Details Matter in Structure-based Drug Design.

Successful structure-based drug design (SBDD) requires the optimization of interactions with the target protein and the minimization of ligand strain. Both factors are often modulated by small changes in the chemical structure which can lead to profound changes in the preferred conformation and interaction preferences of the ligand. We draw from examples of a Roche project targeting phosphodiesterase 10 to highlight that details matter in SBDD. Data mining in crystal structure databases can help to identify these sometimes subtle effects, but it is also a great resource to learn about molecular recognition in general and can be used as part of molecular design tools. We illustrate the use of the Cambridge Structural Database for identifying preferred structural motifs for intramolecular hydrogen bonding and of the Protein Data Bank for deriving propensities for protein-ligand interactions.

Bias and Precision in Magnetic Resonance Imaging-Based Estimates of Renal Blood Flow: Assessment by Triangulation.

BACKGROUND: Renal blood flow (RBF) can be measured with dynamic contrast enhanced-MRI (DCE-MRI) and arterial spin labeling (ASL). Unfortunately, individual estimates from both methods vary and reference-standard methods are not available. A potential solution is to include a third, arbitrating MRI method in the comparison. PURPOSE: To compare RBF estimates between ASL, DCE, and phase contrast (PC)-MRI. STUDY TYPE: Prospective. POPULATION: Twenty-five patients with type-2 diabetes (36% female) and five healthy volunteers (HV, 80% female). FIELD STRENGTH/SEQUENCES: A 3 T; gradient-echo 2D-DCE, pseudo-continuous ASL (pCASL) and cine 2D-PC. ASSESSMENT: ASL, DCE, and PC were acquired once in all patients. ASL and PC were acquired four times in each HV. RBF was estimated and split-RBF was derived as (right kidney RBF)/total RBF. Repeatability error (RE) was calculated for each HV, RE = 1.96 × SD, where SD is the standard deviation of repeat scans. STATISTICAL TESTS: Paired t-tests and one-way analysis of variance (ANOVA) were used for statistical analysis. The 95% confidence interval (CI) for difference between ASL/PC and DCE/PC was assessed using two-sample F-test for variances. Statistical significance level was P < 0.05. Influential outliers were assessed with Cook's distance (Di > 1) and results with outliers removed were presented. RESULTS: In patients, the mean RBF (mL/min/1.73m2 ) was 618 ± 62 (PC), 526 ± 91 (ASL), and 569 ± 110 (DCE). Differences between measurements were not significant (P = 0.28). Intrasubject agreement was poor for RBF with limits-of-agreement (mL/min/1.73m2 ) [-687, 772] DCE-ASL, [-482, 580] PC-DCE, and [-277, 460] PC-ASL. The difference PC-ASL was significantly smaller than PC-DCE, but this was driven by a single-DCE outlier (P = 0.31, after removing outlier). The difference in split-RBF was comparatively small. In HVs, mean RE (±95% CI; mL/min/1.73 m2 ) was significantly smaller for PC (79 ± 41) than for ASL (241 ± 85). CONCLUSIONS: ASL, DCE, and PC RBF show poor agreement in individual subjects but agree well on average. Triangulation with PC suggests that the accuracy of ASL and DCE is comparable. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

A high quality, industrial data set for binding affinity prediction: performance comparison in different early drug discovery scenarios.

We release a new, high quality data set of 1162 PDE10A inhibitors with experimentally determined binding affinities together with 77 PDE10A X-ray co-crystal structures from a Roche legacy project. This data set is used to compare the performance of different 2D- and 3D-machine learning (ML) as well as empirical scoring functions for predicting binding affinities with high throughput. We simulate use cases that are relevant in the lead optimization phase of early drug discovery. ML methods perform well at interpolation, but poorly in extrapolation scenarios-which are most relevant to a real-world application. Moreover, we find that investing into the docking workflow for binding pose generation using multi-template docking is rewarded with an improved scoring performance. A combination of 2D-ML and 3D scoring using a modified piecewise linear potential shows best overall performance, combining information on the protein environment with learning from existing SAR data.

Advanced real-time recordings of neuronal activity with tailored patch pipettes, diamond multi-electrode arrays and electrochromic voltage-sensitive dyes.

To understand the working principles of the nervous system is key to figure out its electrical activity and how this activity spreads along the neuronal network. It is therefore crucial to develop advanced techniques aimed to record in real time the electrical activity, from compartments of single neurons to populations of neurons, to understand how higher functions emerge from coordinated activity. To record from single neurons, a technique will be presented to fabricate patch pipettes able to seal on any membrane with a single glass type and whose shanks can be widened as desired. This dramatically reduces access resistance during whole-cell recording allowing fast intracellular and, if required, extracellular perfusion. To simultaneously record from many neurons, biocompatible probes will be described employing multi-electrodes made with novel technologies, based on diamond substrates. These probes also allow to synchronously record exocytosis and neuronal excitability and to stimulate neurons. Finally, to achieve even higher spatial resolution, it will be shown how voltage imaging, employing fast voltage-sensitive dyes and two-photon microscopy, is able to sample voltage oscillations in the brain spatially resolved and voltage changes in dendrites of single neurons at millisecond and micrometre resolution in awake animals.

Ligand channel in pharmacologically stabilized rhodopsin.

In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa.

Asymmetric Biocatalytic Synthesis of 1-Aryltetrahydro-ß-carbolines Enabled by "Substrate Walking".

Stereoselective catalysts for the Pictet-Spengler reaction of tryptamines and aldehydes may allow a simple and fast approach to chiral 1-substituted tetrahydro-ß-carbolines. Although biocatalysts have previously been employed for the Pictet-Spengler reaction, not a single one accepts benzaldehyde and its substituted derivatives. To address this challenge, a combination of substrate walking and transfer of beneficial mutations between different wild-type backbones was used to develop a strictosidine synthase from Rauvolfia serpentina (RsSTR) into a suitable enzyme for the asymmetric Pictet-Spengler condensation of tryptamine and benzaldehyde derivatives. The double variant RsSTR V176L/V208A accepted various ortho-, meta- and para-substituted benzaldehydes and produced the corresponding chiral 1-aryl-tetrahydro-ß-carbolines with up to 99 % enantiomeric excess.

Identification of Noncompetitive Protein-Ligand Interactions for Structural Optimization.

For efficient structure-guided drug design, it is important to have an excellent understanding of the quality of interactions between the target receptor and bound ligands. Identification and characterization of poor intermolecular contacts offers the possibility to focus design efforts directly on ligand regions with suboptimal molecular recognition. To enable a more straightforward identification of these in a structural model, we use a suitably enhanced version of our previously introduced statistical ratio of frequencies (RF) approach. This allows us to highlight protein-ligand interactions and geometries that occur much less often in the Protein Data Bank than would be expected from the exposed surface areas of the interacting atoms. We provide a comprehensive overview of such noncompetitive interactions and geometries for a set of common ligand substituents. Through retrospective case studies on congeneric series and single-point mutations for several pharmaceutical targets, we illustrate how knowledge of noncompetitive interactions could be exploited in the drug design process.

Nonuniform Fourier-decomposition MRI for ventilation- and perfusion-weighted imaging of the lung.

PURPOSE: To improve the robustness of pulmonary ventilation- and perfusion-weighted imaging with Fourier decomposition (FD) MRI in the presence of respiratory and cardiac frequency variations by replacing the standard fast Fourier transform with the more general nonuniform Fourier transform. THEORY AND METHODS: Dynamic coronal single-slice MRI of the thorax was performed in 11 patients and 5 healthy volunteers on a 1.5T whole-body scanner using a 2D ultra-fast balanced steady-state free-precession sequence with temporal resolutions of 4-9 images/s. For the proposed nonuniform Fourier-decomposition (NUFD) approach, the original signal with variable physiological frequencies that was acquired with constant sampling rate was retrospectively transformed into a signal with (ventilation or perfusion) frequency-adapted sampling rate. For that purpose, frequency tracking was performed with the synchro-squeezed wavelet transform. Ventilation- and perfusion-weighted NUFD amplitude and signal delay maps were generated and quantitatively compared with regularly sampled FD maps based on their signal-to-noise ratio (SNR). RESULTS: Volunteers and patients showed statistically significant increases of SNR in frequency-adapted NUFD results compared to regularly sampled FD results. For ventilation data, the mean SNR increased by 43.4%±25.3% and 24.4%±31.9% in volunteers and patients, respectively; for perfusion data, SNR increased by 93.0%±36.1% and 75.6%±62.8% . Two patients showed perfusion signal in pulmonary areas with NUFD that could not be imaged with FD. CONCLUSION: This study demonstrates that using nonuniform Fourier transform in combination with frequency tracking can significantly increase SNR and reduce frequency overlaps by collecting the signal intensity onto single frequency bins.

Motion-corrected multiparametric renal arterial spin labelling at 3 T: reproducibility and effect of vasodilator challenge.

OBJECTIVES: We investigated the feasibility and reproducibility of free-breathing motion-corrected multiple inversion time (multi-TI) pulsed renal arterial spin labelling (PASL), with general kinetic model parametric mapping, to simultaneously quantify renal perfusion (RBF), bolus arrival time (BAT) and tissue T1. METHODS: In a study approved by the Health Research Authority, 12 healthy volunteers (mean age, 27.6 ± 18.5 years; 5 male) gave informed consent for renal imaging at 3 T using multi-TI ASL and conventional single-TI ASL. Glyceryl trinitrate (GTN) was used as a vasodilator challenge in six subjects. Flow-sensitive alternating inversion recovery (FAIR) preparation was used with background suppression and 3D-GRASE (gradient and spin echo) read-out, and images were motion-corrected. Parametric maps of RBF, BAT and T1 were derived for both kidneys. Agreement was assessed using Pearson correlation and Bland-Altman plots. RESULTS: Inter-study correlation of whole-kidney RBF was good for both single-TI (r2 = 0.90), and multi-TI ASL (r2 = 0.92). Single-TI ASL gave a higher estimate of whole-kidney RBF compared to multi-TI ASL (mean bias, 29.3 ml/min/100 g; p <0.001). Using multi-TI ASL, the median T1 of renal cortex was shorter than that of medulla (799.6 ms vs 807.1 ms, p = 0.01), and mean whole-kidney BAT was 269.7 ± 56.5 ms. GTN had an effect on systolic blood pressure (p < 0.05) but the change in RBF was not significant. CONCLUSIONS: Free-breathing multi-TI renal ASL is feasible and reproducible at 3 T, providing simultaneous measurement of renal perfusion, haemodynamic parameters and tissue characteristics at baseline and during pharmacological challenge. KEY POINTS: • Multiple inversion time arterial spin labelling (ASL) of the kidneys is feasible and reproducible at 3 T. • This approach allows simultaneous mapping of renal perfusion, bolus arrival time and tissue T 1 during free breathing. • This technique enables repeated measures of renal haemodynamic characteristics during pharmacological challenge.

Chemotherapy response of pancreatic cancer by diffusion-weighted imaging (DWI) and intravoxel incoherent motion DWI (IVIM-DWI) in an orthotopic mouse model.

PURPOSE: To investigate the value of using diffusion-weighted imaging (DWI) and intravoxel incoherent motion DWI (IVIM-DWI) to assess the chemotherapy response of pancreatic cancer in an orthotopic mouse model. MATERIALS AND METHODS: Twenty-four BALB/C nu/nu mice in two groups (n = 12/group) with human pancreatic adenocarcinoma xenografts were dosed intravenously with saline (group 1) and gemcitabine (group 2). DWI with 3 b values (b = 50, 400 and 800 s/mm2) and IVIM-DWI with multiple b values (b = 0, 25, 50, 80, 100, 300, 500, 800 s/mm2) were performed on the day before and 1 and 10 days after the treatment. Regions of interest (ROI) were drawn and tumor ADC, Dslow, Dfast and fp values derived from the DWI and IVIM-DWI were compared between the two groups. At the day 28 after the treatment, the tumors were harvested for histologic analyses. RESULTS: The values of ADC and Dslow in the entire tumor region were significantly increased in gemcitabine-treated group in contrast to saline-untreated group at day 1 (1.88 ± 0.34 × 10-3 s/mm2 vs 1.45 ± 0.16 × 10-3 s/mm2, P = 0.028, and 1.74 ± 0.29 × 10-3 s/mm2 vs 1.34 ± 0.26 × 10-3 s/mm2, P =0.030), but Dfast and fp values were not significantly different. Immunohistochemical results showed that cell proliferation was significantly reduced (P < 0.001) and cell apoptosis (P < 0.001) significantly increased in gemcitabine group compared to saline group. MVD was not significantly different. CONCLUSION: Both ADC value and Dslow value can be used as early imaging marker to assess the early chemotherapy response of pancreatic cancer.

D3R Grand Challenge 2: blind prediction of protein-ligand poses, affinity rankings, and relative binding free energies.

The Drug Design Data Resource (D3R) ran Grand Challenge 2 (GC2) from September 2016 through February 2017. This challenge was based on a dataset of structures and affinities for the nuclear receptor farnesoid X receptor (FXR), contributed by F. Hoffmann-La Roche. The dataset contained 102 IC50 values, spanning six orders of magnitude, and 36 high-resolution co-crystal structures with representatives of four major ligand classes. Strong global participation was evident, with 49 participants submitting 262 prediction submission packages in total. Procedurally, GC2 mimicked Grand Challenge 2015 (GC2015), with a Stage 1 subchallenge testing ligand pose prediction methods and ranking and scoring methods, and a Stage 2 subchallenge testing only ligand ranking and scoring methods after the release of all blinded co-crystal structures. Two smaller curated sets of 18 and 15 ligands were developed to test alchemical free energy methods. This overview summarizes all aspects of GC2, including the dataset details, challenge procedures, and participant results. We also consider implications for progress in the field, while highlighting methodological areas that merit continued development. Similar to GC2015, the outcome of GC2 underscores the pressing need for methods development in pose prediction, particularly for ligand scaffolds not currently represented in the Protein Data Bank ( http://www.pdb.org ), and in affinity ranking and scoring of bound ligands.

Efficient Industrial Synthesis of the MDM2 Antagonist Idasanutlin via a Cu(I)-catalyzed [3+2] Asymmetric Cycloaddition.

A concise asymmetric synthesis has been developed to prepare idasanutlin, a small molecule MDM2 antagonist. Idasanutlin is currently being investigated as a potential treatment for various solid tumors and hematologic malignancies. The highly congested pyrrolidine core, containing four contiguous stereocenters, was constructed via a Cu(I)/(R)-BINAP catalyzed [3+2]-cycloaddition reaction. This optimized copper(I)-catalyzed process has been used to produce more than 1500 kg of idasanutlin. The manufacturing process will be described, highlighting the exceptionally selective and consistent cycloaddition/isomerization/hydrolysis sequence. The excellent yields, short cycle times and reduction in waste streams result in a sustainable production process with low environmental impact.

Substituent Effects in Parallel-Displaced π-π Stacking Interactions: Distance Matters.

Host-guest systems with Rebek imide type receptors and a 2,6-di(isobutyramido)pyridine ligand were employed to investigate substituent effects in parallel-displaced π-π stacking interactions. Changing the intermolecular distance between the para substituent on the aromatic platform of the receptor and the pyridine ring of the guest results in a strongly different substituent effect. With a short ethyne-1,2-diyl spacer between the Rebek imide and the aromatic platform, partial overlap of substituent and guest stabilizes the π-π stacking interactions independent of the electronic nature of the substituent (Wheeler-Houk model). When the substituent is shifted further away by using a buta-1,3-diyne-1,4-diyl spacer, direct, through-space interactions between substituent and guest are prevented. A linear correlation between logKa (Ka =association constant) and the Hammett substituent constant σpara is observed, confirming predictions by the Hunter-Sanders model experimentally.

Structural and Conformational Aspects of Equatorial and Axial Trifluoromethyl, Difluoromethyl, and Monofluoromethyl Groups.

The X-ray crystal structures of cis- and trans-1-(indol-3-yl)-4-methyl cyclohexane and its congeners with stepwise fluorination of the methyl group are reported. The trans-configured compounds adopted diequatorial conformations, whereas the cis analogues adopted regular cyclohexane chair conformations with the methyl group preferentially assuming the axial position, even in the case of the CF3 group. Surprisingly, although the axial CF3 derivative displayed distinct valence deformations in the cyclohexane moiety, the observed structural changes were relatively modest. The cis derivatives with axial mono- and difluorinated methyl groups exhibited conformational disorder in the crystals with significant population levels for the staggered conformations that had one fluorine atom in the endo position; their respective trans counterparts adopted unique conformations, but again with one fluorine atom in the endo position. Theoretical calculations for a series of cis- and trans-1,4-dimethyl cyclohexane model compounds with stepwise fluorination of one equatorial or axial methyl group reproduced the experimentally observed structural response patterns very well, reproduced the experimentally determined nonlinear correlation of the axial-equatorial energy difference with the degree of methyl fluorination in a satisfactory manner, and provided further insights into important conformational aspects of partially fluorinated methyl groups.

Addressing the Glycine-Rich Loop of Protein Kinases by a Multi-Facetted Interaction Network: Inhibition of PKA and a PKB Mimic.

Protein kinases continue to be hot targets in drug discovery research, as they are involved in many essential cellular processes and their deregulation can lead to a variety of diseases. A series of 32 enantiomerically pure inhibitors was synthesized and tested towards protein kinase A (PKA) and protein kinase B mimic PKAB3 (PKA triple mutant). The ligands bind to the hinge region, ribose pocket, and glycine-rich loop at the ATP site. Biological assays showed high potency against PKA, with Ki values in the low nanomolar range. The investigation demonstrates the significance of targeting the often neglected glycine-rich loop for gaining high binding potency. X-ray co-crystal structures revealed a multi-facetted network of ligand-loop interactions for the tightest binders, involving orthogonal dipolar contacts, sulfur and other dispersive contacts, amide-π stacking, and H-bonding to organofluorine, besides efficient water replacement. The network was analyzed in a computational approach.

Design and synthesis of selective, dual fatty acid binding protein 4 and 5 inhibitors.

Dual inhibition of fatty acid binding proteins 4 and 5 (FABP4 and FABP5) is expected to provide beneficial effects on a number of metabolic parameters such as insulin sensitivity and blood glucose levels and should protect against atherosclerosis. Starting from a FABP4 selective focused screening hit, biostructure information was used to modulate the selectivity profile in the desired way and to design potent dual FABP4/5 inhibitors with good selectivity against FABP3. With very good pharmacokinetic properties and no major safety alerts, compound 12 was identified as a suitable tool compound for further in vivo investigations.

Synthesis of annulated pyridines as inhibitors of aldosterone synthase (CYP11B2).

A series of cyclopenta[c]pyridine aldosterone synthase (AS) inhibitors were conveniently accessed using batch or continuous flow Kondrat'eva reactions. Preparation of the analogous cyclohexa[c]pyridines led to the identification of a potent and more selective AS inhibitor. The structure-activity-relationship (SAR) in this new series was rationalized using binding mode models in the crystal structure of AS.

NMDA spike/plateau potentials in dendrites of thalamocortical neurons.

Dendritic NMDA spike/plateau potentials, first discovered in cortical pyramidal neurons, provide supralinear integration of synaptic inputs on thin and distal dendrites, thereby increasing the impact of these inputs on the soma. The more specific functional role of these potentials has been difficult to clarify, partly due to the complex circuitry of cortical neurons. Thalamocortical (TC) neurons in the dorsal lateral geniculate nucleus participate in simpler circuits. They receive their primary afferent input from retina and send their output to visual cortex. Cortex, in turn, regulates this output through massive feedback to distal dendrites of the TC neurons. The TC neurons can operate in two modes related to behavioral states: burst mode prevailing during sleep, when T-type calcium bursts largely disrupt the transfer of signals from retina to cortex, and tonic mode, which provides reliable transfer of retinal signals to cortex during wakefulness. We studied dendritic potentials in TC neurons with combined two-photon calcium imaging and whole-cell recording of responses to local dendritic glutamate iontophoresis in acute brain slices from mice. We found that NMDA spike/plateaus can be elicited locally at distal dendrites of TC neurons. We suggest that these dendritic potentials have important functions in the cortical regulation of thalamocortical transmission. NMDA spike/plateaus can induce shifts in the functional mode from burst to tonic by blockade of T-type calcium conductances. Moreover, in tonic mode, they can facilitate the transfer of retinal signals to cortex by depolarization of TC neurons.

Functional analysis and transcriptional output of the Göttingen minipig genome.

BACKGROUND: In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development. RESULTS: Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies. CONCLUSIONS: Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed.

Intravoxel incoherent motion diffusion-weighted MR imaging of the liver: effect of triggering methods on regional variability and measurement repeatability of quantitative parameters.

PURPOSE: To compare the influence of triggering methods for diffusion-weighted imaging (DWI) on apparent diffusion coefficient (ADC) and intravoxel incoherent motion (IVIM) parameters in the liver, as well as regional variability and measurement repeatability. MATERIALS AND METHODS: In this institutional review board-approved prospective study, 12 healthy volunteers (six women, six men; mean age, 30 years) underwent 1.5-T DWI of the liver by using nine b values twice with free breathing (FB) without triggering (mean acquisition time ± standard deviation, 3.7 minutes ± 0), respiratory triggering (RT) (mean acquisition time, 6.8 minutes ± 1.4), and echocardiography triggering (ET) (mean acquisition time, 8.3 minutes ± 2.0) after providing written informed consent. ADC and IVIM parameters, including pure diffusion coefficient (D), perfusion fraction (f), and perfusion-related diffusion coefficient (D*), were measured by using 15 regions of interest (ROIs). Regional variability of ADC and IVIM parameters and measurement repeatability were evaluated by using the coefficient of variation (CV) across ROIs and within-subject CV, respectively. RESULTS: ET DWI (range of CV across ROIs, 6.69%-20.0%) resulted in significantly decreased regional variability of ADC, D, and f, compared with FB DWI (13.86%-35.8%) and RT DWI (15.15%-35.91%, P ≤. 049). ET DWI showed better repeatability of ADC measurement (within-subject CV range, 3.17%-4.12% for ET DWI; 4.15%-4.74% for FB DWI; and 2.33%-6.96% for RT DWI), D (4.05%-5.34% for ET DWI, 4.11%-12.51% for FB DWI, and 3.19%-16.17% for RT DWI), and f (7.6%-9.86% for ET DWI, 13.83%-16.81% for FB DWI, and 10.05%-12.10% for RT DWI), compared with FB DWI and RT DWI, with significant differences in within-subject CV for D in the left hepatic lobe compared with RT DWI (P = .023) and for f compared with FB DWI (P ≤ .032). For all three imaging techniques, D* showed the worst repeatability (within-subject CV, 57.05%-156.61%) among ADC and IVIM parameters. CONCLUSION: ET DWI is more effective for decreasing regional variability of ADC and IVIM parameters than FB DWI or RT DWI; it may improve measurement repeatability by reducing cardiac motion-induced measurement error.