Selectivity Studies and Free Energy Calculations of AKT Inhibitors.

Protein kinase B (PKB) or AKT protein is an important target for cancer treatment. Significant advances have been made in developing ATP-competitive inhibitors and allosteric binders targeting AKT1. However, adverse effects or toxicities have been found, and the cutaneous toxicity was found to be linked to the inhibition of AKT2. Thus, selective inhibition of AKT inhibitors is of significance. Our work, using the Schrödinger Covalent Dock (CovDock) program and the Movable Type (MT)-based free energy calculation (ΔG), yielded small mean errors for the experimentally derived binding free energy (ΔG). The docking data suggested that AKT1 binding may require residues Asn54, Trp80, Tyr272, Asp274, and Asp292, whereas AKT2 binding would expect residues Phe163 and Glu279, and AKT3 binding would favor residues Glu17, Trp79, Phe306, and Glu295. These findings may help guide AKT1-selective or AKT3-selective molecular design while sparing the inhibition of AKT2 to minimize the cutaneous toxicity.

Docking and Selectivity Studies of Covalently Bound Janus Kinase 3 Inhibitors.

The Janus kinases (JAKs) are a family of non-receptor cytosolic protein kinases critical for immune signaling. Many covalently bound ligands of JAK3 inhibitors have been reported. To help design selective JAK inhibitors, in this paper, we used five model proteins to study the subtype selectivity of and the mutational effects on inhibitor binding. We also compared the Covalent Dock programs from the Schrodinger software suite and the MOE software suite to determine which method to use for the drug design of covalent inhibitors. Our results showed that the docking affinity from 4Z16 (JAK3 wild-type model), 4E4N (JAK1), 4D1S (JAK2), and 7UYT (TYK2) from the Schrödinger software suite agreed well with the experimentally derived binding free energies with small predicted mean errors. However, the data from the mutant 5TTV model using the Schrödinger software suite yielded relatively large mean errors, whereas the MOE Covalent Dock program gave small mean errors in both the wild-type and mutant models for our model proteins. The docking data revealed that Leu905 of JAK3 and the hydrophobic residue at the same position in different subtypes (Leu959 of JAK1, Leu932 of JAK2, and Val981 of TYK2) is important for ligand binding to the JAK proteins. Arg911 and Asp912 of JAK3, Asp939 of JAK2, and Asp988 of TYK2 can be used for selective binding over JAK1, which contains Lys965 and Glu966 at the respective positions. Asp1021, Asp1039, and Asp1042 can be utilized for JAK1-selective ligand design, whereas Arg901 and Val981 may help guide TYK2-selective molecule design.

Novel curcumin analog (cis-trans curcumin) as ligand to adenosine receptors A2A and A2B: potential for therapeutics.

All four of the adenosine receptor (AR) subtypes mediate pain and have been targeted by pharmacologists to generate new therapeutics for chronic pain. The vanilloid phytochemicals, which include curcumin, capsaicin, and gingerol, have been shown to alleviate pain. However, there is little to no literature on the interaction of vanilloid phytochemicals with ARs. In this study, photochemical methods were used to generate a novel isomer of curcumin (cis-trans curcumin or CTCUR), and the interactions of both curcumin and CTCUR with the two Gs-linked AR subtypes were studied. Competitive binding assays, docking analysis, and confocal fluorescence microscopy were performed to measure binding affinity; cell survival assays were used to measure toxicity; and cAMP assays were performed to measure receptor activation. Competitive binding results indicated that CTCUR binds to both AR A2A and AR A2B with Ki values of 5 µM and 7 µM, respectively, which is consistent with our docking results. Fluorescence microscopy data also shows binding for A2B and A2A. Cell survival results show that CTCUR and CUR are nontoxic at the tested concentrations in these cell lines. Overall, our results suggest that vanilloid phytochemicals may be slightly modified to increase interaction with Gs-ARs, and thereby can be further explored to provide a novel class of non-opioid antinociceptives.

Virtual screening and biological evaluation of PPARγ antagonists as potential anti-prostate cancer agents.

The peroxisome proliferator-activated receptor gamma (PPARγ) was identified as an oncogene and it plays a key role in prostate cancer (PC) development and progression. PPARγ antagonists have been shown to inhibit PC cell growth. Herein, we describe a virtual screening-based approach that led to the discovery of novel PPARγ antagonist chemotypes that bind at the allosteric pocket. Arg288, Lys367, and His449 appear to be important for PPARγ antagonist binding.

Ligand-based design of GLUT inhibitors as potential antitumor agents.

Glucose transporters (GLUTs) regulate glucose uptake and are often overexpressed in several human tumors. To identify new chemotypes targeting GLUT1, we built a pharmacophore model and searched against a NCI compound database. Sixteen hit molecules with good docking scores were screened for GLUT1 inhibition and antiproliferative activities. From these, we identified that compounds 2, 5, 6 and 13 inhibited the cell viability in a dose-dependent manner and that the IC50s of 2 and 6 are<10 µM concentration in the HCT116 colon cancer cell line. Lead compound 13 (NSC295720) was a GLUT1 inhibitor. Docking studies show that GLUT1 residues Phe291, Phe379, Glu380, Trp388, and Trp412 were important for inhibitor binding.

Molecular Modeling Studies on the Binding Mode of the PD-1/PD-L1 Complex Inhibitors.

The programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) is an immune checkpoint (ICP) overexpressed in various types of tumors; thus, it has been considered as an important target for cancer therapy. To determine important residues for ligand binding, we applied molecular docking studies to PD-1/PD-L1 complex inhibitors against the PD-L1 protein. Our data revealed that the residues Tyr56, Asp122, and Lys124 play critical roles in ligand binding to the PD-L1 protein and they could be used to design ligands that are active against the PD-1/PD-L1 complex. The formation of H-bonds with Arg125 of the PD-L1 protein may enhance the potency of the PD-1/PD-L1 binding.

Conformational Studies of Glucose Transporter 1 (GLUT1) as an Anticancer Drug Target.

Glucose transporter 1 (GLUT1) is a facilitative glucose transporter overexpressed in various types of tumors; thus, it has been considered as an important target for cancer therapy. GLUT1 works through conformational switching from an outward-open (OOP) to an inward-open (IOP) conformation passing through an occluded conformation. It is critical to determine which conformation is preferred by bound ligands because the success of structure-based drug design depends on the appropriate starting conformation of the target protein. To find out the most favorable GLUT 1 conformation for ligand binding, we ran systemic molecular docking studies for different conformations of GLUT1 using known GLUT1 inhibitors. Our data revealed that the IOP is the preferred conformation and that residues Phe291, Phe379, Glu380, Trp388, and Trp412 may play critical roles in ligand binding to GLUT1. Our data suggests that conformational differences in these five amino acids in the different conformers of GLUT1 may be used to design ligands that inhibit GLUT1.

Free-Energy-Based Protein Design: Re-Engineering Cellular Retinoic Acid Binding Protein II Assisted by the Moveable-Type Approach.

How to fine-tune the binding free energy of a small-molecule to a receptor site by altering the amino acid residue composition is a key question in protein engineering. Indeed, the ultimate solution to this problem, to chemical accuracy (±1 kcal/mol), will result in profound and wide-ranging applications in protein design. Numerous tools have been developed to address this question using knowledge-based models to more computationally intensive molecular dynamics simulations-based free energy calculations, but while some success has been achieved there remains room for improvement in terms of overall accuracy and in the speed of the methodology. Here we report a fast, knowledge-based movable-type (MT)-based approach to estimate the absolute and relative free energy of binding as influenced by mutations in a small-molecule binding site in a protein. We retrospectively validate our approach using mutagenesis data for retinoic acid binding to the Cellular Retinoic Acid Binding Protein II (CRABPII) system and then make prospective predictions that are borne out experimentally. The overall performance of our approach is supported by its success in identifying mutants that show high or even sub-nano-molar binding affinities of retinoic acid to the CRABPII system.

Cannabidiol promotes intestinal cholesterol uptake mediated by Pregnane X receptor.

Background: Cannabidiol (CBD), a non-psychoactive phytocannabinoid of cannabis, is therapeutically used as an analgesic, anti-convulsant, anti-inflammatory, and anti-psychotic drug. There is a growing concern about the adverse side effects posed by CBD usage. Pregnane X receptor (PXR) is a nuclear receptor activated by a variety of dietary steroids, pharmaceutical agents, and environmental chemicals. In addition to the role in xenobiotic metabolism, the atherogenic and dyslipidemic effects of PXR have been revealed in animal models. CBD has a low affinity for cannabinoid receptors, thus it is important to elucidate the molecular mechanisms by which CBD activates cellular signaling and to assess the possible adverse impacts of CBD on pro-atherosclerotic events in cardiovascular system, such as dyslipidemia. Objective: Our study aims to explore the cellular and molecular mechanisms by which exposure to CBD activates human PXR and increases the risk of dyslipidemia. Methods: Both human hepatic and intestinal cells were used to test if CBD was a PXR agonist via cell-based transfection assay. The key residues within PXR's ligand-binding pocket that CBD interacted with were investigated using computational docking study together with site-directed mutagenesis assay. The C57BL/6 wildtype mice were orally fed CBD in the presence of PXR antagonist resveratrol (RES) to determine how CBD exposure could change the plasma lipid profiles in a PXR-dependent manner. Human intestinal cells were treated with CBD and/or RES to estimate the functions of CBD in cholesterol uptake. Results: CBD was a selective agonist of PXR with higher activities on human PXR than rodents PXRs and promoted the dissociation of human PXR from nuclear co-repressors. The key amino acid residues Met246, Ser247, Phe251, Phe288, Trp299, and Tyr306 within PXR's ligand binding pocket were identified to be necessary for the agonistic effects of CBD. Exposure to CBD increased the circulating total cholesterol levels in mice which was partially caused by the induced expression levels of the key intestinal PXR-regulated lipogenic genes. Mechanistically, CBD induced the gene expression of key intestinal cholesterol transporters, which led to the increased cholesterol uptake by intestinal cells. Conclusion: CBD was identified as a selective PXR agonist. Exposure to CBD activated PXR signaling and increased the atherogenic cholesterol levels in plasma, which partially resulted from the ascended cholesterol uptake by intestinal cells. Our study provides potential evidence for the future risk assessment of CBD on cardiovascular disease, such as dyslipidemia.

Targeting the EGFR/RAS/RAF signaling pathway in anticancer research: a recent update on inhibitor design and clinical trials (2020-2023).

INTRODUCTION: Recent years have seen significant strides in drug developmenttargeting the EGFR/RAS/RAF signaling pathway which is critical forcell growth and proliferation. Protein-protein interaction networksamong EGFR, RAS, and RAF proteins offer insights for drug discovery. This review discusses the drug design and development efforts ofinhibitors targeting these proteins over the past 3 years, detailingtheir structures, selectivity, efficacy, and combination therapy.Strategies to combat drug resistance and minimize toxicities areexplored, along with future research directions. AREA COVERED: This review encompasses clinical trials and patents on EGFR, KRAS,and BRAF inhibitors from 2020 to 2023, including advancements indesign and synthesis of proteolysis targeting chimeras (PROTACs) forprotein degradation. EXPERT OPINION: To tackle drug resistance, designing allosteric fourth-generationEGFR inhibitors is vital. Covalent, allosteric, or combinationaltherapies, along with PROTAC degraders, are key methods to addressresistance and toxicity in KRAS and BRAF inhibitors.

Targeting the PI3K/AKT signaling pathway in anticancer research: a recent update on inhibitor design and clinical trials (2020-2023).

INTRODUCTION: Recent years have witnessed great achievements in drug design and development targeting the phosphatidylinositol 3-kinase/protein kinase-B (PI3K/AKT) signaling pathway, a pathway central to cell growth and proliferation. The nearest neighbor protein-protein interaction networks for PI3K and AKT show the interplays between these target proteins which can be harnessed for drug discovery. In this review, we discuss the drug design and clinical development of inhibitors of PI3K/AKT in the past three years. We review in detail the structures, selectivity, efficacy, and combination therapy of 35 inhibitors targeting these proteins, classified based on the target proteins. Approaches to overcoming drug resistance and to minimizing toxicities are discussed. Future research directions for developing combinational therapy and PROTACs of PI3K and AKT inhibitors are also discussed. AREA COVERED: This review covers clinical trial reports and patent literature on inhibitors of PI3K and AKT published between 2020 and 2023. EXPERT OPINION: To address drug resistance and drug toxicity of inhibitors of PI3K and AKT, it is highly desirable to design and develop subtype-selective PI3K inhibitors or subtype-selective AKT1 inhibitors to minimize toxicity or to develop allosteric drugs that can form covalent bonds. The development of PROTACs of PI3Kα or AKT helps to reduce off-target toxicities.

Curcumin Stereoisomer, Cis-Trans Curcumin, as a Novel Ligand to A1 and A3 Adenosine Receptors.

Adenosine receptors (ARs) are being explored to generate non-opioid pain therapeutics. Vanilloid compounds, curcumin, capsaicin, and vanillin possess antinociceptive properties through their interactions with the transient receptor potential channel family. However, their binding with adenosine receptors has not been well studied. The hypothesis in this study was that a vanilloid compound, cis-trans curcumin (CTCUR), binds to each of the two Gi-linked AR subtypes (A1AR and A3AR). CTCUR was synthesized from curcumin (CUR) using the cavitand-mediated photoisomerization technique. The cell lines transfected with the specific receptor (A1AR or A3AR) were treated with CTCUR or CUR and the binding was analyzed using competitive assays, confocal microscopy, and docking. The binding assays and molecular docking indicated that CTCUR had Ki values of 306 nM (A1AR) and 400 nM (A3AR). These values suggest that CTCUR is selective for Gi-linked ARs (A1AR or A3AR) over Gs-linked ARs (A2AAR or A2BAR), based on our previous published research. In addition, the docking showed that CTCUR binds to the toggle switch domain of ARs. Curcumin (CUR) did not exhibit binding at any of these receptors. In summary, CTCUR and other modifications of CUR can be developed as novel therapeutic ligands for the Gi-linked ARs (A1AR and A3AR) involved with pain and cancer.

Binding and selectivity studies of phosphatidylinositol 3-kinase (PI3K) inhibitors.

Overexpression of the Phosphatidylinositol 3-kinase (PI3K) proteins have been observed in cancer cells. Targeting the phosphatidylinositol 3-kinase (PI3K) signaling transduction pathway by inhibition of the PI3K substrate recognition sites has been proved to be an effective approach to block cancer progression. Many PI3K inhibitors have been developed. Seven drugs have been approved by the US FDA with a mechanism of targeting the phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. In this study, we used docking tools to investigate selective binding of ligands toward four different subtypes of PI3Ks (PI3Kα, PI3Kß, PI3Kγ and PI3Kδ). The affinity predicted from both the Glide dock and the Movable-Type (MT)-based free energy calculations agreed well with the experimental data. The validation of our predicted methods with a large dataset of 147 ligands showed very small mean errors. We identified residues that may dictate the subtype-specific binding. Particularly, residues Asp964, Ser806, Lys890 and Thr886 of PI3Kγ might be utilized for PI3Kγ-selective inhibitor design. Residues Val828, Trp760, Glu826 and Tyr813 may be important for PI3Kδ-selective inhibitor binding.

Targeting BRD4 and PI3K signaling pathways for the treatment of medulloblastoma.

Medulloblastoma (MB) is a malignant pediatric brain tumor which shows upregulation of MYC and sonic hedgehog (SHH) signaling. SHH inhibitors face acquired resistance, which is a major cause of relapse. Further, direct MYC oncogene inhibition is challenging, inhibition of MYC upstream insulin-like growth factor/ phosphatidylinositol-4,5-bisphosphate 3-kinase (IGF/PI3K) is a promising alternative. While PI3K inhibition activates resistance mechanisms, simultaneous inhibition of bromodomain-containing protein 4 (BRD4) and PI3K can overcome resistance. We synthesized a new molecule 8-(2,3-dihydrobenzo[b] [1, 4] dioxin-6-yl)-2-morpholino-4H-chromen-4-one (MDP5) that targets both BRD4 and PI3K pathways. We used X-ray crystal structures and a molecular modeling approach to confirm the interactions between MDP5 with bromo domains (BDs) from both BRD2 and BRD4, and molecular modeling for PI3K binding. MDP5 was shown to inhibit target pathways and MB cell growth in vitro and in vivo. MDP5 showed higher potency in DAOY cells (IC50 5.5 µM) compared to SF2523 (IC50 12.6 µM), and its IC50 values in HD-MB03 cells were like SF2523. Treatment of MB cells with MDP5 significantly decreased colony formation, increased apoptosis, and halted cell cycle progression. Further, MDP5 was well tolerated in NSG mice bearing either xenograft or orthotopic MB tumors at the dose of 20 mg/kg, and significantly reduced tumor growth and prolonged animal survival.

Biological evaluation and docking studies of recently identified inhibitors of phosphoinositide-3-kinases.

The alpha isoform of the phosphatidylinositol-3-kinases (PI3Kα) is often mutated, amplified and overexpressed in human tumors. In an effort to develop new inhibitors targeting this enzyme, we carried out a pharmacophore model study based on six PI3Kα-selective compounds. The pharmacophore searching identified three structurally novel inhibitors of PI3Kα and its H1047R mutant. Our biological studies show that two of our hit molecules suppressed the formation of pAKT, a downstream effector of PI3Kα, and induced apoptosis in the HCT116 colon cancer cell line. QPLD-based docking showed that residues Asp933, Glu849, Val851, and Gln859 appeared to be key binding residues for active inhibitors.

Binding selectivity studies of phosphoinositide 3-kinases using free energy calculations.

Phosphoinositide 3-kinases (PI3Ks) and their phosphatidylinositol 3,4,5-triphosphate (PIP(3)) products regulate a variety of cellular processes. Of these, PI3Kα is an attractive target for anticancer drug design. Mutations in the PI3Kα kinase domain alter the mobility of the activation loop resulting in gain of function. We employed molecular dynamics (MD) simulations-based energetic analysis using molecular mechanics/generalized born surface area (MM/GBSA) for PI3Kα and -γ. MD simulations were carried out for PI3K models based on the RESP (restrained electrostatic potential) and quantum mechanics (QM)-polarized ligand docking (QPLD)-derived partial charges. Computational alanine scanning was also used to evaluate the contributions of key binding residues to ligand binding. Our results show that both RESP and QPLD charge models of PI3Kα and PI3Kγ provide similar performance in MD simulations. For example, the predicted RESP and QPLD free energies of -9.5 and -9.3 kcal/mol for LY294002 binding to PI3Kγ and -10.9 and -11.7 kcal/mol for wortmannin binding to PI3Kα are in good agreement with experimental values. A significant loss in binding free energy was observed when hydrophobic residues were mutated to alanine, suggesting that specific hydrophobic interactions are important to optimal ligand binding. MM/GBSA calculations suggested that residues Ser774, Gln859, and Ile932 of PI3Kα might be used to design H1047R mutant-specific ligands, whereas Lys890 of PI3Kγ can be used for ligand design targeting PI3Kγ.

N-Phenyl-4-hydroxy-2-quinolone-3-carboxamides as selective inhibitors of mutant H1047R phosphoinositide-3-kinase (PI3Kα).

This work describes our efforts to optimize the lead PI3Kα inhibitor N-benzyl 4-hydroxy-2-quinolone-3-carboxamide using structure-based design and molecular docking. We identified a series of N-phenyl 4-hydroxy-2-quinolone-3-carboxamides as selective inhibitors of mutant H1047R versus wild-type PI3Kα and we also showed that the cell growth inhibition by these compounds likely occurs by inhibiting the formation of pAKT and induction of apoptosis.

Randomized, double-blind, parallel-controlled clinical trial on correspondence of prescriptions and traditional Chinese medicine syndrome of ischemic stroke.

OBJECTIVE: To investigate the effect of correspondence of prescriptions and the traditional Chinese medicine (TCM) syndrome of ischemic stroke. METHODS: Based on standard syndrome diagnosis and a series of effective formulas, a randomized, double-blind, parallel-controlled clinical trial on correspondence of prescriptions and the TCM syndrome of ischemic stroke was conducted. The formulas were dynamically changed according to the patient's TCM syndrome. RESULTS: The treatment was composed of a series of formulas changed daily according to the TCM syndrome. Treatments which were composed of a series of formulas had similar short-term (1 month after onset) effects in improving mild and moderate neurological deficits compared with treatments composed of only one formula. However, patients treated with a series of formulas showed improved subjective symptoms in the short-term and a decreased risk of stroke recurrence within three months after onset. CONCLUSION: Complete (series of formulas) and incomplete (only one formula) correspondence of prescriptions and TCM syndrome have similar effects on the central clinical manifestation of ischemic stroke, such as neurological deficit and activities in daily living. However, they have significant differences regarding individual dysfunctions, such as subjective symptoms.

Molecular Modeling of Allosteric Site of Isoform-Specific Inhibition of the Peroxisome Proliferator-Activated Receptor PPARγ.

The peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor and controls a number of gene expressions. The ligand binding domain (LBD) of PPARγ is large and involves two binding sites: orthosteric and allosteric binding sites. Increased evidence has shown that PPARγ is an oncogene and thus the PPARγ antagonists have potential as anticancer agents. In this paper, we use Glide Dock approach to determine which binding site, orthosteric or allosteric, would be a preferred pocket for PPARγ antagonist binding, though antidiabetic drugs such as thiazolidinediones (TZDs) bind to the orthosteric site. The Glide Dock results show that the binding of PPARγ antagonists at the allosteric site yielded results that were much closer to the experimental data than at the orthosteric site. The PPARγ antagonists seem to selectively bind to residues Lys265, Ser342 and Arg288 at the allosteric binding site, whereas PPARγ agonists would selectively bind to residues Leu228, Phe363, and His449, though Phe282 and Lys367 may also play a role for agonist binding at the orthosteric binding pocket. This finding will provide new perspectives in the design and optimization of selective and potent PPARγ antagonists or agonists.

Synthesis and optical properties of triphenylene-based dendritic donor perylene diimide acceptor systems.

A donor-acceptor charge transfer system based on two discotic mesogens has been synthesized. The donor is either a triphenylene (POG0) or a triphenylene-based conjugated dendron (POG1), while the acceptor is a perylene diimide (PDI) core. The donors are covalently linked to the bay positions of the PDI core through an ether linkage. In chloroform, due to the short donor-acceptor distance and the matching frontier orbital levels, photoinduced charge transfer from either the donor excitation or the acceptor excitation are both thermodynamically and kinetically favored, resulting in efficient quenching of both donor and acceptor fluorescence. In a less polar solvent, hexane, while charge transfer is still the dominant mechanism for decay of the excited electronic state of POG1, photoinduced charge transfer is no longer energetically favorable for POG0 when the acceptor PDI core is excited, making the PDI core of POG0 weakly fluorescent in chloroform but strongly so in hexane. In solid film, POG0 is highly aggregated through both PDI-PDI and triphenylene-triphenylene homotopic stacking. POG1, on the other hand, aggregates through triphenylene dendrons with limited PDI-PDI core stacking, presumably due to the steric hindrance caused by bulky triphenylene moieties which block the access to the PDI core. The efficient photoinduced charge transfer, coupled with the homotopic stacking that forms separated electron-transporting PDI-stacked columns and hole transporting triphenylene-stacked columns, suggests that the reported donor-acceptor systems based on dual-discotic mesogens are potentially new efficient photovoltaic materials.