public health. The lack of specific treatments against this infection and the success of viral protease inhibitors-based treatments against HIV, Hepatitis C, and SARS-CoV-2, brought the monkeypox virus I7L protease under the spotlight as a potential target for the development of specific and compelling drugs against this emerging disease. In the present work, the structure of the monkeypox virus I7L protease was modeled and thoroughly characterized through a dedicated computational study. Furthermore, structural information gathered in the first part of the study was exploited to virtually screen the DrugBank database, consisting of drugs approved by the Food and Drug Administration (FDA) and clinical-stage drug candidates, in search for readily repurposable compounds with similar binding features as TTP-6171, the only non-covalent I7L protease inhibitor reported in the literature. The virtual screening resulted in the identification of 14 potential inhibitors of the monkeypox I7L protease. Finally, based on data collected within the present work, some considerations on developing allosteric modulators of the I7L protease are reported.

Thermal Titration Molecular Dynamics (TTMD): Not Your Usual Post-Docking Refinement.

Molecular docking is one of the most widely used computational approaches in the field of rational drug design, thanks to its favorable balance between the rapidity of execution and the accuracy of provided results. Although very efficient in exploring the conformational degrees of freedom available to the ligand, docking programs can sometimes suffer from inaccurate scoring and ranking of generated poses. To address this issue, several post-docking filters and refinement protocols have been proposed throughout the years, including pharmacophore models and molecular dynamics simulations. In this work, we present the first application of Thermal Titration Molecular Dynamics (TTMD), a recently developed method for the qualitative estimation of protein-ligand unbinding kinetics, to the refinement of docking results. TTMD evaluates the conservation of the native binding mode throughout a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein-ligand interaction fingerprints. The protocol was successfully applied to retrieve the native-like binding pose among a set of decoy poses of drug-like ligands generated on four different pharmaceutically relevant biological targets, including casein kinase 1δ, casein kinase 2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease.

Discovery of the 3-Amino-1,2,4-triazine-Based Library as Selective PDK1 Inhibitors with Therapeutic Potential in Highly Aggressive Pancreatic Ductal Adenocarcinoma.

Pyruvate dehydrogenase kinases (PDKs) are serine/threonine kinases, that are directly involved in altered cancer cell metabolism, resulting in cancer aggressiveness and resistance. Dichloroacetic acid (DCA) is the first PDK inhibitor that has entered phase II clinical; however, several side effects associated with weak anticancer activity and excessive drug dose (100 mg/kg) have led to its limitation in clinical application. Building upon a molecular hybridization approach, a small library of 3-amino-1,2,4-triazine derivatives has been designed, synthesized, and characterized for their PDK inhibitory activity using in silico, in vitro, and in vivo assays. Biochemical screenings showed that all synthesized compounds are potent and subtype-selective inhibitors of PDK. Accordingly, molecular modeling studies revealed that a lot of ligands can be properly placed inside the ATP-binding site of PDK1. Interestingly, 2D and 3D cell studies revealed their ability to induce cancer cell death at low micromolar doses, being extremely effective against human pancreatic KRAS mutated cancer cells. Cellular mechanistic studies confirm their ability to hamper the PDK/PDH axis, thus leading to metabolic/redox cellular impairment, and to ultimately trigger apoptotic cancer cell death. Remarkably, preliminary in vivo studies performed on a highly aggressive and metastatic Kras-mutant solid tumor model confirm the ability of the most representative compound 5i to target the PDH/PDK axis in vivo and highlighted its equal efficacy and better tolerability profile with respect to those elicited by the reference FDA approved drugs, cisplatin and gemcitabine. Collectively, the data highlights the promising anticancer potential of these novel PDK-targeting derivatives toward obtaining clinical candidates for combatting highly aggressive KRAS-mutant pancreatic ductal adenocarcinomas.

Qualitative Estimation of Protein-Ligand Complex Stability through Thermal Titration Molecular Dynamics Simulations.

The prediction of ligand efficacy has long been linked to thermodynamic properties such as the equilibrium dissociation constant, which considers both the association and the dissociation rates of a defined protein-ligand complex. In the last 15 years, there has been a paradigm shift, with an increased interest in the determination of kinetic properties such as the drug-target residence time since they better correlate with ligand efficacy compared to other parameters. In this article, we present thermal titration molecular dynamics (TTMD), an alternative computational method that combines a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein-ligand interaction fingerprints for the qualitative estimation of protein-ligand-binding stability. The protocol has been applied to four different pharmaceutically relevant test cases, including protein kinase CK1δ, protein kinase CK2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease, on a variety of ligands with different sizes, structures, and experimentally determined affinity values. In all four cases, TTMD was successfully able to distinguish between high-affinity compounds (low nanomolar range) and low-affinity ones (micromolar), proving to be a useful screening tool for the prioritization of compounds in a drug discovery campaign.

From the Wuhan-Hu-1 strain to the XD and XE variants: is targeting the SARS-CoV-2 spike protein still a pharmaceutically relevant option against COVID-19?

Since the outbreak of the COVID-19 pandemic in December 2019, the SARS-CoV-2 genome has undergone several mutations. The emergence of such variants has resulted in multiple pandemic waves, contributing to sustaining to date the number of infections, hospitalisations, and deaths despite the swift development of vaccines, since most of these mutations are concentrated on the Spike protein, a viral surface glycoprotein that is the main target for most vaccines. A milestone in the fight against the COVID-19 pandemic has been represented by the development of Paxlovid, the first orally available drug against COVID-19, which acts on the Main Protease (Mpro). In this article, we analyse the structural features of both the Spike protein and the Mpro of the recently reported SARS-CoV-2 variant XE, as well the closely related XD and XF ones, discussing their impact on the efficacy of existing treatments against COVID-19 and on the development of future ones.

Bat coronaviruses related to SARS-CoV-2: what about their 3CL proteases (MPro)?

Despite a huge effort by the scientific community to determine the animal reservoir of SARS-CoV-2, which led to the identification of several SARS-CoV-2-related viruses both in bats and in pangolins, the origin of SARS-CoV-2 is still not clear. Recently, Temmam et al. reported the discovery of bat coronaviruses with a high degree of genome similarity with SARS-CoV-2, especially concerning the RBDs of the S protein, which mediates the capability of such viruses to enter and therefore infect human cells through a hACE2-dependent pathway. These viruses, especially the one named BANAL-236, showed a higher affinity for the hACE2 compared to the original strain of SARS-CoV-2. In the present work, we analyse the similarities and differences between the 3CL protease (main protease, Mpro) of these newly reported viruses and SARS-CoV-2, discussing their relevance relative to the efficacy of existing therapeutic approaches against COVID-19, particularly concerning the recently approved orally available Paxlovid, and the development of future ones.

The Multifaceted Role of GPCRs in Amyotrophic Lateral Sclerosis: A New Therapeutic Perspective?

Amyotrophic lateral sclerosis (ALS) is a degenerating disease involving the motor neurons, which causes a progressive loss of movement ability, usually leading to death within 2 to 5 years from the diagnosis. Much effort has been put into research for an effective therapy for its eradication, but still, no cure is available. The only two drugs approved for this pathology, Riluzole and Edaravone, are onlyable to slow down the inevitable disease progression. As assessed in the literature, drug targets such as protein kinases have already been extensively examined as potential drug targets for ALS, with some molecules already in clinical trials. Here, we focus on the involvement of another very important and studied class of biological entities, G protein-coupled receptors (GPCRs), in the onset and progression of ALS. This workaimsto give an overview of what has been already discovered on the topic, providing useful information and insights that can be used by scientists all around the world who are putting efforts into the fight against this very important neurodegenerating disease.

Supervised Molecular Dynamics (SuMD) Insights into the mechanism of action of SARS-CoV-2 main protease inhibitor PF-07321332.

The chemical structure of PF-07321332, the first orally available Covid-19 clinical candidate, has recently been revealed by Pfizer. No information has been provided about the interaction pattern between PF-07321332 and its biomolecular counterpart, the SARS-CoV-2 main protease (Mpro). In the present work, we exploited Supervised Molecular Dynamics (SuMD) simulations to elucidate the key features that characterise the interaction between this drug candidate and the protease, emphasising similarities and differences with other structurally related inhibitors such as Boceprevir and PF-07304814. The structural insights provided by SuMD will hopefully be able to inspire the rational discovery of other potent and selective protease inhibitors.

A Computational Workflow for the Identification of Novel Fragments Acting as Inhibitors of the Activity of Protein Kinase CK1δ.

Fragment-Based Drug Discovery (FBDD) has become, in recent years, a consolidated approach in the drug discovery process, leading to several drug candidates under investigation in clinical trials and some approved drugs. Among these successful applications of the FBDD approach, kinases represent a class of targets where this strategy has demonstrated its real potential with the approved kinase inhibitor Vemurafenib. In the Kinase family, protein kinase CK1 isoform δ (CK1δ) has become a promising target in the treatment of different neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In the present work, we set up and applied a computational workflow for the identification of putative fragment binders in large virtual databases. To validate the method, the selected compounds were tested in vitro to assess the CK1δ inhibition.

Emergency department attendance for injury and behaviours suggestive of attention deficit hyperactivity disorder (ADHD): a cross-sectional study.

BACKGROUND: The study aimed to investigate if the behaviours suggestive of ADHD were more frequent in a population of children attending the Emergency Department (ED) for injuries, rather than for other causes. METHODS: A cross-sectional study was carried out. Patients, aged 6 to 17 years, attending the ED for acute injuries and other causes were considered cases and controls, respectively. We used a questionnaire, which investigates the presence in the child of inattention, hyperactivity, and impulsivity. The primary outcome was the number of children with behaviours suggestive of ADHD in cases and controls. RESULTS: Five hundred forty-five children were enrolled, 251 with injuries and 294 with other complains. Twenty two out of two hundred fifty one (9%) children visited for injuries, and 30 out of 294 (10%) visited for other causes had behaviours suggestive of ADHD (p = 0.661). Among these cases, children with evocative ADHD scores had a higher probability (OR 4.52; 95% CI 1.45-14.04; p = 0.009) of having had more than five previous ED accesses due to injury, compared to the others. CONCLUSIONS: This study did non shown a difference in behaviours suggestive of ADHD between cases and controls, but identified a population of children with behaviours suggestive of ADHD who more frequently access the ED for injuries.

Measuring empathy in pediatrics: validation of the Visual CARE measure.

BACKGROUND: Empathy is a key element of "Patient and Family Centered Care", a clinical approach recommended by the American Academy of Pediatrics. However, there is a lack of validated tools to evaluate paediatrician empathy. This study aimed to validate the Visual CARE Measure, a patient rated questionnaire measuring physician empathy, in the setting of a Pediatric Emergency Department (ED). METHODS: The empathy of physicians working in the Pediatric ED of the University Hospital of Udine, Italy, was assessed using an Italian translation of the Visual Care Measure. This test has three versions suited to different age groups: the 5Q questionnaire was administered to children aged 7-11, the 10Q version to those older than 11, and the 10Q-Parent questionnaire to parents of children younger than 7. The internal reliability, homogeneity and construct validity of the 5Q and 10Q/10Q-Parent versions of the Visual Care Measure, were separately assessed. The influence of family background on the rating of physician empathy and satisfaction with the clinical encounter was also evaluated. RESULTS: Seven physicians and 416 children and their parents were included in the study. Internal consistency measured by Cronbach's alpha was 0.95 for the 10Q/10Q-Parent versions and 0.88 for the 5Q version. The item-total correlation was > 0.75 for each item. An exploratory factor analysis showed that all the items load onto the first factor. Physicians' empathy scores correlated with patients' satisfaction for both the 10Q and 10Q-Parent questionnaires (Spearman's rho = 0.7189; p < 0.001) and for the 5Q questionnaire (Spearman's rho = 0.5968; p < 0,001). Trust in the consulting physician was lower among immigrant parents (OR 0.43. 95% CI 0.20-0.93). CONCLUSIONS: The Visual Care Measure is a reliable second-person test of physician empathy in the setting of a Pediatric Emergency Room. More studies are needed to evaluate the reliability of this instrument in other pediatric settings distinct from the Emergency Room and to further evaluate its utility in measuring the impact of communication and empathy training programmes for healthcare professionals working in pediatrics.

Gradient and shim pre-emphasis by inversion of a linear time-invariant system model.

PURPOSE: The goal of this contribution is to enhance the fidelity and switching speed of gradient and shim fields by advancing pre-emphasis toward broadband and full cross-term correction. THEORY AND METHODS: The proposed approach is based on viewing gradient and shim chains as linear, time-invariant (LTI) systems. Pre-emphasis is accomplished by inversion of a broadband digital system model. In the multiple-channel case, it amounts to a matrix of broadband filters that perform concerted self- and cross-term correction. This approach is demonstrated with gradients and shims up to the third order in a 7 Tesla whole-body MR system. RESULTS: Pre-emphasis by LTI model inversion is first verified by studying settling speeds and response behavior without and with the correction. It is then demonstrated for rapid shim updating, achieving substantially enhanced fidelity of field dynamics and shim settling within approximately 1 ms. In single-shot echo-planar imaging (EPI) acquisitions in vivo, this benefit is shown to translate into enhanced geometric fidelity. CONCLUSIONS: The fidelity of gradient and shim dynamics can be greatly enhanced by pre-emphasis based on inverting a general LTI system model. Permitting shim settling on the millisecond scale, broadband multiple-channel pre-emphasis promises to render higher-order shimming fully versatile at the level of MRI sequence design. Magn Reson Med 78:1607-1622, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Fast iterative pre-emphasis calibration method enabling third-order dynamic shim updated fMRI.

PURPOSE: To calibrate a pre-emphasis to sufficiently compensate eddy currents for application of dynamic shim updating to fMRI without extension of scan times. METHODS: Eddy current effects induced into all shim terms up to third-order were characterized by spatiotemporal field monitoring, using a third-order field camera. Pre-emphasis settings were derived from the measurements and iteratively evaluated and refined. The calibrated pre-emphasis was applied to slice-wise dynamic shim updating in combination with a dynamic excitation frequency (F0) determination and a slice-wise B0 optimization routine for in vivo echo planar imaging and resting-state functional MRI. RESULTS: The described method for pre-emphasis calibration led to settling times of remaining eddy current effects below 2 ms, allowing for the application of dynamic shim updating to fMRI without extension of scan times or induction of eddy current related artifacts. A dynamic F0 determination compensates frequency shifts induced by the superposition of different shim fields, and therefore, prevents an image shift within the field of view. Hardware limitations necessitate the reduction of the maximum applicable B0 shim field amplitudes and restrict the shim performance. CONCLUSION: The proposed method enables accurate pre-emphasis calibration, and therefore, the application of dynamic shim updating to fMRI.

Exploring the bandwidth limits of ZTE imaging: Spatial response, out-of-band signals, and noise propagation.

PURPOSE: Zero echo time (ZTE) imaging with single-pulse excitation is a fast, robust, and silent three-dimensional (3D) method for MRI of short T2 tissues. In this technique, algebraic reconstruction serves to fill gaps in the center of k-space due to finite acquisition dead time. The purpose of this study was to investigate the effect of this operation on depiction characteristics, noise behavior, and achievable bandwidth. METHODS: The spatial response function (SRF) and noise covariance resulting from ZTE reconstruction were studied using formal analysis, simulations, and phantom experiments. RESULTS: Three prominent limiting phenomena were identified: SRF behavior within the field of view, heightened sensitivity to out-of-band signal sources, and noise amplification. The related errors all appear as image distortions of low spatial frequency and are strongly attenuated upon the transition from one-dimensional projections to 3D image data. Relying on these observations, ZTE imaging was accomplished with a previously unreached gap size, permitting the depiction of a solid sample with T2 ≈ 25 µs at a bandwidth of 500 kHz. CONCLUSION: The tightest bandwidth limits in ZTE arise from background signal and radiofrequency (RF) switching transients. Significant advances in ZTE performance will be afforded by faster transmit-receive (T/R) switching with negligible transients and RF coils free of background signal.

Field camera measurements of gradient and shim impulse responses using frequency sweeps.

PURPOSE: Applications of dynamic shimming require high field fidelity, and characterizing the shim field dynamics is therefore necessary. Modeling the system as linear and time-invariant, the purpose of this work was to measure the impulse response function with optimal sensitivity. THEORY AND METHODS: Frequency-swept pulses as inputs are analyzed theoretically, showing that the sweep speed is a key factor for the measurement sensitivity. By adjusting the sweep speed it is possible to achieve any prescribed noise profile in the measured system response. Impulse response functions were obtained for the third-order shim system of a 7 Tesla whole-body MR scanner. Measurements of the shim fields were done with a dynamic field camera, yielding also cross-term responses. RESULTS: The measured shim impulse response functions revealed system characteristics such as response bandwidth, eddy currents and specific resonances, possibly of mechanical origin. Field predictions based on the shim characterization were shown to agree well with directly measured fields, also in the cross-terms. CONCLUSION: Frequency sweeps provide a flexible tool for shim or gradient system characterization. This may prove useful for applications involving dynamic shimming by yielding accurate estimates of the shim fields and a basis for setting shim pre-emphasis.