Allosteric Binding Research Articles

Active and allosteric site binding MM-QM studies of Methylidene tetracyclo derivative in PCSK9 protein intended to make a safe antilipidemic agent

Interaction of low-density lipoprotein receptors with proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a vital part in causing atherosclerosis. It is the hidden precursor of clinical myocardial infarction (MI), stroke, CVD and estimates 60% of deaths worldwide. The current need is to design small molecules to prevent the interaction between PCSK9 and LDL receptors. This study aims to evaluate the interaction between Methylidene tetracyclo derivative and PCSK9 protein through conceptual studies and compare the same with the interaction of the standard atorvastatin. Also, a comparative study was performed to analyze the interaction of molecules inside the active and allosteric sites of PCSK9. The RCSB downloaded pdb file 7S5H and the above said ligands were optimized to the level of local minima energy and configured inside the active and allosteric sites. The stability of non-bonded interactions of the complexes were analyzed using Desmond MD simulation studies. The results of docking showed that the Methylidene tetracyclo molecule possesses a two-fold higher affinity of −10.894 kcal/mol in the active site and −10.884 kcal/mol in the allosteric site. The Phe379 amino acid enabled the Methylidene tetracyclo molecule to orient inside the active site. Nine H-bonds with 6 amino acids of allosteric site increased the binding affinity compared to Atorvastatin. The MD simulation studies confirmed the stability of the nonbonded interaction of Methylidene tetracyclo molecule throughout 100 ns. This confirms that the Methylidene tetracyclo molecule will be the better hit as well as the lead molecule to modulate the behavior of PCSK9 protein. Communicated by Ramaswamy H. Sarma

Fluorescent Ligands Enable Target Engagement Studies for the Intracellular Allosteric Binding Site of the Chemokine Receptor CXCR2.

Herein, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of CXC chemokine receptor 2 (CXCR2), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in oncology and inflammation. Starting from the cocrystallized intracellular CXCR2 antagonist 00767013 (1), tetramethylrhodamine (TAMRA)-labeled CXCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CXCR2. By means of these studies, we developed Mz438 (9a) as a high-affinity and selective fluorescent CXCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and high-throughput manner. Further, we show that 9a can be used as a tool to visualize intracellular target engagement for CXCR2 via fluorescence microscopy. Thus, our small-molecule-based fluorescent CXCR2 ligand 9a represents a promising tool for future studies of CXCR2 pharmacology.

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain.

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

Discovery of VH domains that allosterically inhibit ENPP1

Ectodomain phosphatase/phosphodiesterase-1 (ENPP1) is overexpressed on cancer cells and functions as an innate immune checkpoint by hydrolyzing extracellular cyclic guanosine monophosphate adenosine monophosphate (cGAMP). Biologic inhibitors have not yet been reported and could have substantial therapeutic advantages over current small molecules because they can be recombinantly engineered into multifunctional formats and immunotherapies. Here we used phage and yeast display coupled with in cellulo evolution to generate variable heavy (VH) single-domain antibodies against ENPP1 and discovered a VH domain that allosterically inhibited the hydrolysis of cGAMP and adenosine triphosphate (ATP). We solved a 3.2 Å-resolution cryo-electron microscopy structure for the VH inhibitor complexed with ENPP1 that confirmed its new allosteric binding pose. Finally, we engineered the VH domain into multispecific formats and immunotherapies, including a bispecific fusion with an anti-PD-L1 checkpoint inhibitor that showed potent cellular activity.

NLRP3 monomer functional dynamics: From the effects of allosteric binding to implications for drug design

The protein NLRP3 and its complexes are associated with an array of inflammatory pathologies, among which neurodegenerative, autoimmune, and metabolic diseases. Targeting the NLRP3 inflammasome represents a promising strategy for easing the symptoms of pathologic neuroinflammation. When the inflammasome is activated, NLRP3 undergoes a conformational change triggering the production of pro-inflammatory cytokines IL-1β and IL-18, as well as cell death by pyroptosis. NLRP3 nucleotide-binding and oligomerization (NACHT) domain plays a crucial role in this function by binding and hydrolysing ATP and is primarily responsible, together with conformational transitions involving the PYD domain, for the complex-assembly process. Allosteric ligands proved able to induce NLRP3 inhibition.Herein, we examine the origins of allosteric inhibition of NLRP3. Through the use of molecular dynamics (MD) simulations and advanced analysis methods, we provide molecular-level insights into how allosteric binding affects protein structure and dynamics, remodelling of the conformational ensembles populated by the protein, with key reverberations on how NLRP3 is preorganized for assembly and ultimately function. The data are used to develop a Machine Learning model to define the protein as Active or Inactive, only based on the analysis of its internal dynamics. We propose this model as a novel tool to select allosteric ligands.

Structural Modifications Introduced by NS2B Cofactor Binding to the NS3 Protease of the Kyasanur Forest Disease Virus

Kyasanur Forest Disease virus (KFDV), a neglected human pathogenic virus, is a Flavivirus that causes severe hemorrhagic fever in humans. KFDV is transmitted to humans by the bite of the hard tick (Haemaphysalis spinigera), which acts as a reservoir of KFDV. The recent expansion of the endemic area of KFDV is of concern and requires the development of new preventive measures against KFDV. Currently, there is no antiviral therapy against KFDV, and the existing vaccine has limited efficacy. To develop a new antiviral therapy against KFDV, we focused on the nonstructural proteins NS2B and NS3 of KFDV, which are responsible for serine protease activity. Viral proteases have shown to be suitable therapeutic targets in the development of antiviral drugs against many diseases. However, success has been limited in flaviviruses, mainly because of the important features of the active site, which is flat and highly charged. In this context, the present study focuses on the dynamics of NS2B and NS3 to identify potential allosteric sites in the NS2B/NS3 protease of KDFV. To our knowledge, there are no reports on the dynamics of NS2B and NS3 in KFDV, and the crystal structure of the NS2B/NS3 protease of KFDV has not yet been solved. Overall, we created the structure of the NS2B/NS3 protease of KFDV using AlphaFold and performed molecular dynamics simulations with and without NS2B cofactor to investigate structural rearrangements due to cofactor binding and to identify alternative allosteric sites. The identified allosteric site is promising due to its geometric and physicochemical properties and druggability and can be used for new drug development. The applicability of the proposed allosteric binding sites was verified for the best-hit molecules from the virtual screening and MD simulations.

Molecular Dynamics Simulations Combined with Markov Model to Explore the Effect of Allosteric Inhibitor Binding on Bromodomain-Containing Protein 4

Bromodomain-Containing Protein 4 (BRD4) can play an important role in gene transcriptional regulation of tumor development and survival by participating in histone modification epigenetic mechanism. Although it has been reported that novel allosteric inhibitors such as ZL0590 have a high affinity with target protein BRD4 and good efficacy, their inhibitory mechanism has not been studied further. The aim of this study was to reveal the inhibition mechanism of allosteric inhibitor ZL0590 on Free-BRD4 and BRD4 binding MS436 (orthosteric inhibitor) by molecular dynamics simulation combined with a Markov model. Our results showed that BRD4-ZL0590 led to α-helices formation of 100–105 compared with Free-BRD4; the combination of MS436 caused residues 30–40 and 95–105 to form α-helices, while the combination of allosteric inhibitors untangled the α-helices formed by the MS436. The results of Markov flux analysis showed that the binding process of inhibitors mainly involved changes in the degree of α-helices at ZA loop. The binding of ZL0590 reduced the distance between ZA loop and BC loop, blocked the conformation at the active site, and inhibited the binding of MS436. After the allosteric inhibitor binding, the MS436 that could normally penetrate into the interior of the pocket was floating on the edge of the active pocket and did not continue to penetrate into the active pocket as expected. In summary, we provide a theoretical basis for the inhibition mechanism of ZL0590 against BRD4, which can be used as a reference for improving the development of drug targets for cancer therapy.

Role of Conserved Tyrosine Lid Residues in the Activation of the M2 Muscarinic Acetylcholine Receptor.

The development of subtype selective small molecule drugs for the muscarinic acetylcholine receptor (mAChR) family has been challenging. The design of more selective ligands can be improved by understanding the structure and function of key amino acid residues that line ligand binding sites. Here we study the role of three conserved key tyrosine residues [Y1043.33, Y4036.51, and Y4267.39 (Ballesteros and Weinstein numbers in superscript)] at the human M2 mAChR, located at the interface between the orthosteric and allosteric binding sites of the receptor. We specifically focused on the role of the three tyrosine hydroxyl groups in the transition between the inactive and active conformations of the receptor by making phenylalanine point mutants. Single-point mutation at either of the three positions was sufficient to reduce the affinity of agonists by ∼100-fold for the M2 mAChR, whereas the affinity of antagonists remained largely unaffected. In contrast, neither of the mutations affected the efficacy of orthosteric agonists. When mutations were combined into double and triple M2 mAChR mutants, the affinity of antagonists was reduced by more than 100-fold compared with the wild-type M2 receptor. In contrast, the affinity of allosteric modulators, either negative or positive, was retained at all single and multiple mutations, but the degree of allosteric effect exerted on the endogenous ligand acetylcholine was affected at all mutants containing Y4267.39F. These findings will provide insights to consider when designing future mAChR ligands. SIGNIFICANCE STATEMENT: Structural studies demonstrated that three tyrosine residues between the orthosteric and allosteric sites of the M2 muscarinic acetylcholine receptor (mAChR) had different hydrogen bonding networks in the inactive and active conformations. The role of hydroxyl groups of the tyrosine residues on orthosteric and allosteric ligand pharmacology was unknown. We found that hydroxyl groups of the tyrosine residues differentially affected the molecular pharmacology of orthosteric and allosteric ligands. These results provide insights to consider when designing future mAChR ligands.

Beta-Secretase 1 Recruits Amyloid-Beta Precursor Protein to ROCK2 Kinase, Resulting in Erroneous Phosphorylation and Beta-Amyloid Plaque Formation.

The amyloidogenic processing of APP depends on two events: its phosphorylation by ROCK2 (at Thr654) and the phosphorylation of the APP-cleaving enzyme BACE1 (at Ser498). However, the mechanisms and structural details of APP-ROCK2 and BACE1-ROCK2 binding are unknown. Using direct physical methods in combination with an in silico approach, we found that BACE1 binds into the substrate-binding groove of ROCK2 with a low affinity (Kd = 18 µM), while no binding of APP to ROCK2 alone could be detected. On the other hand, a strong association (Kd = 3.5 nM) of APP to the weak ROCK2-BACE1 complex was observed, although no stable ternary complex was detected, i.e., BACE1 was displaced by APP. We constructed a sequential functional model: (1) BACE1 weakly binds to ROCK2 and induces an allosteric conformational change in ROCK2; (2) APP strongly binds to the ROCK2-BACE1 complex, and BACE1 is released; and (3) ROCK2 phosphorylates APP at Thr654 (leading to a longer stay in the early endosome during APP processing). Direct fluorescence titration experiments showed that the APP646-664 or APP665-695 fragments did not bind separately to the ROCK2-BACE1 complex. Based on these observations, we conclude that two binding sites are involved in the ROCK2-APP interaction: (1) the substrate-binding groove, where the APP646-664 sequence containing Thr654 sits and (2) the allosteric binding site, where the APP665-695 sequence binds. These results open the way to attack the allosteric site to prevent APP phosphorylation at Thr654 by ROCK2 without inhibiting the activity of ROCK2 towards its other substrates.

Inter-domain interaction of ferredoxin-NADP+ reductase important for the negative cooperativity by ferredoxin and NADP(H).

Ferredoxin-NADP+ reductase (FNR) in plants receives electrons from ferredoxin (Fd) and converts NADP+ to NADPH. The affinity between FNR and Fd is weakened by the allosteric binding of NADP(H) on FNR, which is considered as a part of negative cooperativity. We have been investigating the molecular mechanism of this phenomenon and proposed that the NADP(H)-binding signal is transferred to the Fd-binding region across the two domains of FNR, NADP(H)-binding domain and FAD-binding domain. In this study, we analyzed the effect of altering the inter-domain interaction of FNR on the negative cooperativity. Four site-directed FNR mutants at the inter-domain region were prepared, and their NADPH-dependent changes in the Km for Fd and physical binding ability to Fd were investigated. Two mutants, in which an inter-domain hydrogen bond was changed to a disulfide bond (FNR D52C/S208C) and an inter-domain salt bridge was lost (FNR D104N), were shown to suppress the negative cooperativity by using kinetic analysis and Fd-affinity chromatography. These results showed that the inter-domain interaction of FNR is important for the negative cooperativity, suggesting that the allosteric NADP(H)-binding signal is transferred to Fd-binging region by conformational changes involving inter-domain interactions of FNR.

Feedback Inhibition of Bacterial Nucleotidyltransferases by Rare Nucleotide l-Sugars Restricts Substrate Promiscuity.

Bacterial glycomes are rich in prokaryote-specific or "rare" sugars that are absent in mammals. Like common sugars found across organisms, rare sugars are typically activated as nucleoside diphosphate sugars (NDP-sugars) by nucleotidyltransferases. In bacteria, the nucleotidyltransferase RmlA initiates the production of several rare NDP-sugars, which in turn regulate downstream glycan assembly through feedback inhibition of RmlA via binding to an allosteric site. In vitro, RmlA activates a range of common sugar-1-phosphates to produce NDP-sugars for biochemical and synthetic applications. However, our ability to probe bacterial glycan biosynthesis is hindered by limited chemoenzymatic access to rare NDP-sugars. We postulate that natural feedback mechanisms impact nucleotidyltransferase utility. Here, we use synthetic rare NDP-sugars to identify structural features required for regulation of RmlA from diverse bacterial species. We find that mutation of RmlA to eliminate allosteric binding of an abundant rare NDP-sugar facilitates the activation of noncanonical rare sugar-1-phosphate substrates, as products no longer affect turnover. In addition to promoting an understanding of nucleotidyltransferase regulation by metabolites, this work provides new routes to access rare sugar substrates for the study of important bacteria-specific glycan pathways.

Front Cover: Allosteric Tuning of Caspase‐7: Establishing the Nexus of Structure and Catalytic Power (Chem. Eur. J. 40/2023)

Mapping the constellation of structural and dynamic changes concurrent with allosteric inhibitor binding to caspase-7, also known as allosteric structure–activity relationships (ASARs), reveals the nexus between the active site and the remote allosteric pocket. These ASARs also elucidate the link between structure and catalytic power for caspase-7. Our insights from exploration of caspase-7 allostery offer a new path for navigating allosteric drug discovery. More information can be found in the Research Article by M. A. Spies and co-workers (DOI: 10.1002/chem.202300872).

Structural modification of resveratrol analogue exhibits anticancer activity against lung cancer stem cells via suppression of Akt signaling pathway

BackgroundCompound with cancer stem cell (CSC)-suppressing activity is promising for the improvement of lung cancer clinical outcomes. Toward this goal, we discovered the CSC-targeting activity of resveratrol (RES) analog moscatilin (MOS). With slight structural modification from RES, MOS shows dominant cytotoxicity and CSC-suppressive effect.MethodsThree human lung cancer cell lines, namely H23, H292, and A549, were used to compare the effects of RES and MOS. Cell viability and apoptosis were determined by the MTT assay and Hoechst33342/PI double staining. Anti-proliferative activity was determined by colony formation assay and cell cycle analysis. Intracellular reactive oxygen species (ROS) were measured by fluorescence microscopy using DCFH2-DA staining. CSC-rich populations of A549 cells were generated, and CSC markers, and Akt signaling were determined by Western blot analysis and immunofluorescence. Molecular docking and molecular dynamics (MD) simulations were used to predict the possible binding of the compound to Akt protein.ResultsIn this study, we evaluated the effects of RES and MOS on lung cancer and its anti-CSC potential. Compared with RES, its analog MOS more effectively inhibited cell viability, colony formation, and induced apoptosis in all lung cancer cell lines (H23, H292, and A549). We further investigated the anti-CSC effects on A549 CSC-rich populations and cancer adherent cells (A549 and H23). MOS possesses the ability to suppress CSC-like phenotype of lung cancer cells more potent than RES. Both MOS and RES repressed lung CSCs by inhibiting the viability, proliferation, and lung CSC-related marker CD133. However, only MOS inhibits the CSC marker CD133 in both CSC-rich population and adherent cells. Mechanistically, MOS exerted its anti-CSC effects by inhibiting Akt and consequently restored the activation of glycogen synthase kinase 3β (GSK-3β) and decreased the pluripotent transcription factors (Sox2 and c-Myc). Thus, MOS inhibits CSC-like properties through the repression of the Akt/GSK-3β/c-Myc pathway. Moreover, the superior inhibitory effects of MOS compared to RES were associated with the improved activation of various mechanism, such as cell cycle arrest at G2/M phase, production of ROS-mediated apoptosis, and inhibition of Akt activation. Notably, the computational analysis confirmed the strong interaction between MOS and Akt protein. MD simulations revealed that the binding between MOS and Akt1 was more stable than RES, with MM/GBSA binding free energy of − 32.8245 kcal/mol at its allosteric site. In addition, MOS interacts with Trp80 and Tyr272, which was a key residue in allosteric inhibitor binding and can potentially alter Akt activity.ConclusionsKnowledge about the effect of MOS as a CSC-targeting compound and its interaction with Akt is important for the development of drugs for the treatment of CSC-driven cancer including lung cancer.

Long Time Scale Ensemble Methods in Molecular Dynamics: Ligand-Protein Interactions and Allostery in SARS-CoV-2 Targets.

We subject a series of five protein-ligand systems which contain important SARS-CoV-2 targets, 3-chymotrypsin-like protease (3CLPro), papain-like protease, and adenosine ribose phosphatase, to long time scale and adaptive sampling molecular dynamics simulations. By performing ensembles of ten or twelve 10 μs simulations for each system, we accurately and reproducibly determine ligand binding sites, both crystallographically resolved and otherwise, thereby discovering binding sites that can be exploited for drug discovery. We also report robust, ensemble-based observation of conformational changes that occur at the main binding site of 3CLPro due to the presence of another ligand at an allosteric binding site explaining the underlying cascade of events responsible for its inhibitory effect. Using our simulations, we have discovered a novel allosteric mechanism of inhibition for a ligand known to bind only at the substrate binding site. Due to the chaotic nature of molecular dynamics trajectories, regardless of their temporal duration individual trajectories do not allow for accurate or reproducible elucidation of macroscopic expectation values. Unprecedentedly at this time scale, we compare the statistical distribution of protein-ligand contact frequencies for these ten/twelve 10 μs trajectories and find that over 90% of trajectories have significantly different contact frequency distributions. Furthermore, using a direct binding free energy calculation protocol, we determine the ligand binding free energies for each of the identified sites using long time scale simulations. The free energies differ by 0.77 to 7.26 kcal/mol across individual trajectories depending on the binding site and the system. We show that, although this is the standard way such quantities are currently reported at long time scale, individual simulations do not yield reliable free energies. Ensembles of independent trajectories are necessary to overcome the aleatoric uncertainty in order to obtain statistically meaningful and reproducible results. Finally, we compare the application of different free energy methods to these systems and discuss their advantages and disadvantages. Our findings here are generally applicable to all molecular dynamics based applications and not confined to the free energy methods used in this study.

Generation of nanobodies acting as silent and positive allosteric modulators of the α7 nicotinic acetylcholine receptor

The α7 nicotinic acetylcholine receptor (nAChR), a potential drug target for treating cognitive disorders, mediates communication between neuronal and non-neuronal cells. Although many competitive antagonists, agonists, and partial-agonists have been found and synthesized, they have not led to effective therapeutic treatments. In this context, small molecules acting as positive allosteric modulators binding outside the orthosteric, acetylcholine, site have attracted considerable interest. Two single-domain antibody fragments, C4 and E3, against the extracellular domain of the human α7-nAChR were generated through alpaca immunization with cells expressing a human α7-nAChR/mouse 5-HT3A chimera, and are herein described. They bind to the α7-nAChR but not to the other major nAChR subtypes, α4β2 and α3β4. E3 acts as a slowly associating positive allosteric modulator, strongly potentiating the acetylcholine-elicited currents, while not precluding the desensitization of the receptor. An E3–E3 bivalent construct shows similar potentiating properties but displays very slow dissociation kinetics conferring quasi-irreversible properties. Whereas, C4 does not alter the receptor function, but fully inhibits the E3-evoked potentiation, showing it is a silent allosteric modulator competing with E3 binding. Both nanobodies do not compete with α-bungarotoxin, localizing at an allosteric extracellular binding site away from the orthosteric site. The functional differences of each nanobody, as well as the alteration of functional properties through nanobody modifications indicate the importance of this extracellular site. The nanobodies will be useful for pharmacological and structural investigations; moreover, they, along with the extracellular site, have a direct potential for clinical applications.

Potential Binding Sites of Pharmacological Chaperone NCGC00241607 on Mutant β-Glucocerebrosidase and Its Efficacy on Patient-Derived Cell Cultures in Gaucher and Parkinson's Disease.

Mutations in the GBA1 gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), cause Gaucher disease (GD) and are the most common genetic risk factor for Parkinson's disease (PD). Pharmacological chaperones (PCs) are being developed as an alternative treatment approach for GD and PD. To date, NCGC00241607 (NCGC607) is one of the most promising PCs. Using molecular docking and molecular dynamics simulation we identified and characterized six allosteric binding sites on the GCase surface suitable for PCs. Two sites were energetically more preferable for NCGC607 and located nearby to the active site of the enzyme. We evaluated the effects of NCGC607 treatment on GCase activity and protein levels, glycolipids concentration in cultured macrophages from GD (n = 9) and GBA-PD (n = 5) patients as well as in induced human pluripotent stem cells (iPSC)-derived dopaminergic (DA) neurons from GBA-PD patient. The results showed that NCGC607 treatment increased GCase activity (by 1.3-fold) and protein levels (by 1.5-fold), decreased glycolipids concentration (by 4.0-fold) in cultured macrophages derived from GD patients and also enhanced GCase activity (by 1.5-fold) in cultured macrophages derived from GBA-PD patients with N370S mutation (p < 0.05). In iPSC-derived DA neurons from GBA-PD patients with N370S mutation NCGC607 treatment increased GCase activity and protein levels by 1.1-fold and 1.7-fold (p < 0.05). Thus, our results showed that NCGC607 could bind to allosteric sites on the GCase surface and confirmed its efficacy on cultured macrophages from GD and GBA-PD patients as well as on iPSC-derived DA neurons from GBA-PD patients.

Allosteric Tuning of Caspase-7: Establishing the Nexus of Structure and Catalytic Power.

Caspase-7 (C7), a cysteine protease involved in apoptosis, is a valuable drug target for its role in human diseases (e.g. Parkinson's, Alzheimer's, sepsis). The C7 allosteric site has great potential for small molecule targeting, but numerous drug discovery efforts have identified precious few allosteric inhibitors. Here we present the first selective, drug-like inhibitor of C7 along with several other improved inhibitors based on our previous fragment hit. We also provide a rational basis for the impact of allosteric binding on the C7 catalytic cycle using an integrated approach including X-ray crystallography, stopped-flow kinetics, and molecular dynamics simulations. Our findings suggest allosteric binding disrupts C7 pre-acylation via neutralization of the catalytic dyad, displacement of substrate from the oxyanion hole, and altered dynamics of substrate binding loops. This work advances drug targeting efforts and bolsters our understanding of allosteric structure activity relationships (ASARs).

Enhancement of Inhibitory Activity by Combining Allosteric Inhibitors Putatively Binding to Different Allosteric Sites on Cathepsin K.

Cathepsin K, which is involved in bone resorption, is a good target for treating osteoporosis, but no clinically approved medicine has been developed. Recently, allosteric inhibitors with high specificity and few side effects have been attracting attention for use in new medicines. Cathepsin K inhibitors were isolated from the methanol extract of Chamaecrista nomame (Leguminosae) using cathepsin K inhibition activity-assisted multi-step chromatography. Standard kinetic analysis was employed to examine the mechanism of cathepsin K inhibition when an isolated inhibitor and its derivative were used. The allosteric binding of these cathepsin K inhibitors was supported by a docking study using AutoDock vina. Combinations of allosteric cathepsin K inhibitors expected to bind to different allosteric sites were examined by means of cathepsin K inhibition assay. Two types of cathepsin K inhibitors were identified in the methanol extract of Chamaecrista nomame. One type consisted of cassiaoccidentalin B and torachrysone 8-β-gentiobioside, and inhibited both cathepsin K and B with similar inhibitory potential, while the other type of inhibitor consisted of pheophytin a, and inhibited cathepsin K but not cathepsin B, suggesting that pheophytin a binds to an allosteric site of cathepsin K. Kinetic analysis of inhibitory activity suggested that pheophytin a and its derivative, pheophorbide b, bind allosterically to cathepsin K. This possibility was supported by a docking study on cathepsin K. The cathepsin K inhibitory activity of pheophytin a and pheophorbide b was enhanced by combining them with the allosteric inhibitors NSC 13345 and NSC94914, which bind to other allosteric sites on cathepsin K. Different allosteric inhibitors that bind to different sites in combination, as shown in this study, may be useful for designing new allosteric inhibitory drugs with high specificity and few side effects.

Functional DNA Superstructures Exhibit Positive Homotropic Allostery in Ligand Binding

AbstractInspired by intrinsically disordered proteins in nature, DNA aptamers can be engineered to display strongly homotropic allosteric (or cooperative) ligand binding, representing a unique feature that could be of great utility in applications such as biosensing, imaging and drug delivery. The use of an intrinsic disorder mechanism, however, comes with an inherent drawback of significantly reduced overall binding affinity. We hypothesize that it could be addressed via the design of multivalent supramolecular aptamers. We built functional DNA superstructures (denoted as 3D DNA), made of long‐chain DNA containing tandem repeating DNA aptamers (or concatemeric aptamers). The 3D DNA systems exhibit highly cooperative binding to both small molecules and proteins, without loss of binding affinities of their parent aptamers. We further produced a highly responsive sensor for fluorescence imaging of glutamate stimulation‐evoked adenosine triphosphate (ATP) release in neurons, as well as force stimulus‐triggered ATP release in astrocytes.