Plasma metabolites as mediators in immune cell-pancreatic cancer risk: insights from Mendelian randomization.

Background: Immune cells play a crucial role in the development and progression of pancreatic cancer, yet the causal relationship remains uncertain due to complex immune microenvironments and conflicting research findings. Mendelian randomization (MR), this study aims to delineate the causal relationships between immune cells and pancreatic cancer while identifying intermediary factors. Methods: The genome-wide association study (GWAS) data on immune cells, pancreatic cancer, and plasma metabolites are derived from public databases. In this investigation, inverse variance weighting (IVW) as the primary analytical approach to investigate the causal relationship between exposure and outcome. Furthermore, this study incorporates MR-Egger, simple mode, weighted median, and weighted mode as supplementary analytical approaches. To ensure the reliability of our findings, we further assessed horizontal pleiotropy and heterogeneity and evaluated the stability of MR results using the Leave-one-out method. In conclusion, this study employed mediation analysis to elucidate the potential mediating effects of plasma metabolites. Results: Our investigation revealed a causal relationship between immune cells and pancreatic cancer, highlighting the pivotal roles of CD11c+ monocytes (odds ratio, ORIVW=1.105; 95% confidence interval, 95%CI: 1.002-1.218; P=0.045), HLA DR+ CD4+ antigen-presenting cells (ORIVW=0.920; 95%CI: 0.873-0.968; P=0.001), and HLA DR+ CD8br T cells (ORIVW=1.058; 95%CI: 1.002-1.117; P=0.041) in pancreatic cancer progression. Further mediation analysis indicated that oxalate (proportion of mediation effect in total effect: -11.6%, 95% CI: -89.7%, 66.6%) and the mannose to trans-4-hydroxyproline ratio (-19.4, 95% CI: -136%, 96.8%) partially mediate the relationship between HLA DR+ CD8br T cells and pancreatic cancer in nature. In addition, our analysis indicates that adrenate (-8.39%, 95% CI: -18.3%, 1.54%) plays a partial mediating role in the association between CD11c+ monocyte and pancreatic cancer, while cortisone (-26.6%, 95% CI: 138%, -84.8%) acts as a partial mediator between HLA DR+ CD4+ AC and pancreatic cancer. Conclusion: This MR investigation provides evidence supporting the causal relationship between immune cell and pancreatic cancer, with plasma metabolites serving as mediators. Identifying immune cell phenotypes with potential causal effects on pancreatic cancer sheds light on its underlying mechanisms and suggests novel therapeutic targets.

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors.

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.

Numerical Investigation of Mechanical Performance and Micro-Structure Failure of Polymer-Fiber Reinforced Sand.

Natural sand has a loose and porous structure with low strength, and is prone to many geoengineering problems that cause huge losses. In this study, an organic polymer-polymer-fiber blend was used to improve the strength of sand. Using a series of laboratory and numerical simulation tests, researchers have investigated the microdamage behavior of an organic polymer and fiber-treated sand in various types of mechanical tests and explored the improvement mechanism. The results showed that the polymer- and fiber-treated sand enhanced the integrity and exhibited differential damage responses under different test conditions. The increase in polymer content induced uniform force transfer, leading to a wider range of particle motion and crack initiation, whereas the fibers adhered and confined the surrounding particles, inducing an arching force chain and dispersive/buckling cracking. Polymer- and fiber-treated sands increased their energy-carrying capacity and improved their energy release, which affected the damage characteristics. Organic polymers, fibers, and sand particles were wrapped around each other to form an effective interlocking structure, which enhances the integrity and mechanical properties of sand. This study provides novel ideas and methods in the polymer-fiber composite treatment of sand in the microscopic field.

An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.

Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.

Efficient Screening of Target-Specific Selected Compounds in Mixtures by 19 F NMR Binding Assay with Predicted 19 F NMR Chemical Shifts.

Ligand-based 19 F NMR screening is a highly effective and well-established hit-finding approach. The high sensitivity to protein binding makes it particularly suitable for fragment screening. Different criteria can be considered for generating fluorinated fragment libraries. One common strategy is to assemble a large, diverse, well-designed and characterized fragment library which is screened in mixtures, generated based on experimental 19 F NMR chemical shifts. Here, we introduce a complementary knowledge-based 19 F NMR screening approach, named 19 Focused screening, enabling the efficient screening of putative active molecules selected by computational hit finding methodologies, in mixtures assembled and on-the-fly deconvoluted based on predicted 19 F NMR chemical shifts. In this study, we developed a novel approach, named LEFshift, for 19 F NMR chemical shift prediction using rooted topological fluorine torsion fingerprints in combination with a random forest machine learning method. A demonstration of this approach to a real test case is reported.

Lycopene Regulates Dietary Dityrosine-Induced Mitochondrial-Lipid Homeostasis by Increasing Mitochondrial Complex Activity.

SCOPE: Dityrosine (DT), a marker of protein oxidation, is widely found in many high-protein foods. Dietary intake of DT induces myocardial oxidative stress injury and impairs energy metabolism. Lycopene is a common dietary supplement with antioxidant and mitochondrial-lipid homeostasis modulating abilities. This study aimed to examine the effects of lycopene on DT-induced disturbances in myocardial function and energy metabolism. METHODS AND RESULTS: Four-week-old C57BL/6J mice received intragastric administration of either tyrosine (420 µg kg-1 BW), DT (420 µg kg-1 BW), or lycopene at high (10 mg kg-1 BW) and low (5 mg kg-1 BW) doses for 35 days. Lycopene administration effectively reduced oxidative stress, cardiac fatty acid accumulation, and cardiac hypertrophy and improved mitochondrial performance in DT-induced mice. In vitro experiments in H9c2 cells showed that DT directly inhibited the activity of the respiratory chain complex, whereas oxidative phosphorylation and ß-oxidation gene expression is upregulated. Lycopene enhanced the activity of the complexes and inhibited ROS production caused by compensatory regulation. CONCLUSION: Lycopene improves DT-mediated myocardial energy homeostasis disorder by promoting the activity of respiratory chain complexes I and IV and alleviates the accumulation of cardiac fatty acids and myocardial hypertrophy.

Differences in energy metabolism and mitochondrial redox status account for the differences in propensity for developing obesity in rats fed on high-fat diet.

Obesity is a metabolic disease that is accompanied by oxidative stress. Mitochondrial dysfunction is closely associated with the occurrence and development of obesity. However, it is unclear if there are differences in mitochondrial redox homeostasis and energy metabolism between obesity-prone (OP) and obesity-resistant (OR) individuals and if these differences account for the different susceptibilities to developing obesity. The present study aimed to compare the regulation of energy metabolism between OP and OR rats during high-fat diet (HFD)-induced oxidative stress. Male Sprague Dawley rats were randomly divided into the control group and the HFD group. The HFD group was further divided into the OP and OR groups based on body weight gain (upper 1/3 for OP; lower 1/3 for OR) after eight weeks on HFD. Rats were sacrificed at the 8th and 20th week, and serum and organs were collected. At 8 weeks, HFD decreased mitochondrial antioxidant enzyme activity and increased the production of ROS in the OP rats, which was accompanied by unusual mitochondrial oxidative phosphorylation, reduced mitochondrial membrane potential (MMP), and decreased ATP production. When the feeding period was extended beyond the 8 weeks, the energy expenditure of the OP rats reduced further, resulting in elevated blood lipids and glucose levels and increased body weight. In contrast, the OR rats had higher mitochondrial antioxidant enzyme activity and normal redox homeostasis throughout the period, which was beneficial in energy utilization and ATP production. Thus, the increase in energy expenditure in the OR rats reduced the HFD-induced weight gain. Mitochondrial function and antioxidant defense might be involved in the different propensities for developing obesity. Consequently, the ability of OR rats to resist obesity may be attributed to their ability to maintain mitochondrial function and redox balance.

A comparison study of the influence of milk protein versus whey protein in high-protein diets on adiposity in rats.

High-protein diets are known to reduce weight and fat deposition. However, there have been only a few studies on the efficacy of different types of high-protein diets in preventing obesity. Therefore, the emphasis of this study lies in comparing the efficacy of two high-protein diets (milk protein and whey protein) in preventing obesity and exploring specific mechanisms. Eighty Sprague Dawley rats were divided into two groups and fed with milk protein concentrate (MPC) and whey protein concentrate (WPC) for 12 weeks. Each group was divided into four levels: two low-fat regimens with either low or high protein content (L-14%, L-40%) and two high-fat regimens with either low or high protein content (H-14%, H-40%). The studies we have performed showed that rats treated with MPC at the 40% protein level had significantly reduced body weight, fat weight and fat ratio gain induced by a high-fat diet, while the protein level in the WPC group had no effect on body weight or body fat in rats fed with a high-fat diet. What is more, rats fed with MPC at the H-40% energy level showed a significant decrease in plasma triglyceride, total cholesterol and low-density lipoprotein cholesterol levels and a significant increase in plasma high-density lipoprotein cholesterol levels compared with the H-14% energy level group. In contrast, in the WPC groups, increasing the protein content in high-fat diets had no significant influence on plasma lipid levels. The results of the amino acid composition of the two proteins and plasma showed that the MPC diet of 40% protein level increased the transsulfuration pathway in rats, thereby increasing the level of H2S. This research work has shown that not all types of high-protein diets can effectively prevent obesity induced by high-fat diets, as effectiveness depends on the amino acid composition of the protein.

Dietary Dityrosine Induces Mitochondrial Dysfunction by Diminished Thyroid Hormone Function in Mouse Myocardia.

Oxidized tyrosine products (OTP) have been detected in commercial foods with high protein content, such as meat and milk products. OTP intake induces tissue oxidative stress and affects the normal activity of the hypothalamic-pituitary-thyroid axis (HPT). This study aims to investigate the effects of OTP and their main product, dityrosine (Dityr), on mouse myocardial function and myocardial energy metabolism. Mice received daily intragastric administration of either tyrosine (Tyr; 420 µg/kg body weight), Dityr (420 µg/kg body weight), or OTP (1909 µg/kg body weight) for 35 days. Additionally, H9c2 cells were incubated with various concentrations of Dityr for 72 h. We found that OTP and pure Dityr induced oxidative stress in growing mice and in H9c2 cells, resulting in a redox state imbalance, myocardial injury, mitochondrial dysfunction, and energy metabolism disorder. Dityr interferes with T3 regulation of the myocardium via the PI3K/AKT/GSK3ß pathway, leading to myocardial mitochondrial damage and energy metabolism disorders. Food-borne OTP, especially Dityr, can disrupt thyroid hormone function in mouse myocardia leading to mitochondrial dysfunction, energy metabolism disorder, and oxidative stress.

An Artificial Intelligence Approach to Proactively Inspire Drug Discovery with Recommendations.

Artificial intelligence (AI) is becoming established in drug discovery. For example, many in the industry are applying machine learning approaches to target discovery or to optimize compound synthesis. While our organization is certainly applying these sorts of approaches, we propose an additional approach: using AI to augment human intelligence. We have been working on a series of recommendation systems that take advantage of our existing laboratory processes, both wet and computational, in order to provide inspiration to our chemists, suggest next steps in their work, and automate existing workflows. We will describe five such systems in various stages of deployment within the Novartis Institutes for BioMedical Research. While each of these systems addresses different stages of the discovery pipeline, all of them share three common features: a trigger that initiates the recommendation, an analysis that leverages our existing systems with AI, and the delivery of a recommendation. The goal of all of these systems is to inspire and accelerate the drug discovery process.

Structural and Biological Basis of Small Molecule Inhibition of Escherichia coli LpxD Acyltransferase Essential for Lipopolysaccharide Biosynthesis.

LpxD, acyl-ACP-dependent N-acyltransferase, is the third enzyme of lipid A biosynthesis in Gram-negative bacteria. A recent probe-based screen identified several compounds, including 6359-0284 (compound 1), that inhibit the enzymatic activity of Escherichia coli (E. coli) LpxD. Here, we use these inhibitors to chemically validate LpxD as an attractive antibacterial target. We first found that compound 1 was oxidized in solution to the more stable aromatized tetrahydro-pyrazolo-quinolinone compound 1o. From the Escherichia coli strain deficient in efflux, we isolated a mutant that was less susceptible to compound 1o and had an lpxD missense mutation (Gly268Cys), supporting the cellular on-target activity. Using surface plasma resonance, we showed direct binding to E. coli LpxD for compound 1o and other reported LpxD inhibitors in vitro. Furthermore, we determined eight cocrystal structures of E. coli LpxD/inhibitor complexes. These costructures pinpointed the 4'-phosphopantetheine binding site as the common ligand binding hotspot, where hydrogen bonds to Gly269 and/or Gly287 were important for inhibitor binding. In addition, the LpxD/compound 1o costructure rationalized the reduced activity of compound 1o in the LpxDGly268Cys mutant. Moreover, we obtained the LpxD structure in complex with a previously reported LpxA/LpxD dual targeting peptide inhibitor, RJPXD33, providing structural rationale for the unique dual targeting properties of this peptide. Given that the active site residues of LpxD are conserved in multidrug resistant Enterobacteriaceae, this work paves the way for future LpxD drug discovery efforts combating these Gram-negative pathogens.

Prediction of pKa Using Machine Learning Methods with Rooted Topological Torsion Fingerprints: Application to Aliphatic Amines.

The acid-base dissociation constant, pKa, is a key parameter to define the ionization state of a compound and directly affects its biopharmaceutical profile. In this study, we developed a novel approach for pKa prediction using rooted topological torsion fingerprints in combination with five machine learning (ML) methods: random forest, partial least squares, extreme gradient boosting, lasso regression, and support vector regression. With a large and diverse set of 14 499 experimental pKa values, pKa models were developed for aliphatic amines. The models demonstrated consistently good prediction statistics and were able to generate accurate prospective predictions as validated with an external test set of 726 pKa values (RMSE 0.45, MAE 0.33, and R2 0.84 by the top model). The factors that may affect prediction accuracy and model applicability were carefully assessed. The results demonstrated that rooted topological torsion fingerprints coupled with ML methods provide a promising approach for developing accurate pKa prediction models.

Spatial Learning and Memory Impairment in Growing Mice Induced by Major Oxidized Tyrosine Product Dityrosine.

This study focused on the effects of oxidized tyrosine products (OTPs) and major component dityrosine (DT) on the brain and behavior of growing mice. Male and female mice were treated with daily intragastric administration of either tyrosine (Tyr; 420 µg/kg body weight), DT (420 µg/kg body weight), or OTPs (1909 µg/kg body weight) for 35 days. We found that pure DT and OTPs caused redox state imbalance, elevated levels of inflammatory factors, hippocampal oxidative damage, and neurotransmitter disorders while activating the mitochondrial apoptosis pathway in the hippocampus and downregulating the genes associated with learning and memory. These events eventually led to growing mice learning and memory impairment, lagging responses, and anxiety-like behaviors. Furthermore, the male mice exhibited slightly more oxidative damage than the females. These findings imply that contemporary diets and food-processing strategies of the modern world should be modified to reduce oxidized protein intake.

Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria.

The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.

Discovery and Optimization of Phosphopantetheine Adenylyltransferase Inhibitors with Gram-Negative Antibacterial Activity.

In the preceding manuscript [ Moreau et al. 2018 , 10.1021/acs.jmedchem.7b01691 ] we described a successful fragment-based lead discovery (FBLD) strategy for discovery of bacterial phosphopantetheine adenylyltransferase inhibitors (PPAT, CoaD). Following several rounds of optimization two promising lead compounds were identified: triazolopyrimidinone 3 and 4-azabenzimidazole 4. Here we disclose our efforts to further optimize these two leads for on-target potency and Gram-negative cellular activity. Enabled by a robust X-ray crystallography system, our structure-based inhibitor design approach delivered compounds with biochemical potencies 4-5 orders of magnitude greater than their respective fragment starting points. Additional optimization was guided by observations on bacterial permeability and physicochemical properties, which ultimately led to the identification of PPAT inhibitors with cellular activity against wild-type E. coli.

Design, Synthesis, and Properties of a Potent Inhibitor of Pseudomonas aeruginosa Deacetylase LpxC.

Over the past several decades, the frequency of antibacterial resistance in hospitals, including multidrug resistance (MDR) and its association with serious infectious diseases, has increased at alarming rates. Pseudomonas aeruginosa is a leading cause of nosocomial infections, and resistance to virtually all approved antibacterial agents is emerging in this pathogen. To address the need for new agents to treat MDR P. aeruginosa, we focused on inhibiting the first committed step in the biosynthesis of lipid A, the deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by the enzyme LpxC. We approached this through the design, synthesis, and biological evaluation of novel hydroxamic acid LpxC inhibitors, exemplified by 1, where cytotoxicity against mammalian cell lines was reduced, solubility and plasma-protein binding were improved while retaining potent anti-pseudomonal activity in vitro and in vivo.

Rationally Designed PI3Kα Mutants to Mimic ATR and Their Use to Understand Binding Specificity of ATR Inhibitors.

ATR, a protein kinase in the PIKK family, plays a critical role in the cell DNA-damage response and is an attractive anticancer drug target. Several potent and selective inhibitors of ATR have been reported showing significant antitumor efficacy, with most advanced ones entering clinical trials. However, due to the absence of an experimental ATR structure, the determinants contributing to ATR inhibitors' potency and specificity are not well understood. Here we present the mutations in the ATP-binding site of PI3Kα to progressively transform the pocket to mimic that of ATR. The generated PI3Kα mutants exhibit significantly improved affinity for selective ATR inhibitors in multiple chemical classes. Furthermore, we obtained the X-ray structures of the PI3Kα mutants in complex with the ATR inhibitors. The crystal structures together with the analysis on the inhibitor affinity profile elucidate the roles of individual amino acid residues in the binding of ATR inhibitors, offering key insights for the binding mechanism and revealing the structure features important for the specificity of ATR inhibitors. The ability to obtain structural and binding data for these PI3Kα mutants, together with their ATR-like inhibitor binding profiles, makes these chimeric PI3Kα proteins valuable model systems for structure-based inhibitor design.

Upregulation of miR-21 expression is a valuable predicator of advanced clinicopathological features and poor prognosis in patients with renal cell carcinoma through the p53/p21­cyclin E2­Bax/caspase-3 signaling pathway.

In the present study, we investigated whether the upregulation of miR-21 expression is a predictor of advanced clinicopathological features and poor prognosis in patients with renal cell carcinoma (RCC). It was found that the expression of miR-21 not only had a significant effect on the clinicopathological features and survival in patients with RCC, but also exerted an important influence on the occurrence rate of RCC. In addition, miR-21 expression was lower in the human kidney HRPTEpiC cell line than that noted in RCC A-498 cells. Meanwhile, the overexpression of miR-21 not only increased cell proliferation, inhibited apoptosis and reduced caspase-3 activity in the A-498 cells, but also suppressed p53, CDKN1A p21, cyclin E2 and Bax protein expression in the A-498 cells. In contrary, the downregulation of miR-21 expression promoted the protein expression of p53, CDKN1A p21 and cyclin E2, and decreased Bax protein expression and caspase-3 activity in the A-498 cells. Our data indicate a clear correlation between miR-21 expression and clinicopathological features and poor prognosis in patients with RCC through the p53/p21-cyclin E2-Bax/caspase-3 signaling pathway.

Benefit of Retraining pKa Models Studied Using Internally Measured Data.

The ionization state of drugs influences many pharmaceutical properties such as their solubility, permeability, and biological activity. It is therefore important to understand the structure property relationship for the acid-base dissociation constant pKa during the lead optimization process to make better-informed design decisions. Computational approaches, such as implemented in MoKa, can help with this; however, they often predict with too large error especially for proprietary compounds. In this contribution, we look at how retraining helps to greatly improve prediction error. Using a longitudinal study with data measured over 15 years in a drug discovery environment, we assess the impact of model training on prediction accuracy and look at model degradation over time. Using the MoKa software, we will demonstrate that regular retraining is required to address changes in chemical space leading to model degradation over six to nine months.

Structure-Based Drug Design of Novel Potent and Selective Tetrahydropyrazolo[1,5-a]pyrazines as ATR Inhibitors.

A saturation strategy focused on improving the selectivity and physicochemical properties of ATR inhibitor HTS hit 1 led to a novel series of highly potent and selective tetrahydropyrazolo[1,5-a]pyrazines. Use of PI3Kα mutants as ATR crystal structure surrogates was instrumental in providing cocrystal structures to guide the medicinal chemistry designs. Detailed DMPK studies involving cyanide and GSH as trapping agents during microsomal incubations, in addition to deuterium-labeled compounds as mechanistic probes uncovered the molecular basis for the observed CYP3A4 TDI in the series.