Structural Features Affecting the Interactions and Transportability of LAT1-Targeted Phenylalanine Drug Conjugates.

L-type amino acid transporter 1 (LAT1) transfers essential amino acids across cell membranes. Owing to its predominant expression in the blood-brain barrier and tumor cells, LAT1 has been exploited for drug delivery and targeting to the central nervous system (CNS) and various cancers. Although the interactions of amino acids and their mimicking compounds with LAT1 have been extensively investigated, the specific structural features for an optimal drug scaffold have not yet been determined. Here, we evaluated a series of LAT1-targeted drug-phenylalanine conjugates (ligands) by determining their uptake rates by in vitro studies and investigating their interaction with LAT1 via induced-fit docking. Combining the experimental and computational data, we concluded that although LAT1 can accommodate various types of structures, smaller compounds are preferred. As the ligand size increased, its flexibility became more crucial in determining the compound's transportability and interactions. Compounds with linear or planar structures exhibited reduced uptake; those with rigid lipophilic structures lacked interactions and likely utilized other transport mechanisms for cellular entry. Introducing polar groups between aromatic structures enhanced interactions. Interestingly, compounds with a carbamate bond in the aromatic ring's para-position displayed very good transport efficiencies for the larger compounds. Compared to the ester bond, the corresponding amide bond had superior hydrogen bond acceptor properties and increased interactions. A reverse amide bond was less favorable than a direct amide bond for interactions with LAT1. The present information can be applied broadly to design appropriate CNS or antineoplastic drug candidates with a prodrug strategy and to discover novel LAT1 inhibitors used either as direct or adjuvant cancer therapy.

Synthesis, Structural and Behavioral Studies of Indole Derivatives D2AAK5, D2AAK6 and D2AAK7 as Serotonin 5-HT1A and 5-HT2A Receptor Ligands.

Serotonin receptors are involved in a number of physiological functions and regulate aggression, anxiety, appetite, cognition, learning, memory, mood, nausea, sleep, and thermoregulation. Here we report synthesis and detailed structural and behavioral studies of three indole derivatives: D2AAK5, D2AAK6, and D2AAK7 as serotonin 5-HT1A and 5-HT2A receptor ligands. X-ray studies revealed that the D2AAK5 compound crystallizes in centrosymmetric triclinic space group with one molecule in the asymmetric unit. The main interaction between the ligands and the receptors is the salt bridge between the protonatable nitrogen atom of the ligands and the conserved Asp (3.32) of the receptors. The complexes were stable in the molecular dynamic simulations. MD revealed that the studied ligands are relatively stable in their binding sites, with the exception of D2AAK7 in the serotonin 5-HT1A receptor. D2AAK7 exerts anxiolytic activity in the EPM test, while D2AAK5 has a beneficial effect on the memory processes in the PA test.

In Vitro Identification and In Vivo Confirmation of Inhibitors for Sweet Potato Chlorotic Stunt Virus RNA Silencing Suppressor, a Viral RNase III.

Sweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses' synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases.IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.

SARS-CoV-2-host proteome interactions for antiviral drug discovery.

Treatment options for COVID-19, caused by SARS-CoV-2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS-CoV-2-host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS-CoV-2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP-MS) and the complementary proximity-based labeling MS method (BioID-MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS-CoV-2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image-based drug screen with infectious SARS-CoV-2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein-protein interactions.

Synthesis and evaluation of 1,2,3-dithiazole inhibitors of the nucleocapsid protein of feline immunodeficiency virus (FIV) as a model for HIV infection.

We disclose a series of potent anti-viral 1,2,3-dithiazoles, accessed through a succinct synthetic approach from 4,5-dichloro-1,2,3-dithiazolium chloride (Appel's salt). A series of small libraries of compounds were screened against feline immunodeficiency virus (FIV) infected cells as a model for HIV. This approach highlighted new structure activity relationship understanding and led to the development of sub-micro molar anti-viral compounds with reduced toxicity. In addition, insight into the mechanistic progress of this system is provided via advanced QM-MM modelling. The 1,2,3-dithiazole represents a versatile scaffold with potential for further development to treat both FIV and HIV.

Identification of Key Amino Acids that Impact Organic Solute Transporter α/ß (OSTα/ß).

Organic solute transporter α/ß (OSTα/ß) is a bidirectional bile acid transporter localized on the basolateral membrane of hepatic, intestinal, and renal epithelial cells. OSTα/ß plays a critical role in intestinal bile acid reabsorption and is upregulated in hepatic diseases characterized by elevated bile acids, whereas genetic variants in SLC51A/B have been associated with clinical cholestasis. OSTα/ß also transports and is inhibited by commonly used medications. However, there is currently no high-resolution structure of OSTα/ß, and structure-function data for OSTα, the proposed substrate-binding subunit, are lacking. The present study addressed this knowledge gap and identified amino acids in OSTα that are important for bile acid transport. This was accomplished using computational modeling and site-directed mutagenesis of the OSTα subunit to generate OSTα/ß mutant cell lines. Out of the 10 OSTα/ß mutants investigated, four (S228K, T229S, Q269E, Q269K) exhibited decreased [3H]-taurocholate (TCA) uptake (ratio of geometric means relative to OSTα/ß wild type (WT) of 0.76, 0.75, 0.79, and 0.13, respectively). Three OSTα/ß mutants (S228K, Q269K, E305A) had reduced [3H]-TCA efflux % (ratio of geometric means relative to OSTα/ß WT of 0.86, 0.65, and 0.79, respectively). Additionally, several OSTα/ß mutants demonstrated altered expression and cellular localization when compared with OSTα/ß WT. In summary, we identified OSTα residues (Ser228, Thr229, Gln269, Glu305) in predicted transmembrane domains that affect expression of OSTα/ß and may influence OSTα/ß-mediated bile acid transport. These data advance our understanding of OSTα/ß structure/function and can inform future studies designed to gain further insight into OSTα/ß structure or to identify additional OSTα/ß substrates and inhibitors. SIGNIFICANCE STATEMENT: OSTα/ß is a clinically important transporter involved in enterohepatic bile acid recycling with currently no high-resolution protein structure and limited structure-function data. This study identified four OSTα amino acids (Ser228, Thr229, Gln269, Glu305) that affect expression of OSTα/ß and may influence OSTα/ß-mediated bile acid transport. These data can be utilized to inform future investigation of OSTα/ß structure and refine molecular modeling approaches to facilitate the identification of substrates and/or inhibitors of OSTα/ß.

Structural Characterization of LsrK as a Quorum Sensing Target and a Comparison between X-ray and Homology Models.

Quorum sensing is being investigated as an alternative therapeutic strategy in antibacterial drug discovery programs aimed at combatting bacterial resistance. LsrK is an autoinducer-2 kinase (belongs to the sugar kinase family), playing a key role in the phosphorylation of the autoinducer-2 (AI-2) signaling molecules involved in quorum sensing. Inhibiting LsrK could result in reduced pathogenicity by interfering with quorum sensing signaling. Previously, we have generated homology models to employ in structure-based virtual screening and successfully identified the first class of LsrK inhibitors. While conducting these studies, the crystal structure of LsrK was released, providing us with an opportunity to evaluate the reliability and quality of our models. A comparative structural analysis of the crystal structure and homology models revealed consistencies among them in the overall structural fold and binding site. Furthermore, the binding characteristics and conformational changes of LsrK have been investigated using molecular dynamics to inspect whether LsrK undergoes similar conformational changes as that of sugar kinases. These studies revealed the flexibility of the LsrK C-terminal domain (Domain II) attributing to the conformational changes in LsrK resulting in open and closed states during the phosphorylation. Further, simulations provided us with insights into the flexibility of a loop in Domain I that can influence the ligand accessibility to the LsrK binding site.

Molecular characteristics supporting l-Type amino acid transporter 1 (LAT1)-mediated translocation.

l-Type amino acid transporter 1 (LAT1) is an interesting protein due to its peculiar expression profile. It can be utilized not only as a carrier for improved or targeted drug delivery, e.g., into the brain but also as a target protein by which amino acid supply can be restricted, e.g., from the cancer cells. The recognition and binding processes of LAT1-ligands, such as amino acids and clinically used small molecules, including l-dopa, gabapentin, and melphalan, are today well-known. Binding to LAT1 is crucial, particularly when designing the LAT1-inhibitors. However, it will not guarantee effective translocation across the cell membrane via LAT1, which is a definite requirement for LAT1-substrates, such as drugs that elicit their pharmacological effects inside the cells. Therefore, in the present study, the accumulation of known LAT1-utilizing compounds into the selected LAT1-expressing cancer cells (MCF-7) was explored experimentally over a time period. The differences found among the transport efficiency and affinity of the studied compounds for LAT1 were subsequently explained by docking the ligands into the human LAT1 model (based on the recent cryo-electron microscopy structure). Thus, the findings of this study clarify the favorable structural requirements of the size, shape, and polarity of the ligands that support the translocation and effective transport across the cell membrane via LAT1. This knowledge can be applied in future drug design to attain improved or targeted drug delivery and hence, successful LAT1-utilizing drugs with increased therapeutic effects.

Docking-Based 3D-QSAR Studies for 1,3,4-oxadiazol-2-one Derivatives as FAAH Inhibitors.

This work aimed to construct 3D-QSAR CoMFA and CoMSIA models for a series of 31 FAAH inhibitors, containing the 1,3,4-oxadiazol-2-one moiety. The obtained models were characterized by good statistical parameters: CoMFA Q2 = 0.61, R2 = 0.98; CoMSIA Q2 = 0.64, R2 = 0.93. The CoMFA model field contributions were 54.1% and 45.9% for steric and electrostatic fields, respectively. In the CoMSIA model, electrostatic, steric, hydrogen bond donor, and hydrogen acceptor properties were equal to 34.6%, 23.9%, 23.4%, and 18.0%, respectively. These models were validated by applying the leave-one-out technique, the seven-element test set (CoMFA r2test-set = 0.91; CoMSIA r2test-set = 0.91), a progressive scrambling test, and external validation criteria developed by Golbraikh and Tropsha (CoMFA r20 = 0.98, k = 0.95; CoMSIA r20 = 0.98, k = 0.89). As the statistical significance of the obtained model was confirmed, the results of the CoMFA and CoMSIA field calculation were mapped onto the enzyme binding site. It gave us the opportunity to discuss the structure-activity relationship based on the ligand-enzyme interactions. In particular, examination of the electrostatic properties of the established CoMFA model revealed fields that correspond to the regions where electropositive substituents are not desired, e.g., in the neighborhood of the 1,3,4-oxadiazol-2-one moiety. This highlights the importance of heterocycle, a highly electronegative moiety in this area of each ligand. Examination of hydrogen bond donor and acceptor properties contour maps revealed several spots where the implementation of another hydrogen-bond-donating moiety will positively impact molecules' binding affinity, e.g., in the neighborhood of the 1,3,4-oxadiazol-2-one ring. On the other hand, there is a large isopleth that refers to the favorable H-bond properties close to the terminal phenoxy group of a ligand, which means that, generally speaking, H-bond acceptors are desired in this area.

Synthesis and Evaluation of Novel 1,2,6-Thiadiazinone Kinase Inhibitors as Potent Inhibitors of Solid Tumors.

A focused series of substituted 4H-1,2,6-thiadiazin-4-ones was designed and synthesized to probe the anti-cancer properties of this scaffold. Insights from previous kinase inhibitor programs were used to carefully select several different substitution patterns. Compounds were tested on bladder, prostate, pancreatic, breast, chordoma, and lung cancer cell lines with an additional skin fibroblast cell line as a toxicity control. This resulted in the identification of several low single digit micro molar compounds with promising therapeutic windows, particularly for bladder and prostate cancer. A number of key structural features of the 4H-1,2,6-thiadiazin-4-one scaffold are discussed that show promising scope for future improvement.

New Insights into 4-Anilinoquinazolines as Inhibitors of Cardiac Troponin I-Interacting Kinase (TNNi3K).

We report the synthesis of several related 4-anilinoquinazolines as inhibitors of cardiac troponin I-interacting kinase (TNNi3K). These close structural analogs of 3-((6,7-dimethoxyquinazolin-4-yl)amino)-4-(dimethylamino)-N-methylbenzenesulfonamide (GSK114) provide new understanding of structure-activity relationships between the 4-anilinoquinazoline scaffold and TNNi3K inhibition. Through a small focused library of inhibitors, we observed that the N-methylbenzenesulfonamide was driving the potency in addition to the more traditional quinazoline hinge-binding motif. We also identified a compound devoid of TNNi3K kinase activity due to the addition of a methyl group in the hinge binding region. This compound could serve as a negative control in the study of TNNi3K biology. Small molecule crystal structures of several quinazolines have been solved, supporting observations made about overall conformation and TNNi3K inhibition.

Quinazoline-Based Antivirulence Compounds Selectively Target Salmonella PhoP/PhoQ Signal Transduction System.

The rapid emergence of multidrug resistance among bacterial pathogens has become a significant challenge to human health in our century. Therefore, development of next-generation antibacterial compounds is an urgent need. Two-component signal transduction systems (TCS) are stimulus-response coupling devices that allow bacteria to sense and elaborate adaptive responses to changing environmental conditions, including the challenges that pathogenic bacteria face inside the host. The differential presence of TCS, present in bacteria but absent in the animal kingdom, makes them attractive targets in the search for new antibacterial compounds. In Salmonella enterica, the PhoP/PhoQ two-component system controls the expression of crucial phenotypes that define the ability of the pathogen to establish infection in the host. We now report the screening of 686 compounds from a GlaxoSmithKline published kinase inhibitor set in a high-throughput whole-cell assay that targets Salmonella enterica serovar Typhimurium PhoP/PhoQ. We identified a series of quinazoline compounds that showed selective and potent downregulation of PhoP/PhoQ-activated genes and define structural attributes required for their efficacy. We demonstrate that their bioactivity is due to repression of the PhoQ sensor autokinase activity mediated by interaction with its catalytic domain, acting as competitive inhibitors of ATP binding. While noncytotoxic, the hit molecules exhibit antivirulence effect by blockage of S Typhimurium intramacrophage replication. Together, these features make these quinazoline compounds stand out as exciting leads to develop a therapeutic intervention to fight salmonellosis.

Assessment of mutation probabilities of KRAS G12 missense mutants and their long-timescale dynamics by atomistic molecular simulations and Markov state modeling.

A mutated KRAS protein is frequently observed in human cancers. Traditionally, the oncogenic properties of KRAS missense mutants at position 12 (G12X) have been considered as equal. Here, by assessing the probabilities of occurrence of all KRAS G12X mutations and KRAS dynamics we show that this assumption does not hold true. Instead, our findings revealed an outstanding mutational bias. We conducted a thorough mutational analysis of KRAS G12X mutations and assessed to what extent the observed mutation frequencies follow a random distribution. Unique tissue-specific frequencies are displayed with specific mutations, especially with G12R, which cannot be explained by random probabilities. To clarify the underlying causes for the nonrandom probabilities, we conducted extensive atomistic molecular dynamics simulations (170 µs) to study the differences of G12X mutations on a molecular level. The simulations revealed an allosteric hydrophobic signaling network in KRAS, and that protein dynamics is altered among the G12X mutants and as such differs from the wild-type and is mutation-specific. The shift in long-timescale conformational dynamics was confirmed with Markov state modeling. A G12X mutation was found to modify KRAS dynamics in an allosteric way, which is especially manifested in the switch regions that are responsible for the effector protein binding. The findings provide a basis to understand better the oncogenic properties of KRAS G12X mutants and the consequences of the observed nonrandom frequencies of specific G12X mutations.

Synthesis and comparison of substituted 1,2,3-dithiazole and 1,2,3-thiaselenazole as inhibitors of the feline immunodeficiency virus (FIV) nucleocapsid protein as a model for HIV infection.

We report the first biological evaluation the 1,2,3-thiaselenazole class of compound and utilising a concise synthetic approach of sulfur extrusion, selenium insertion of the 1,2,3-dithiazoles. We created a small diverse library of compounds to contrast the two ring systems. This approach has highlighted new structure activity relationship insights and lead to the development of sub-micro molar anti-viral compounds with reduced toxicity. The 1,2,3-thiaselenazole represents a new class of potential compounds for the treatment of FIV and HIV.

Novel epidithiodiketopiperazines as anti-viral zinc ejectors of the Feline Immunodeficiency Virus (FIV) nucleocapsid protein as a model for HIV infection.

Focused libraries of multi-substituted epidithiodiketopiperazines (ETP) were prepared and evaluated for efficacy of inhibiting the nucleocapsid protein function of the Feline Immunodeficiency Virus (FIV) as a model for HIV. This activity was compared and contrasted to observed toxicity utilising an in-vitro cell culture approach. This resulted in the identification of several promising lead compounds with nanomolar potency in cells with low toxicity and a favorable therapeutic index.

In Vitro and in Silico Evaluation of Bikaverin as a Potent Inhibitor of Human Protein Kinase CK2.

Protein kinase CK2 is an emerging target for therapeutic intervention in human diseases, particularly in cancer. Inhibitors of this enzyme are currently in clinical trials, indicating the druggability of human CK2. By virtual screening of the ZINC database, we found that the natural compound bikaverin can fit well in the ATP binding site of the target enzyme CK2. By further in vitro evaluation using CK2 holoenzyme, bikaverin turned to be a potent inhibitor with an IC50 value of 1.24 µM. In this work, the cell permeability of bikaverin was determined using a Caco-2 cell permeability assay as a prerequisite for cellular evaluation and the compound turned out to be cell permeable with a Papp- value of 4.46 × 10-6 cm/s. Bikaverin was tested for its effect on cell viability using a MTT assay and cell proliferation using an EdU assay in different cancer cell lines (MCF7, A427 and A431 cells). Cell viability and cell proliferation were reduced dramatically after treatment with 10 µM bikaverin for 24 h. Additionally the IncuCyte® live-cell imaging system was applied for monitoring the cytotoxicity of bikaverin in the three tested cancer cell lines. Finally, molecular dynamic studies were performed to clarify the ligand binding mode of bikaverin at the ATP binding site of CK2 and to identify the amino acids involved.

Towards the Development of an In vivo Chemical Probe for Cyclin G Associated Kinase (GAK).

SGC-GAK-1 (1) is a potent, selective, cell-active chemical probe for cyclin G-associated kinase (GAK). However, 1 was rapidly metabolized in mouse liver microsomes by cytochrome P450-mediated oxidation, displaying rapid clearance in liver microsomes and in mice, which limited its utility in in vivo studies. Chemical modifications of 1 that improved metabolic stability, generally resulted in decreased GAK potency. The best analog in terms of GAK activity in cells was 6-bromo-N-(1H-indazol-6-yl)quinolin-4-amine (35) (IC50 = 1.4 µM), showing improved stability in liver microsomes while still maintaining a narrow spectrum activity across the kinome. As an alternative to scaffold modifications we also explored the use of the broad-spectrum cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) to decrease intrinsic clearance of aminoquinoline GAK inhibitors. Taken together, these approaches point towards the development of an in vivo chemical probe for the dark kinase GAK.

The Effects of Sequence Variation on Genome-wide NRF2 Binding--New Target Genes and Regulatory SNPs.

Transcription factor binding specificity is crucial for proper target gene regulation. Motif discovery algorithms identify the main features of the binding patterns, but the accuracy on the lower affinity sites is often poor. Nuclear factor E2-related factor 2 (NRF2) is a ubiquitous redox-activated transcription factor having a key protective role against endogenous and exogenous oxidant and electrophile stress. Herein, we decipher the effects of sequence variation on the DNA binding sequence of NRF2, in order to identify both genome-wide binding sites for NRF2 and disease-associated regulatory SNPs (rSNPs) with drastic effects on NRF2 binding. Interactions between NRF2 and DNA were studied using molecular modelling, and NRF2 chromatin immunoprecipitation-sequence datasets together with protein binding microarray measurements were utilized to study binding sequence variation in detail. The binding model thus generated was used to identify genome-wide binding sites for NRF2, and genomic binding sites with rSNPs that have strong effects on NRF2 binding and reside on active regulatory elements in human cells. As a proof of concept, miR-126-3p and -5p were identified as NRF2 target microRNAs, and a rSNP (rs113067944) residing on NRF2 target gene (Ferritin, light polypeptide, FTL) promoter was experimentally verified to decrease NRF2 binding and result in decreased transcriptional activity.

1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors.

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.

Structural review of PPARγ in complex with ligands: Cartesian- and dihedral angle principal component analyses of X-ray crystallographic data.

Two decades of research into the ligand-dependent modulation of the activity of the peroxisome proliferator-activated receptor γ (PPARγ) have demonstrated the heterogeneous modes of action of PPARγ ligands, in terms of their interaction surfaces in the ligand-binding pocket, binding stoichiometry and ability to interact with functionally important parts of the receptor, through both direct and allosteric mechanisms. These findings signal the complex mechanistic bases of the distinct biological effects of different classes of PPARγ ligands. Today, the development of PPARγ ligands focuses on partial- and non-agonists as opposed to classical agonists, due to the severe side effects observed with PPARγ classical agonists as therapeutic agents. To aid this development, we performed principal component analyses of the atomic (Cartesian) coordinates (cPCA) and dihedral angles (dPCA) of the structures of human PPARγ from X-ray crystallography, available in the public domain, seeking to reveal ligand-induced trends. In the cPCA, projections of the structures along the principal components (PCs) demonstrated a moderate correlation between cPC1 and structural parameters related to the stabilization of helix 12, which is central to the transcriptional activation by PPARγ classical agonists. Consequently, the presented cPCA mapping of the PPARγ-ligand complexes may guide in silico drug discovery programs seeking to avoid stabilization of helix 12 in their development of partial- and non-agonistic PPARγ ligands. Notably, while the dPCA could identify key regions of dihedral fluctuation in the structural ensemble, the distributions along dPC1 - 2 could not be classified according to the same parameters as the distribution along cPC1. Proteins 2017; 85:1684-1698. © 2017 Wiley Periodicals, Inc.