Identifying Potential Ligand Binding Sites on Glycogen Synthase Kinase 3 Using Atomistic Cosolvent Simulations.

Glycogen synthase kinase 3 ß (GSK3ß) is a serine/threonine kinase that phosphorylates several protein substrates in crucial cell signaling pathways. Owing to its therapeutic importance, there is a need to develop GSK3ß inhibitors with high specificity and potency. One approach is to find small molecules that can allosterically bind to the GSK3ß protein surface. We have employed fully atomistic mixed-solvent molecular dynamics (MixMD) simulations to identify three plausible allosteric sites on GSK3ß that can facilitate the search for allosteric inhibitors. Our MixMD simulations narrow down the allosteric sites to precise regions on the GSK3ß surface, thereby improving upon the previous predictions of the locations of these sites.

Mechanistic insights into MARK4 inhibition by galantamine toward therapeutic targeting of Alzheimer's disease.

Introduction: Hyperphosphorylation of tau is an important event in Alzheimer's disease (AD) pathogenesis, leading to the generation of "neurofibrillary tangles," a histopathological hallmark associated with the onset of AD and related tauopathies. Microtubule-affinity regulating kinase 4 (MARK4) is an evolutionarily conserved Ser-Thr (S/T) kinase that phosphorylates tau and microtubule-associated proteins, thus playing a critical role in AD pathology. The uncontrolled neuronal migration is attributed to overexpressed MARK4, leading to disruption in microtubule dynamics. Inhibiting MARK4 is an attractive strategy in AD therapeutics. Methods: Molecular docking was performed to see the interactions between MARK4 and galantamine (GLT). Furthermore, 250 ns molecular dynamic studies were performed to investigate the stability and conformational dynamics of the MARK4-GLT complex. We performed fluorescence binding and isothermal titration calorimetry studies to measure the binding affinity between GLT and MARK4. Finally, an enzyme inhibition assay was performed to measure the MARK4 activity in the presence and absence of GLT. Results: We showed that GLT, an acetylcholinesterase inhibitor, binds to the active site cavity of MARK4 with an appreciable binding affinity. Molecular dynamic simulation for 250 ns demonstrated the stability and conformational dynamics of the MARK4-GLT complex. Fluorescence binding and isothermal titration calorimetry studies suggested a strong binding affinity. We further show that GLT inhibits the kinase activity of MARK4 significantly (IC50 = 5.87 µM). Conclusion: These results suggest that GLT is a potential inhibitor of MARK4 and could be a promising therapeutic target for AD. GLT's inhibition of MARK4 provides newer insights into the mechanism of GLT's action, which is already used to improve cognition in AD patients.

Inhibition of microtubule affinity regulating kinase 4 by an acetylcholinesterase inhibitor, Huperzine A: Computational and experimental approaches.

Microtubule affinity regulating kinase 4 (MARK4), 752 amino acids long, belonging to the AMPK superfamily, plays a vital role in regulating microtubules due to its potential to phosphorylate microtubule-associated proteins (MAP's) and thus, MARK4 plays a key role in Alzheimer's disease (AD) pathology. MARK4 is a druggable target for cancer, neurodegenerative diseases, and metabolic disorders. In this study, we have evaluated the MARK4 inhibitory potential of Huperzine A (HpA), an acetylcholinesterase inhibitor (AChEI), a potential AD drug. Molecular docking revealed the key residues governing the MARK4-HpA complex formation. The structural stability and conformational dynamics of the MARK4-HpA complex was assessed by employing Molecular dynamics (MD) simulation. The results suggested that the binding of HpA with MARK4 leads to minimal structural alterations in the native conformation of MARK4, implying the stability of the MARK4-HpA complex. Isothermal titration calorimetry (ITC) studies deciphered that HpA binds to MARK4 spontaneously. Moreover, the kinase assay depicted significant inhibition of MARK by HpA (IC50 = 4.91 µM), implying it to be a potent MARK4 inhibitor that can be implicated in the treatment of MARK4-directed diseases.

Sunsetting Binding MOAD with its last data update and the addition of 3D-ligand polypharmacology tools.

Binding MOAD is a database of protein-ligand complexes and their affinities with many structured relationships across the dataset. The project has been in development for over 20 years, but now, the time has come to bring it to a close. Currently, the database contains 41,409 structures with affinity coverage for 15,223 (37%) complexes. The website BindingMOAD.org provides numerous tools for polypharmacology exploration. Current relationships include links for structures with sequence similarity, 2D ligand similarity, and binding-site similarity. In this last update, we have added 3D ligand similarity using ROCS to identify ligands which may not necessarily be similar in two dimensions but can occupy the same three-dimensional space. For the 20,387 different ligands present in the database, a total of 1,320,511 3D-shape matches between the ligands were added. Examples of the utility of 3D-shape matching in polypharmacology are presented. Finally, plans for future access to the project data are outlined.

Correction: Ashraf et al. Inhibition of Microtubule Affinity Regulating Kinase 4 by Metformin: Exploring the Neuroprotective Potential of Antidiabetic Drug through Spectroscopic and Computational Approaches. Molecules 2022, 27, 4652.

The updated affiliation information can be seen in the affiliation part of this correction [...].

Targeting inhibition of microtubule affinity regulating kinase 4 by Harmaline: Strategy to combat Alzheimer's disease.

Microtubule-affinity regulating kinase 4 (MARK4) is linked with the development of cancer, diabetes and neurodegenerative diseases. Due to its direct role in the hyperphosphorylation of tau protein, MARK4 is considered as an attractive target to fight Alzheimer's disease (AD) and neuroinflammation. In the present study, we have selected Harmaline (HAR), an alkaloid of Paganum harmala, to investigate its MARK4 inhibitory potential and its binding mechanism. Molecular docking and fluorescence binding studies were carried out to estimate the binding affinity of the HAR with the MARK4. We observed an excellent binding affinity of HAR to the MARK4 (K = 107 M-1), further complemented by isothermal titration calorimetric measurements. In addition, HAR significantly inhibits the kinase activity of MARK4 (IC50 value of 4.46 µM). Structural investigations suggested that HAR binds to the active site pocket and forms several non-covalent interactions with biologically important residues of MARK4. All-atom molecular dynamics simulation studies further advocated that the MARK4-HAR complex is stabilized throughout the trajectory of 200 ns and causes a little conformational change. All these findings suggest that HAR is a potential MARK4 inhibitor that can be implicated in managing MARK4-associated diseases, including AD.

Role of lisinopril in the therapeutic management of cardiovascular disease by targeting microtubule affinity regulating kinase 4: molecular docking and molecular dynamics simulation approaches.

Cardiovascular diseases (CVDs) are a major cause of premature adult death. Various factors contribute to the development of CVDs, such as atherosclerosis leading to myocardial infarction (MI), and compromised cardiac function after MI leads to chronic heart failure with systemic health complications and a high mortality rate. Microtubule detyrosination has rapidly evolved as an essential mechanism to regulate cardiomyocyte contractility. Microtubule affinity regulating kinase 4 (MARK4) regulates cardiomyocyte contractility in a way that it promotes phosphorylation of microtubule-associated protein 4, thereby facilitating the access of vasohibin 2-a tubulin carboxypeptidase-to microtubules for the detyrosination of α-tubulin. Lisinopril, a drug belonging to the class of angiotensin-converting enzyme inhibitors, is used to treat high blood pressure. This is also used to treat heart failure, which plays a vital role in improving the survival rate post-heart attack. In this study, we will evaluate the MARK4 inhibitory potential of lisinopril employing molecular docking and molecular dynamics (MD) simulation approaches. Molecular docking analysis suggested that lisinopril binds to MARK4 with a significant binding affinity forming interactions with functionally essential residues of MARK4. Additionally, MD simulation deciphered the structural dynamics and stability of the MARK4-lisinopril complex. The findings of MD studies established that minimal structural deviations are observed during simulation, affirming the stability of the MARK4-lisinopril complex. Altogether, this study demonstrates lisinopril's crucial role in the therapeutic management of CVD by targeting MARK4.Communicated by Ramaswamy H. Sarma.

Inhibition of Microtubule Affinity Regulating Kinase 4 by Metformin: Exploring the Neuroprotective Potential of Antidiabetic Drug through Spectroscopic and Computational Approaches.

Microtubule affinity regulating kinase 4 (MARK4) regulates the mechanism of microtubules by its ability to phosphorylate the microtubule-associated proteins (MAP's). MARK4 is known for its major role in tau phosphorylation via phosphorylating Ser262 residue in the KXGS motif, which results in the detachment of tau from microtubule. In lieu of this vital role in tau pathology, a hallmark of Alzheimer's disease (AD), MARK4 is a druggable target to treat AD and other neurodegenerative disorders (NDs). There is growing evidence that NDs and diabetes are connected with many pieces of literature demonstrating a high risk of developing AD in diabetic patients. Metformin (Mtf) has been a drug in use against type 2 diabetes mellitus (T2DM) for a long time; however, recent studies have established its therapeutic effect in neurodegenerative diseases (NDs), namely AD, Parkinson's disease (PD) and amnestic mild cognitive impairment. In this study, we have explored the MARK4 inhibitory potential of Mtf, employing in silico and in vitro approaches. Molecular docking demonstrated that Mtf binds to MARK4 with a significant affinity of -6.9 kcal/mol forming interactions with binding pocket's critical residues. Additionally, molecular dynamics (MD) simulation provided an atomistic insight into the binding of Mtf with MARK4. ATPase assay of MARK4 in the presence of Mtf shows that it inhibits MARK4 with an IC50 = 7.05 µM. The results of the fluorescence binding assay demonstrated significant binding of MARK4 with a binding constant of 0.6 × 106 M-1. The present study provides an additional axis towards the utilization of Mtf as MARK4 inhibitor targeting diabetes with NDs.

Inhibition of MARK4 by serotonin as an attractive therapeutic approach to combat Alzheimer's disease and neuroinflammation.

Mitogen-activated protein kinases (MAPKs) govern various cellular programs and crucial intermediate pathways in signaling. Microtubule affinity-regulating kinase 4 (MARK4) is a part of the kinase family recognized for actively phosphorylating neural microtubule-associated proteins (MAPs) like MAP2, MAP4 and most importantly, tau. The Ser/Thr kinase MARK4 overexpression is associated with various life-threatening conditions such as neurodegenerative disorders, diabetic neuropathy, and cancer. Functionally, MARK4 is correlated with many important signaling cascades and transcription factors contributing to neurodegeneration and cancer onset and progression. Serotonin is a key molecule associated with regulating mood, stress, and various behavioral aspects. Low serotonin levels promote the progression of neurological and psychotic disorders, which is also a consequence of tau accumulation. MARK4 being a major contributor to phosphorylating tau, leading to its accumulation, and contributing to tauopathy, is targeted for inhibition by serotonin. The study deals with the inhibition of MARK4 by serotonin using combined computational and experimental studies. The results presented in this paper provide strong evidence for the direct physical binding of serotonin to recombinant MARK4 and subsequent inhibition of its kinase activity. In addition, we have performed molecular docking, followed by 100 ns MD simulations of MARK4 in the presence of serotonin, to estimate the stability of the protein-ligand complex. Since MARK4 is a potential drug target and can be exploited for drug design and discovery for cancer and neurodegenerative disorders, the results presented here are of interest and may be further exploited for Alzheimer's disease (AD) and other neurodegenerative diseases.

Investigating binding mechanism of thymoquinone to human transferrin, targeting Alzheimer's disease therapy.

Iron deposition in the central nervous system (CNS) is one of the causes of neurodegenerative diseases. Human transferrin (hTf) acts as an iron carrier present in the blood plasma, preventing it from contributing to redox reactions. Plant compounds and their derivatives are frequently being used in preventing or delaying Alzheimer's disease (AD). Thymoquinone (TQ), a natural product has gained popularity because of its broad therapeutic applications. TQ is one of the significant phytoconstituent of Nigella sativa. The binding of TQ to hTf was determined by spectroscopic methods and isothermal titration calorimetry. We have observed that TQ strongly binds to hTf with a binding constant (K) of 0.22 × 106 M-1 and forming a stable complex. In addition, isothermal titration calorimetry revealed the spontaneous binding of TQ with hTf. Molecular docking analysis showed key residues of the hTf that were involved in the binding to TQ. We further performed a 250 ns molecular dynamics simulation which deciphered the dynamics and stability of the hTf-TQ complex. Structure analysis suggested that the binding of TQ doesn't cause any significant alterations in the hTf structure during the course of simulation and a stable complex is formed. Altogether, we have elucidated the mechanism of binding of TQ with hTf, which can be further implicated in the development of a novel strategy for AD therapy.

Computational Identification of Possible Allosteric Sites and Modulators of the SARS-CoV-2 Main Protease.

In this study, we target the main protease (Mpro) of the SARS-CoV-2 virus as it is a crucial enzyme for viral replication. Herein, we report three plausible allosteric sites on Mpro that can expand structure-based drug discovery efforts for new Mpro inhibitors. To find these sites, we used mixed-solvent molecular dynamics (MixMD) simulations, an efficient computational protocol that finds binding hotspots through mapping the surface of unbound proteins with 5% cosolvents in water. We have used normal mode analysis to support our claim of allosteric control for these sites. Further, we have performed virtual screening against the sites with 361 hits from Mpro screenings available through the National Center for Advancing Translational Sciences (NCATS). We have identified the NCATS inhibitors that bind to the remote sites better than the active site of Mpro, and we propose these molecules may be allosteric regulators of the system. After identifying our sites, new X-ray crystal structures were released that show fragment molecules in the sites we found, supporting the notion that these sites are accurate and druggable.

Fungal Endophthalmitis: Analysis of 730 Consecutive Eyes from 7 Tertiary Eye Care Centers in India.

PURPOSE: To evaluate the clinical and microbiological features of a large cohort with culture-confirmed fungal endophthalmitis across India. DESIGN: Cross-sectional, hospital-based, retrospective medical record review. PARTICIPANTS: Seven large tertiary eye care centers from different regions of India. METHODS: Patient data were pooled from electronic or physical medical records of each participating center. Fellowship-trained vitreoretinal specialists clinically managed all patients, and in-house microbiology laboratories performed all microbiological workups. The clinical and microbiological procedures were broadly uniform across all participating centers. The essential treatment consisted of vitreous surgery as well as intravitreal and systemic therapies with antifungal agents. MAIN OUTCOME MEASURES: Clinical outcome of the causative event and causative fungus. RESULTS: In the period from 2005 to 2020, 7 centers treated 3830 cases of culture-proven endophthalmitis, and of these, 19.1% (n = 730) were cases of culture-confirmed fungal endophthalmitis. It included 46.9% cases of postoperative (87.4% postcataract surgery), 35.6% of traumatic, and 17.5% of endogenous endophthalmitis. The fungi included 39.0% of Aspergillus (high prevalence in central, east, and south zones), 15.1% of Candida (high prevalence in west zone), and 15.9% of Fusarium (high prevalence in north and west zones). The time to symptom development was between 1 week and 4 weeks in more than one third of the patients, except in patients with traumatic endophthalmitis. Less than half of the patients had hypopyon on presentation. The presenting visual acuity (PVA) in most patients was <20/400. Nearly all patients needed vitrectomy and an average of 2 intravitreal injections of antifungal agents. At least 10% of eyes needed therapeutic keratoplasty, and up to 7% of eyes were eviscerated. After treatment, the final (best corrected) visual acuity (FVA) was >20/400 in 30.5% (n = 222) of eyes and >20/40 in 7.9% (n = 58) of eyes, and 12% (n = 88) of eyes lost light perception. A post hoc analysis showed the male sex to be significantly more associated with traumatic endophthalmitis than with postoperative (P < 0.0001) and endogenous (P = 0.001) endophthalmitis, more isolation of Candida species in patients with endogenous endophthalmitis than in those with postoperative (P = 0.004) and traumatic (P < 0.0001) endophthalmitis, better PVA in eyes with Candida species infection (P < 0.0001), and poorer FVA in eyes with Aspergillus species infection. CONCLUSIONS: Fungal endophthalmitis is not uncommon in India. The inclusion of antifungal agents with antibiotics as the first empirical intravitreal therapy before microbiological confirmation should be considered when a fungal infection is suspected.

Using artificial intelligence for diabetic retinopathy screening: Policy implications.

Artificial intelligence (AI) has evolved over the last few years; its use in DR screening has been demonstrated in multiple evidences across the globe. However, there are concerns right from the data acquisition, bias in data, difficulty in comparing between different algorithm, challenges in machine learning, its application in different group of population, and human barrier to AI adoption in health care. There are also legal and ethical concerns related to AI. The tension between risks and concerns on one hand versus potential and opportunity on the other have driven a need for authorities to implement policies for AI in DR screening to address these issues. The policy makers should support and facilitate research and development of AI in healthcare, but at the same time, it has to be ensured that the use of AI in healthcare aligns with recognized standards of safety, efficacy, and equity. It is essential to ensure that algorithms, datasets, and decisions are auditable and when applied to medical care (such as screening, diagnosis, or treatment) are clinically validated and explainable. Policy frameworks should require design of AI systems in health care that are informed by real-world workflow and human-centric design. Lastly, it should be ensured that healthcare AI solutions align with all relevant ethical obligations, from design to development to use and to be delivered properly in the real world.

Mixed-solvent molecular dynamics simulation-based discovery of a putative allosteric site on regulator of G protein signaling 4.

Regulator of G protein signaling 4 (RGS4) is an intracellular protein that binds to the Gα subunit ofheterotrimeric G proteins and aids in terminating G protein coupled receptor signaling. RGS4 has been implicated in pain, schizophrenia, and the control of cardiac contractility. Inhibitors of RGS4 have been developed but bind covalently to cysteine residues on the protein. Therefore, we sought to identify alternative druggable sites on RGS4 using mixed-solvent molecular dynamics simulations, which employ low concentrations of organic probes to identify druggable hotspots on the protein. Pseudo-ligands were placed in consensus hotspots, and perturbation with normal mode analysis led to the identification and characterization of a putative allosteric site, which would be invaluable for structure-based drug design of non-covalent, small molecule inhibitors. Future studies on the mechanism of this allostery will aid in the development of novel therapeutics targeting RGS4.

Free Energies and Entropies of Binding Sites Identified by MixMD Cosolvent Simulations.

In our recent efforts to map protein surfaces using mixed-solvent molecular dynamics (MixMD) (Ghanakota, P.; Carlson, H. A. Moving Beyond Active-Site Detection: MixMD Applied to Allosteric Systems. J. Phys. Chem. B 2016, 120, 8685-8695), we were able to successfully capture active sites and allosteric sites within the top-four most occupied hotspots. In this study, we describe our approach for estimating the thermodynamic profile of the binding sites identified by MixMD. First, we establish a framework for calculating free energies from MixMD simulations, and we compare our approach to alternative methods. Second, we present a means to obtain a relative ranking of the binding sites by their configurational entropy. The theoretical maximum and minimum free energy and entropy values achievable under such a framework along with the limitations of the techniques are discussed. Using this approach, the free energy and relative entropy ranking of the top-four MixMD binding sites were computed and analyzed across our allosteric protein targets: Abl Kinase, Androgen Receptor, Pdk1 Kinase, Farnesyl Pyrophosphate Synthase, Chk1 Kinase, Glucokinase, and Protein Tyrosine Phosphatase 1B.

A component analysis of the free energies of folding of 35 proteins: A consensus view on the thermodynamics of folding at the molecular level.

What factors favor protein folding? This is a textbook question. Parsing the experimental free energies of folding/unfolding into diverse enthalpic and entropic components of solute and solvent favoring or disfavoring folding is not an easy task. In this study, we present a computational protocol for estimating the free energy contributors to protein folding semi-quantitatively using ensembles of unfolded and native states generated via molecular dynamics simulations. We tested the methodology on 35 proteins with diverse structural motifs and sizes and found that the calculated free energies correlate well with experiment (correlation coefficient ∼ 0.85), enabling us to develop a consensus view of the energetics of folding. As a more sensitive test of the methodology, we also investigated the free energies of folding of an additional 33 single point mutants and obtained a correlation coefficient of 0.8. A notable observation is that the folding free energy components appear to carry signatures of the fold (SCOP classification) of the protein. © 2017 Wiley Periodicals, Inc.

From Ramachandran Maps to Tertiary Structures of Proteins.

Sequence to structure of proteins is an unsolved problem. A possible coarse grained resolution to this entails specification of all the torsional (Φ, Ψ) angles along the backbone of the polypeptide chain. The Ramachandran map quite elegantly depicts the allowed conformational (Φ, Ψ) space of proteins which is still very large for the purposes of accurate structure generation. We have divided the allowed (Φ, Ψ) space in Ramachandran maps into 27 distinct conformations sufficient to regenerate a structure to within 5 Å from the native, at least for small proteins, thus reducing the structure prediction problem to a specification of an alphanumeric string, i.e., the amino acid sequence together with one of the 27 conformations preferred by each amino acid residue. This still theoretically results in 27(n) conformations for a protein comprising "n" amino acids. We then investigated the spatial correlations at the two-residue (dipeptide) and three-residue (tripeptide) levels in what may be described as higher order Ramachandran maps, with the premise that the allowed conformational space starts to shrink as we introduce neighborhood effects. We found, for instance, for a tripeptide which potentially can exist in any of the 27(3) "allowed" conformations, three-fourths of these conformations are redundant to the 95% confidence level, suggesting sequence context dependent preferred conformations. We then created a look-up table of preferred conformations at the tripeptide level and correlated them with energetically favorable conformations. We found in particular that Boltzmann probabilities calculated from van der Waals energies for each conformation of tripeptides correlate well with the observed populations in the structural database (the average correlation coefficient is ∼0.8). An alpha-numeric string and hence the tertiary structure can be generated for any sequence from the look-up table within minutes on a single processor and to a higher level of accuracy if secondary structure can be specified. We tested the methodology on 100 small proteins, and in 90% of the cases, a structure within 5 Å is recovered. We thus believe that the method presented here provides the missing link between Ramachandran maps and tertiary structures of proteins. A Web server to convert a tertiary structure to an alphanumeric string and to predict the tertiary structure from the sequence of a protein using the above methodology is created and made freely accessible at http://www.scfbio-iitd.res.in/software/proteomics/rm2ts.jsp.