Phenyl Bis-Sulfonamide Keap1-Nrf2 Protein-Protein Interaction Inhibitors with an Alternative Binding Mode.

Inhibitors of Kelch-like ECH-associated protein 1 (Keap1) increase the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) by stalling its ubiquitination and degradation. This enhances the expression of genes encoding proteins involved in drug detoxification, redox homeostasis, and mitochondrial function. Nrf2 activation offers a potential therapeutic approach for conditions including Alzheimer's and Parkinson's diseases, vascular inflammation, and chronic obstructive airway disease. Non-electrophilic Keap1-Nrf2 protein-protein interaction (PPI) inhibitors may have improved toxicity profiles and different pharmacological properties to cysteine-reactive electrophilic inhibitors. Here, we describe and characterize a series of phenyl bis-sulfonamide PPI inhibitors that bind to Keap1 at submicromolar concentrations. Structural studies reveal that the compounds bind to Keap1 in a distinct "peptidomimetic" conformation that resembles the Keap1-Nrf2 ETGE peptide complex. This is different to other small molecule Keap1-Nrf2 PPI inhibitors, including bicyclic aryl bis-sulfonamides, offering a starting point for new design approaches to Keap1 inhibitors.

Biomimetic chromatography-A novel application of the chromatographic principles.

Biomimetic chromatography is the name of the High Performance Liquid Chromatography (HPLC) methods that apply stationary phases containing proteins and phospholipids that can mimic the biological environment where drug molecules distribute. The applied mobile phases are aqueous organic with a pH of 7.4 to imitate physiological conditions that would be encountered in the human body. The calibrated retention of molecules on biomimetic stationary phases reveals a compound's affinity to proteins and phospholipids, which can be used to model the biological and environmental fate of molecules. This technology, when standardised, enables the prediction of in vivo partition and distribution behaviour of compounds and aids the selection of the best compounds for further studies to become a drug molecule. Applying biomimetic chromatographic measurements helps reduce the number of animal experiments during the drug discovery process. New biomimetic stationary phases, such as sphingomyelin and phosphatidylethanolamine, widen the application to the modelling of blood-brain barrier distribution and lung tissue binding. Recently, the measured properties have also been used to predict toxicity, such as phospholipidosis and cardiotoxicity. The aquatic toxicity of drugs and pesticides can be predicted using biomimetic chromatographic data. Biomimetic chromatographic separation methods may also be extended in the future to predict protein and receptor binding kinetics. The development of new biomimetic stationary phases and new prediction models will further accelerate the widespread application of this analytical method.

Biomimetic properties and estimated in vivo distribution of chloroquine and hydroxy-chloroquine enantiomers.

Chloroquine and hydroxy-chloroquine already established as anti-malarial and lupus drugs have recently gained renewed attention in the fight against the Covid-19 pandemic. Bio-mimetic HPLC methods have been used to measure the protein and phospholipid binding of the racemic mixtures of the drugs. The tissue binding and volume of distribution of the enantiomers have been estimated. The enantiomers can be separated using Chiralpak AGP HPLC columns. From the α-1-acid-glycoprotein (AGP) binding, the lung tissue binding can be estimated for the enantiomers. The drugs have a large volume of distribution, showed strong and stereoselective glycoprotein binding, medium-strong phospholipid-binding indicating only moderate phospholipidotic potential, hERG inhibition and promiscuous binding. The drug efficiency of the compounds was estimated to be greater than 2 % which indicates a high level of free biophase concentration relative to dose. The biomimetic properties of the compounds support the well-known tolerability of the drugs.

Prediction of hERG inhibition of drug discovery compounds using biomimetic HPLC measurements.

The major causes of failure of drug discovery compounds in clinics are the lack of efficacy and toxicity. To reduce late-stage failures in the drug discovery process, it is essential to estimate early the probability of adverse effects and potential toxicity. Cardiotoxicity is one of the most often observed problems related to a compound's inhibition of the hERG channel responsible for the potassium cation flux. Biomimetic HPLC methods can be used for the early screening of a compound's lipophilicity, protein binding and phospholipid partition. Based on the published hERG pIC50 data of 90 marketed drugs and their measured biomimetic properties, a model has been developed to predict the hERG inhibition using the measured binding of compounds to alpha-1-acid-glycoprotein (AGP) and immobilised artificial membrane (IAM). A representative test set of 16 compounds was carefully selected. The training set, involving the remaining compounds, served to establish the linear model. The mechanistic model supports the hypothesis that compounds have to traverse the cell membrane and bind to the hERG ion channel to cause the inhibition. The AGP and the hERG ion channel show structural similarity, as both bind positively charged compounds with strong shape selectivity. In contrast, a good IAM partition is a prerequisite for cell membrane traversal. For reasons of comparison, a corresponding model was derived by replacing the measured biomimetic properties with calculated physicochemical properties. The model established with the measured biomimetic binding properties proved to be superior and can explain over 70% of the variance of the hERG pIC50 values.

Revisiting the application of Immobilized Artificial Membrane (IAM) chromatography to estimate in vivo distribution properties of drug discovery compounds based on the model of marketed drugs.

Immobilized Artificial Membrane (IAM) chromatography columns have been used to model the in vivo distribution of drug discovery compounds. Regis Technologies Inc., the manufacturer, had to replace the silica support and consequently introduced a new IAM.PC.DD2 column that shows slightly different selectivity towards acidic and basic compounds. The application of the new IAM.PC.DD2 columns has been evaluated and the in vivo distribution models have been compared with the previous batches of columns. It was found that due to the improved endcapping of the silica, some of the positively charged drug molecules showed shorter retention than previously published. Therefore, the column system suitability data have been updated. However, these differences do not significantly affect the previously published models for the volume of distribution, brain tissue binding and drug efficiency. Therefore, the published models can be used with the new IAM.PC.DD2 columns.

Supreme activity of gramicidin S against resistant, persistent and biofilm cells of staphylococci and enterococci.

Three promising antibacterial peptides were studied with regard to their ability to inhibit the growth and kill the cells of clinical strains of Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. The multifunctional gramicidin S (GS) was the most potent, compared to the membranotropic temporin L (TL), being more effective than the innate-defence regulator IDR-1018 (IDR). These activities, compared across 16 strains as minimal bactericidal and minimal inhibitory concentrations (MIC), are independent of bacterial resistance pattern, phenotype variations and/or biofilm-forming potency. For S. aureus strains, complete killing is accomplished by all peptides at 5 × MIC. For E. faecalis strains, only GS exhibits a rapid bactericidal effect at 5 × MIC, while TL and IDR require higher concentrations. The biofilm-preventing activities of all peptides against the six strains with the largest biofilm biomass were compared. GS demonstrates the lowest minimal biofilm inhibiting concentrations, whereas TL and IDR are consistently less effective. In mature biofilms, only GS completely kills the cells of all studied strains. We compare the physicochemical properties, membranolytic activities, model pharmacokinetics and eukaryotic toxicities of the peptides and explain the bactericidal, antipersister and antibiofilm activities of GS by its elevated stability, pronounced cell-penetration ability and effective utilization of multiple modes of antibacterial action.

Amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is caused by selective and progressive loss of spinal, bulbar and cortical motoneurons and leads to irreversible paralysis, loss of speech, inability to swallow and respiratory malfunctions with the eventual death of the affected individual in a rapid disease course. Several suggested molecular pathways are reviewed including SOD1 gene mutation, protein nitrosylation, phosphorylation and oxidative stress, excitotoxicity, glutamate transporter deprivation, mitochondrial involvement, protein aggregation and motor neuron trophic factors. The role of insulin and its receptor in the brain is described. It is very possible that in 90% of the sporadic ALS cases, the cause of the motor neuron degeneration is different or that multiple mechanisms are involved that would need drugs with multiple mechanisms or action. Several marketed drugs have been selected for clinical trials. Only two drugs have been approved by the FDA as showing positive effect in ALS: Riluzole and Edaravone. Two other drugs that have a significant benefit in ALS are Talampanel and Tamoxifen. The results for modulation of the neurotrophic factor Insulin Growth Factor-1 (IGF1) as a potential treatment are inconclusive. Several compounds are discussed that show a positive effect in the mouse model but which have failed in clinical trials. New approaches using different modalities such as peptides, proteins and stem cells are promising. Our ability to design better drugs would be enhanced by investigating the endogenous factors in neuron death, protein aggregation and oxidative stress that would improve our understanding of the potential pathways that result in neurodegeneration.

Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.

How to identify and eliminate compounds with a risk of high clinical dose during the early phase of lead optimisation in drug discovery.

An alternative approach has been developed to estimate the clinical dose of new drug molecules at an early stage in the drug discovery process. This approach has been compared to traditional methods using the clinical dose as indicated on the drug label of 136 marketed drugs. At the early stages of drug discovery only in silico predictions or some initial in vitro screening data are normally available, typically parameters such as affinity/potency (pXC50)from isolated enzymes or receptors, measured albumin and phospholipid binding using biomimetic HPLC measurements, and in vitro clearance using P450 enzymes or liver microsomes. The combination of the biomimetic HPLC phospholipid and protein binding provides a drug efficiency max parameter described previously (HPLC DEmax), and in vitro potency makes it possible to estimate a clinical dose that would result in an efficacious steady state free concentration at the site of action. The influence of the potential discrepancies between the in vitro and a later stage in vivo DEmax, the whole blood potency, volume of distribution and clearance on the dose estimation has been investigated, using data from a GSK programme profiled during lead optimisation. It was found that drug potency had the greatest influence on estimating the clinical dose. When the estimated dose is low, the impact of other parameters such as the volume of distribution and clearance was much less significant and typically did not affect compound ranking.

Lipophilicity and biomimetic properties measured by HPLC to support drug discovery.

HPLC methods that use chromatographic retention times for gaining information about the properties of compounds for the purpose of designing drug molecules are reviewed. Properties, such as lipophilicity, protein binding, phospholipid binding, and acid/base character can be incorporated in the design of molecules with the right biological distribution and pharmacokinetic profile to become an effective drug. Standardization of various methodologies is suggested in order to obtain data suitable for inter-laboratory comparison. The published HPLC methods for lipophilicity, acid/base character, protein and phospholipid binding are critically reviewed and compared with each other using the solvation equation approach. One of the most important discussion points is how these data can be used in models and how they can influence the drug discovery process. Therefore, the published models for volume of distribution, unbound volume of distribution and drug efficiency are also discussed. The general relationships between the chemical structure and biomimetic HPLC properties are described in view of ranking and selecting putative drug molecules.

Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability.

One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and throughput of analytical techniques have enabled us to develop a high-throughput assay for the measurement of intracellular compound concentration for routine use to support lead optimization. The assay uses a RapidFire-MS based readout of compound concentration in HeLa cells following incubation of cells with test compound. The initial assay validation was performed by ultra-high performance liquid chromatography tandem mass spectrometry, and the assay was subsequently transferred to RapidFire tandem mass spectrometry. Further miniaturization and optimization were performed to streamline the process, increase sample throughput, and reduce cycle time. This optimization has delivered a semi-automated platform with the potential of production scale compound profiling up to 100 compounds per day.

Interrogating the relationship between rat in vivo tissue distribution and drug property data for >200 structurally unrelated molecules.

The ability to explain distribution patterns from drug physicochemical properties and binding characteristics has been explored for more than 200 compounds by interrogating data from quantitative whole body autoradiography studies (QWBA). These in vivo outcomes have been compared to in silico and in vitro drug property data to determine the most influential properties governing drug distribution. Consistent with current knowledge, in vivo distribution was most influenced by ionization state and lipophilicity which in turn affected phospholipid and plasma protein binding. Basic and neutral molecules were generally better distributed than acidic counterparts demonstrating weaker plasma protein and stronger phospholipid binding. The influence of phospholipid binding was particularly evident in tissues with high phospholipid content like spleen and lung. Conversely, poorer distribution of acidic drugs was associated with stronger plasma protein and weaker phospholipid binding. The distribution of a proportion of acidic drugs was enhanced, however, in tissues known to express anionic uptake transporters such as the liver and kidney. Greatest distribution was observed into melanin containing tissues of the eye, most likely due to melanin binding. Basic molecules were consistently better distributed into parts of the eye and skin containing melanin than those without. The data, therefore, suggest that drug binding to macromolecules strongly influences the distribution of total drug for a large proportion of molecules in most tissues. Reducing lipophilicity, a strategy often used in discovery to optimize pharmacokinetic properties such as absorption and clearance, also decreased the influence of nonspecific binding on drug distribution.

The Discovery of in Vivo Active Mitochondrial Branched-Chain Aminotransferase (BCATm) Inhibitors by Hybridizing Fragment and HTS Hits.

The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.

Improving the passive permeability of macrocyclic peptides: Balancing permeability with other physicochemical properties.

A number of methods to improve the passive permeability of a set of cyclic peptides have been investigated using 6- and 7-mer macrocyclic templates. In many cases the peptides were designed by molecular dynamics calculations to evaluate the methods. The aim of this study was not only to improve passive permeability, but also to balance permeability with other physicochemical properties with the goal of understanding and applying the knowledge to develop active cyclic peptides into drug candidates. Evaluation of the methods herein suggest that increasing passive permeability often occurs at the expense of solubility and lipophilicity. Computational methods can be useful when attempting to predict and design features to balance these properties, though limitations were observed.

Predictive approaches to increase absorption of compounds during lead optimisation.

INTRODUCTION: Complex physicochemical and biological processes influence the oral absorption of a drug molecule. Consideration of these processes is an important activity during the optimisation of potential candidate molecules. AREAS COVERED: The authors review the applications of physicochemical and structural requirements for intestinal absorption. Furthermore, they provide examples of how to aid the lead optimisation process through improvement of solubility and permeability. EXPERT OPINION: The physicochemical requirements for absorption are solubility and permeability. Both are influenced by lipophilicity, but in the opposite way. The size of the molecule also affects both solubility and permeability. Several models can be used to estimate oral absorption from chemical structure or from measured physicochemical properties. Thus, logD-cMR model, the 'golden triangle' model, Abraham solvation equations and absorption potential can be used as tools in the lead optimisation process. Measured values of solubility and permeability greatly improve the estimation of in vivo oral absorption of compounds. However, it is important to appreciate that predictions of oral absorption may be confounded by the involvement of active transporters in the gut which may either increase (e.g., active uptake) or decrease (e.g., efflux) the absorption of drug molecules. To evaluate the first-pass metabolism, in vitro clearance measurements using liver microsomes can be used in physiologically based models for the estimation of bioavailability. The general tools discussed in this review are based on the physicochemical property assessment of compound libraries and they help design compounds that occupy desirable property space with increased likelihood of good oral absorption.

In vitro measurement of drug efficiency index to aid early lead optimization.

The concepts of drug efficiency (D(eff) ) and Drug Efficiency Index (DEI) have been recently introduced as useful parameters to optimize the absorption, distribution, metabolism, elimination/excretion, and toxicity properties and in vivo efficacy potential of molecules during lead optimization and at pre-clinical stages. The available free drug concentration relative to dose depends on the compound's bioavailability, clearance, and the nonspecific binding to proteins and phospholipids. In this paper, we have demonstrated, using the data of over 115 known drug molecules, that the nonspecific binding can be determined in vitro very efficiently using biomimetic high-performance liquid chromatography measurements. DEI can therefore be estimated from in vitro measurements. The data show that high in vitro DEI values can be associated with lower efficacious dose. A strategy is described of how to use the DEI parameter during early lead optimization. An example is given to highlight the advantages of optimizing on DEI value rather than on potency alone. In order to facilitate the in silico compound design, correlation between in vitro DEI and in silico ligand efficiency parameters such as ligand lipophilicity efficiency has been revealed, suggesting the potential use of these efficiency-related parameters across lead optimization.

Physicochemical profile of macrolides and their comparison with small molecules.

Macrolides are stereospecific macrolactones of high molecular weights. Herein, 600 mostly semisynthetic macrolides are compared with 50,000 small non-macrolide synthetic molecules in terms of measured physicochemical properties in order to assess the drug-likeness and developability chances of macrolides. The pre-selected set of diverse macrolides is comprised mostly of derivatives of clarithromycin and azithromycin cores. Lipophilicity (CHI logD), affinity for immobilized artificial membranes (CHI IAM), human serum albumin (HSA) and α(1)-acid glycoprotein (AGP) plasma protein bindings (PPB), DMSO precipitative solubility as well as artificial membrane permeability (AMP) have been determined by high-throughput screening methods. It has been found that macrolides and small molecules have similar lipophilicity profiles, though macrolides show weaker PPB and have better solubility than small discovery molecules. However, macrolides are poorly permeable and have high affinity for immobilized artificial membranes signifying their strong interaction with biological phospholipids. In order to retain the drug-like profile, the design of novel macrolide molecules should be focused on optimisation of macrolide cores, that is macrolactone moiety with sugars and other small substituents avoiding large substituents and flexible linkers such as in conjugate derivatives.

Modeling cellular pharmacokinetics of 14- and 15-membered macrolides with physicochemical properties.

Macrolides with 14- and 15-membered ring are characterized by high and extensive tissue distribution, as well as good cellular accumulation and retention. Since macrolide structures do not fit the Lipinski rule of five, macrolide pharmacokinetic properties cannot be successfully predicted by common models based on data for small molecules. Here we describe the development of the first models for macrolide cellular pharmacokinetics. By comparison of cellular accumulation and retention in six human primary cell cultures of leukocytic and lung origin, as well as in lung carcinoma cell line NCI-H292, this cell line was found to be an adequate representative cell type for modeling macrolide cellular pharmacokinetics. Accumulation and retention in the NCI-H292 cells, as well as various physicochemical properties, were determined for a set of 48 rationally designed basic macrolide compounds. Classification models for predicting macrolide cellular accumulation and retention were developed using relatively easily determined and conceptually simple descriptors: experimentally determined physicochemical parameters ChromlogD and CHI IAM, as well as a calculated number of positively charged atoms (POS). The models were further tested and improved by addition of 37 structurally diverse macrolide molecules.

Estimating unbound volume of distribution and tissue binding by in vitro HPLC-based human serum albumin and immobilised artificial membrane-binding measurements.

The in vivo unbound volume of distribution (V(du)) can be used to estimate the free steady-state plasma concentration with a given dose of a drug administered intravenously. We have demonstrated that the calibrated HPLC retention times obtained on biomimetic stationary phases, such as immobilised human serum albumin and phosphatidyl-choline, can be used to estimate compounds' in vivo behaviour. The mechanistic models are based on the assumption that the sum of the albumin and phospholipid binding has the most significant impact on reducing compounds' free concentration both in plasma and in tissues. The model equations were obtained using the literature human volume of distribution and fraction unbound in plasma values of 135 known drug molecules and have been tested on a further 300 in-house compounds. The model can be used to design compounds with low V(du) values and high fraction unbound in tissues which will minimise the required dose to achieve the efficacious free concentration at the target organ (excluding possible active transport processes).

Application of drug efficiency index in drug discovery: a strategy towards low therapeutic dose.

INTRODUCTION: The ultimate objective of optimizing adsorption, distribution, metabolism and excretion (ADME) parameters in drug discovery is to maximize the unbound concentration at the site of action for a given dose level. This has the added benefit of minimizing the efficacious dose, reducing the potential for attrition related to drug burden and direct organ toxicity. The concept of drug efficiency was formulated as a tool to obtain a balanced profile between target affinity and ADME properties during lead optimization. AREAS COVERED: The authors discuss how it is possible to maximize the in vivo pharmacological potential addressing whether drug efficiency adds value to the decision-making process and whether it is possible to introduce a single optimization parameter, the drug efficiency index (DEI), linking target affinity and ADME properties, as a marker of in vivo efficacy. EXPERT OPINION: In the absence of a clear hypothesis-driven approach at the beginning of the program (i.e., pharmacokinetic-pharmacodynamic link), the objective to select molecules with a low therapeutic dose is still a major hurdle in drug discovery. The authors believe that a greater strategic focus on mechanistically relevant measures of the determinants of receptor occupancy would help the optimization and selection process. In this respect, the introduction of the DEI, which can be seen as a correction of target affinity by the in vivo pharmacokinetic potential, may help drug discovery to select and promote those molecules with the highest probability to interact with the biological target and with the best balance between target affinity and ADME properties.