Model of P-Glycoprotein Ligand Binding and Validation with Efflux Substrate Matched Pairs.

The blood-brain barrier (BBB) poses a significant obstacle in developing therapeutics for neurodegenerative diseases and central nervous system (CNS) disorders. P-glycoprotein (P-gp), a multidrug resistance protein, is a critical gatekeeper in the BBB and plays a role in cancer chemoresistance. This paper uses cryo-EM P-gp structures as starting points with an induced fit docking (IFD) model to evaluate 19 pairs of compounds with known P-gp efflux data. The study reveals significant differences in binding energy and sheds light on structural modifications' impact on efflux properties. In the cases examined, fluorine incorporation influences the efflux by altering the molecular conformation rather than proximal heteroatom basicity. Although there are limitations in addressing covalent interactions or when binding extends into the more flexible vestibule region of the protein, the results provide valuable insights and potential strategies to overcome P-gp efflux, contributing to the advancement of drug development for both CNS disorders and cancer therapies.

Effects of MTX-23, a Novel PROTAC of Androgen Receptor Splice Variant-7 and Androgen Receptor, on CRPC Resistant to Second-Line Antiandrogen Therapy.

Although second-line antiandrogen therapy (SAT) is the standard of care in men with castration-resistant prostate cancer (CRPC), resistance inevitably occurs. One major proposed mechanism of resistance to SAT involves the emergence of androgen receptor (AR) splice variant-7, AR-V7. Recently, we developed MTX-23 using the principle of proteolysis targeting chimera (PROTAC) to target both AR-V7 and AR-full length (AR-FL). MTX-23 has been designed to simultaneously bind AR's DNA binding domain (DBD) and the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. Immunoblots demonstrated that MTX-23's degradation concentration 50% (DC50) for AR-V7 and AR-FL was 0.37 and 2 µmol/L, respectively. Further studies revealed that MTX-23 inhibited prostate cancer cellular proliferation and increased apoptosis only in androgen-responsive prostate cancer cells. The antiproliferative effect of MTX-23 was partially reversed when either AR-V7 or AR-FL was overexpressed and was completely abrogated when both were overexpressed. To assess the potential therapeutic value of MTX-23, we next generated 12 human prostate cancer cell lines that are resistant to the four FDA-approved SAT agents-abiraterone, enzalutamide, apalutamide, and darolutamide. When resistant cells were treated with MTX-23, decreased cellular proliferation and reduced tumor growth were observed both in vitro and in mice. These results collectively suggest that MTX-23 is a novel PROTAC small molecule that may be effective against SAT-resistant CRPC by degrading both AR-V7 and AR-FL.

Discovery of 4-Piperazine Isoquinoline Derivatives as Potent and Brain-Permeable Tau Prion Inhibitors with CDK8 Activity.

Tau prions feature in the brains of patients suffering from Alzheimer's disease and other tauopathies. For the development of therapeutics that target the replication of tau prions, a high-content, fluorescence-based cell assay was developed. Using this high-content phenotypic screen for nascent tau prion formation, a 4-piperazine isoquinoline compound (1) was identified as a hit with an EC50 value of 390 nM and 0.04 K p,uu. Analogs were synthesized using a hypothesis-based approach to improve potency and in vivo brain penetration resulting in compound 25 (EC50 = 15 nM; K p,uu = 0.63). We investigated the mechanism of action of this series and found that a small set of active compounds were also CDK8 inhibitors.

Selectivity in the Addition of Electron Deficient Radicals to the C2 Position of Indoles.

The addition of electron deficient radicals to the C2 position of indoles has been described in the literature as opposed to electrophilic addition at the C3 position. Density functional theory calculations were used to understand the switch in regioselectivity from C3 to C2 for indole to undergo radical additions. Electron deficient radicals have a lower barrier for reaction at C2 and a lower energy radical intermediate that benefits from benzylic radical stabilization. Trifluoromethyl radical addition has a lower energy barrier than acetonitrile radical, and the C3 addition transition state is just 0.8 kcal/mol higher than C2. This is supported by experimental observations.

Decreasing the CYP2D6 contribution to metabolism of a CK1ε inhibitor.

Our internal casein kinase 1ε lead inhibitor, compound 1 was partially cleared by the polymorphic cytochrome P450 2D6. CYP2D6 involvement in metabolism implies more extensive clinical trials. We therefore wanted to reduce the contribution to clearance by this enzyme. We utilized metabolism reports for compound 1 performed in recombinant CYP2D6 together with structure-metabolism variation in structures of closely related analogs in order to see if we could incorporate similar substitution patterns in our lead compound. In addition, we utilized a previously established docking method using a modified CYP2D6 crystal structure to see if the metabolism patterns in CYP2D6 could be reproduced to afford the metabolites in the metabolism reports as well as those for the compounds used in the structure-metabolism relationship. All three of these steps, the metabolism report, the establishment of structure-metabolism relationships and the docking, lead to compound 10 where CYP2D6 was not involved in the clearance pathways.

Amelioration of mechanism-based inactivation of CYP3A4 by a H-PGDS inhibitor.

This work describes the rational amelioration of mechanism-based inactivation (MBI) of Cytochrome P450 (CYP) 3A4 in a human hematopoietic prostaglandin D synthase (hH-PGDS) inhibitor (cpd 1). We utilized metabolism reports in order to check if patterns in the metabolism of 1 and similar compounds by CYP3A4 could be deciphered. Then we used structure based design, first modifying the CYP3A4 crystal structure (pdb code: 4NY4) by adding an oxyferryl moiety to the heme, followed by validating the modified structure to obtain the 1' and 4 position oxidation products of midazolam and then recapitulating the metabolism patterns deciphered previously for 1 and analogs. We checked if the pattern deciphered could lead to a putative reactive moiety. Finally we used the docking pose of 1 into this model of the modified CYP3A4 crystal structure to guide transformation of 1 into MBI-free H-PGDS inhibitors.

Amelioration of PXR-mediated CYP3A4 induction by mGluR2 modulators.

This work describes the rational amelioration of Cytochrome P450 4/5 (CYP3A4/5) induction through the Pregnane-X Receptor (PXR) pathway in a series of compounds that modulate the metabotropic glutamate Receptor 2 (mGluR2) via an allosteric mechanism. The compounds were initially shown to induce CYP3A4/5 via the gold-standard induction assay measured in primary human hepatocytes. This was followed up by testing the compounds in a PXR assay which correlated well with the assay in primary cells. Further, one of the compounds was crystallized with PXR (pdb code 6DUP). Analysis of this co-crystal structure, together with previously published PXR co-crystal structures, lead to modification ideas. The compounds synthesized based on these ideas were shown not to be CYP3A4/5 inducers. The mGluR2 activity of the resulting compounds was maintained.

Molecular determinants of loperamide and N-desmethyl loperamide binding in the hERG cardiac K+ channel.

Abuse of the common anti-diarrheal loperamide is associated with QT interval prolongation as well as development of the potentially fatal arrhythmia torsades de pointes. The mechanism underlying this cardiotoxicity is high affinity inhibition of the human ether-a-go-go-related gene (hERG) cardiac K+ channel. N-Desmethyl loperamide is the major metabolite of loperamide and is a close structural relative of the parent molecule. To date no information is available regarding the affinity of N-desmethyl loperamide for human cardiac ion channels. The effects of N-desmethyl loperamide on various cloned human cardiac ion channels including hERG, KvLQT1/mink and Nav1.5 were studied and compared to that of the parent. N-Desmethyl loperamide was a much weaker (7.5-fold) inhibitor of hERG compared to loperamide. However, given the higher plasma levels of the metabolite relative to the parent, it is likely that N-desmethyl loperamide can contribute, at least secondarily, to the cardiotoxicity observed with loperamide abuse. We used the recently solved cryo-EM structure of the hERG channel together with previously published inhibitors, to understand the basis of the interactions as well as the difference that a single methyl plays in the hERG channel blocking affinities of these two compounds.

Proarrhythmic mechanisms of the common anti-diarrheal medication loperamide: revelations from the opioid abuse epidemic.

Loperamide is a µ-opioid receptor agonist commonly used to treat diarrhea and often available as an over-the-counter medication. Recently, numerous reports of QRS widening accompanied by dramatic QT interval prolongation, torsades de pointe arrhythmia, and death have been reported in opioid abusers consuming large amounts of the drug to produce euphoria or prevent opiate withdrawal. The present study was undertaken to determine the mechanisms of this cardiotoxicity. Using whole-cell patch clamp electrophysiology, we tested loperamide on the cloned human cardiac sodium channel (Nav1.5) and the two main repolarizing cardiac K(+) channels cloned from the human heart: KvLQT1/minK and the human ether-a-go-go-related gene (hERG) channel. Loperamide inhibited Nav1.5 with IC50 values of 297 and 239 nM at holding potentials of -90 and -70 mV, respectively. Loperamide was weakly active on KvLQT1/minK producing 17 and 65 % inhibition at concentrations of 1 and 10 µM, respectively. Conversely, loperamide was found to be a very high affinity inhibitor of the hERG channel with an IC50 value of 89 nM at room temperature and 33 nM when measured at physiological temperature. The QRS and QT interval prolongation and the attending arrhythmias, produced by loperamide, derive from high affinity inhibition of Nav1.5 and especially hERG. Since the drug has been widely available and safely used as directed for many years, we believe that the potent inhibition loperamide possesses for cardiac ion channels has only been uncovered because of the excessive misuse of the drug as a consequence of the recent opioid abuse epidemic.

Understanding DP receptor antagonism using a CoMSIA approach.

A Comparative Molecular Similarity Indices Analysis (CoMSIA) was performed for 2,6-substituted-4-monosubstituted aminopyrimidine antagonists of prostaglandin D(2) receptor (DP). Both two-component (Q(2) = 0.63, R(2) = 0.82, SEE = 0.47 pIC(50)) and three-component (Q(2) = 0.70, R(2) = 0.91, SEE = 0.36 pIC(50)) CoMSIA models were established. Two hydrogen-bond acceptors with spatial separation of about 8Å are shown as optimal for binding. A large hydrophobic center that separates the two acceptors confers to the potency of the 2,6-substituted-4-monosubstituted aminopyrimidine. The models were used to predict IC(50) values for compounds which had functional groups different from those in the training set.

A conformationally constrained inhibitor with an enhanced potency for ß-tryptase and stability against semicarbazide-sensitive amine oxidase (SSAO).

A novel ß-tryptase inhibitor with a basic benzylamine P1 group, a piperidine-amide linker, and a substituted indole P4 group was discovered. A substitution at 4-indole position was introduced to constrain the conformational flexibility of the inhibitor to the bioactive conformation exhibited by X-ray structures so that entropic penalty was decreased. More importantly, this constrained conformation limited the accessibility of this molecule to anti-targets, especially SSAO, so that an enhanced metabolic profile was achieved.

Identification of optimum computational protocols for modeling the aryl hydrocarbon receptor (AHR) and its interaction with ligands.

The aryl hydrocarbon receptor (AHR) is one of the principal xenobiotic receptors in living organisms and is responsible for interacting with several drugs and environmental toxins, most notably tetrachlorodibenzodioxin (TCDD). Binding of diverse agonists to AHR initiates an extensive set of downstream gene expression responses and thus identifies AHR among a key set of proteins responsible for mediating interactions between living organisms and foreign molecules. While extensive biochemical investigations on the interaction of AHR with ligands have been carried out, studies comparing the abilities of specific computational algorithms in explaining the potency of known AHR ligands are lacking. In this study we use molecular dynamics simulations to identify a physically realistic conformation of the AHR that is relevant to ligand binding. We then use two sets of existing data on known AHR ligands to evaluate the performance of several docking and scoring protocols in rationalizing the potencies of these ligands. The results identify an optimum set of protocols that could prove useful in future AHR ligand discovery and design as a target or anti-target. Exploration of the details of these protocols sheds light on factors operating in modeling AHR ligand binding.

The challenges of in silico contributions to drug metabolism in lead optimization.

IMPORTANCE OF THE FIELD: The site of metabolism (SOM) predictions by CYP 3A4 are extremely important during the drug discovery process especially during the lead discovery or library design phases. With the ability to rapidly characterize metabolites from these enzymes, the challenges facing in silico contribution change during the drug optimization phase. Some of the challenges are addressed in this article. Some aspects of the SOM prediction software and methodology are discussed in this opinion article and examples of software utility in overcoming metabolic instability in drug optimization are shown. AREAS COVERED IN THIS REVIEW: SOM prediction by various approaches is discussed. Two ways of overcoming metabolic instability, blocking the metabolic softspots and rational modification of the instable molecule to avoid interaction with the CYP pocket, are discussed. The contribution plot in MetaSite and its use are discussed. WHAT THE READER WILL GAIN: The reader will gain an understanding of possible approaches to either blocking the metabolic softspot or rationally modifying the molecule using MetaSite software or docking approaches. Blocking metabolism using fluorination has risks especially introducing multifluorinated benzene rings in the molecule. TAKE HOME MESSAGE: During the lead optimization phase of drug discovery, when metabolic instability is an issue in a series, in silico approaches can be used to modify the molecule in order to decrease clearance due to metabolism, even that due to CYP3A4.

Fragment screening of inhibitors for MIF tautomerase reveals a cryptic surface binding site.

In the course of a fragment screening campaign by in silico docking followed by X-ray crystallography, a novel binding site for migration inhibitory factor (MIF) inhibitors was demonstrated. The site is formed by rotation of the side-chain of Tyr-36 to reveal a surface binding site in MIF that is hydrophobic and surrounded by aromatic side-chain residues. The crystal structures of two small inhibitors that bind to this site and of a quinolinone inhibitor, that spans the canonical deep pocket near Pro-1 and the new surface binding site, have been solved. These results suggest new opportunities for structure-based design of MIF inhibitors.

Discovery of novel inhibitors for DHODH via virtual screening and X-ray crystallographic structures.

Amino-benzoic acid derivatives 1-4 were found to be inhibitors for DHODH by virtual screening, biochemical, and X-ray crystallographic studies. X-ray structures showed that 1 and 2 bind to DHODH as predicted by virtual screening, but 3 and 4 were found to be structurally different from the corresponding compounds initially identified by virtual screening.

On the mechanism of microsomal prostaglandin E synthase type-2--a theoretical study of endoperoxide reaction with MeS(-).

The reaction pathways of deprotonation versus nucleophilic substitution involving mPGES-2 enzyme catalysis were investigated by ab initio molecular orbital theory calculations for the reaction of methylthiolate with the endoperoxide core of PGH(2) and by the combined quantum mechanical molecular mechanical methods. The calculations showed that deprotonation mechanism is energetically more favorable than the nucleophilic substitution pathway.

Discovery of covalent inhibitors for MIF tautomerase via cocrystal structures with phantom hits from virtual screening.

Biochemical and X-ray crystallographic studies confirmed that hydroxyquinoline derivatives identified by virtual screening were actually covalent inhibitors of the MIF tautomerase. Adducts were formed by N-alkylation of the Pro-1 at the catalytic site with a loss of an amino group of the inhibitor.

A crowdsourcing evaluation of the NIH chemical probes.

Between 2004 and 2008, the US National Institutes of Health Molecular Libraries and Imaging initiative pilot phase funded 10 high-throughput screening centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the Molecular Libraries and Imaging initiative output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes.

Discovery of novel 1-arylmethyl pyrrolidin-2-yl ethanol amines as calcium-sensing receptor antagonists.

A 3D quantitative structure-activity relationship study for inhibition of calcium-sensing receptor in the aryloxypropanolamine series predicted that these molecules adopt a U-shaped conformation with pi-stacking between the two aromatic rings. This hypothesis led to the discovery of novel 1-arylmethyl pyrrolidin-2-yl ethanol amines capable of antagonizing the calcium-sensing receptor with potency comparable to that of NPS-2143.

A 3D-QSAR model for CYP2D6 inhibition in the aryloxypropanolamine series.

A comparative molecular similarity index analysis (CoMSiA) has been performed for cytochrome P450 2D6 inhibition on a series of aryloxypropanolamines to determine the factors contributing to this activity. The model is in agreement with a CYP2D6 homology model constructed on the basis of the mammalian CYP2C5 crystal structure. The energy minimized conformations were generated using the systematic search methodology in Sybyl 6.7. The model not only elucidated the relationship between structure and biological activity but, more importantly, provided useful strategies to modulate CYP2D6 affinity in the aryloxypropanolamine series.