Depolarizing Effects in Hydrogen Bond Energy in 310-Helices Revealed by Quantum Chemical Analysis.

Hydrogen-bond (H-bond) energies in 310-helices of short alanine peptides were systematically examined by precise DFT calculations with the negative fragmentation approach (NFA), a modified method based on the molecular tailoring approach. The contribution of each H-bond was evaluated in detail from the 310-helical conformation of total energies (whole helical model, WH3-10 model), and the results were compared with the property of H-bond in α-helix from our previous study. The H-bond energies of the WH3-10 model exhibited tendencies different from those exhibited by the α-helix in that they depended on the helical position of the relevant H-bond pair. H-bond pairs adjacent to the terminal H-bond pairs were observed to be strongly destabilized. The analysis of electronic structures indicated that structural characteristics cause the destabilization of the H-bond in 310-helices. We also found that the longer the helix length, the more stable the H-bond in the terminal pairs of the WH3-10 model, suggesting the action of H-bond cooperativity.

Increased Prevalence of Atopic Dermatitis in Children Aged 0-3 Years Highly Exposed to Parabens.

The prevalence of allergic diseases, such as bronchial asthma, atopic dermatitis, nasal allergies (pollinosis), and food allergies, has been increasing in many countries. The hygiene hypothesis was recently considered from the perspective of exposure to antimicrobial agents and preservatives, such as parabens (CAS number, 94-13-3). It currently remains unclear whether parabens, which are included in many daily consumer products such as cosmetics, shampoos, and personal care products as preservative antimicrobial agents, induce or aggravate allergies. Therefore, the aim of the present study was to examine the relationship between exposure to parabens and the prevalence of allergic diseases in Japanese children. The cross-sectional epidemiology of 236 children aged 0-3 years who underwent health examinations in Shika town in Japan assessed individual exposure to parabens using urinary concentrations of parabens. The results obtained showed that the prevalence of atopic dermatitis was significantly higher in children with high urinary concentrations of parabens than in those with low concentrations (p < 0.001). This relationship remained significant after adjustments for confounding factors, such as age, sex, Kaup's index, and passive smoking (p < 0.001). In conclusion, the present results from a population study suggested a relationship between atopic dermatitis and exposure to parabens. A longitudinal study using a larger sample number and a detailed examination of atopic dermatitis, including EASI scores and exposure to parabens, will be necessary.

Molecular Interaction Mechanism of a 14-3-3 Protein with a Phosphorylated Peptide Elucidated by Enhanced Conformational Sampling.

Enhanced conformational sampling, a genetic-algorithm-guided multidimensional virtual-system coupled molecular dynamics, can provide equilibrated conformational distributions of a receptor protein and a flexible ligand at room temperature. The distributions provide not only the most stable but also semistable complex structures and propose a ligand-receptor binding process. This method was applied to a system consisting of a receptor protein, 14-3-3ε, and a flexible peptide, phosphorylated myeloid leukemia factor 1 (pMLF1). The results present comprehensive binding pathways of pMLF1 to 14-3-3ε. We identified four thermodynamically stable clusters of MLF1 on the 14-3-3ε surface and free-energy barriers among some clusters. The most stable cluster includes two high-density spots connected by a narrow corridor. When pMLF1 passes the corridor, a salt-bridge relay (switching) related to the phosphorylated residue of pMLF1 occurs. Conformations in one high-density spot are similar to the experimentally determined complex structure. Three-dimensional distributions of residues in the intermolecular interface rationally explain the binding constant changes resulting from the alanine mutation experiment for the residues. We also performed a simulation of nonphosphorylated peptide and 14-3-3ε, which demonstrated that the complex structure was unstable, suggesting that phosphorylation of the peptide is crucially important for binding to 14-3-3ε.

Multidimensional virtual-system coupled canonical molecular dynamics to compute free-energy landscapes of peptide multimer assembly.

An enhanced-sampling method termed multidimensional virtual-system coupled canonical molecular dynamics (mD-VcMD) method is developed. In many cases, generalized-ensemble methods realizing enhanced sampling, for example, adaptive umbrella sampling, apply an effective potential, which is derived from temporarily assumed canonical distribution as a function of one or more arbitrarily defined reaction coordinates. However, it is not straightforward to estimate the appropriate canonical distribution, especially for cases applying multiple reaction coordinates. The current method, mD-VcMD, does not rely on the form of the canonical distribution. Therefore, it is practically useful to explore a high-dimensional reaction-coordinate space. In this article, formulation of mD-VcMD and its evaluation with the simple molecular models consisting of three or four alanine peptides are presented. We confirmed that mD-VcMD efficiently searched 2D and 3D reaction-coordinate spaces defined as interpeptide distances. Direct comparisons with results of long-term canonical MD simulations revealed that mD-VcMD produces correct canonical ensembles. © 2019 Wiley Periodicals, Inc.

Hydrogen bond donors and acceptors are generally depolarized in α-helices as revealed by a molecular tailoring approach.

Hydrogen-bond (H-bond) interaction energies in α-helices of short alanine peptides were systematically examined by precise density functional theory calculations, followed by a molecular tailoring approach. The contribution of each H-bond interaction in α-helices was estimated in detail from the entire conformation energies, and the results were compared with those in the minimal H-bond models, in which only H-bond donors and acceptors exist with the capping methyl groups. The former interaction energies were always significantly weaker than the latter energies, when the same geometries of the H-bond donors and acceptors were applied. The chemical origin of this phenomenon was investigated by analyzing the differences among the electronic structures of the local peptide backbones of the α-helices and those of the minimal H-bond models. Consequently, we found that the reduced H-bond energy originated from the depolarizations of both the H-bond donor and acceptor groups, due to the repulsive interactions with the neighboring polar peptide groups in the α-helix backbone. The classical force fields provide similar H-bond energies to those in the minimal H-bond models, which ignore the current depolarization effect, and thus they overestimate the actual H-bond energies in α-helices. © 2019 The Authors. Journal of Computational Chemistry published by Wiley Periodicals, Inc.

Multimodal Structural Distribution of the p53 C-Terminal Domain upon Binding to S100B via a Generalized Ensemble Method: From Disorder to Extradisorder.

Intrinsically disordered regions (IDRs) of a protein employ a flexible binding manner when recognizing a partner molecule. Moreover, it is recognized that binding of IDRs to a partner molecule is accompanied by folding, with a variety of bound conformations often being allowed in formation of the complex. In this study, we investigated a fragment of the disordered p53 C-terminal domain (CTDf) that interacts with one of its partner molecules, S100B, as a representative IDR. Although the 3D structure of CTDf in complex with S100B has been previously reported, the specific interactions remained controversial. To clarify these interactions, we performed generalized ensemble molecular dynamics (MD) simulations (virtual-system coupled multicanonical MD, termed V-McMD), which enable effective conformational sampling beyond that provided by conventional MD. These simulations generated a multimodal structural distribution for our system including CTDf and S100B, indicating that CTDf forms a variety of complex structures upon binding to S100B. We confirmed that our results are consistent with chemical shift perturbations and nuclear Overhauser effects that were observed in previous studies. Furthermore, we calculated the conformational entropy of CTDf in bound and isolated (free) states. Comparison of these CTDf entropies indicated that the disordered CTDf shows further increase in conformational diversity upon binding to S100B. Such entropy gain by binding may comprise an important feature of complex formation for IDRs.

Phosphorylation of an intrinsically disordered region of Ets1 shifts a multi-modal interaction ensemble to an auto-inhibitory state.

Multi-modal interactions are frequently observed in intrinsically disordered regions (IDRs) of proteins upon binding to their partners. In many cases, post-translational modifications in IDRs are accompanied by coupled folding and binding. From both molecular simulations and biochemical experiments with mutational studies, we show that the IDR including a Ser rich region (SRR) of the transcription factor Ets1, just before the DNA-binding core domain, undergoes multi-modal interactions when the SRR is not phosphorylated. In the phosphorylated state, the SRR forms a few specific complex structures with the Ets1 core, covering the recognition helix in the core and drastically reducing the DNA binding affinities as the auto-inhibitory state. The binding kinetics of mutated Ets1 indicates that aromatic residues in the SRR can be substituted with other hydrophobic residues for the interactions with the Ets1 core.

Accurate Prediction of Complex Structure and Affinity for a Flexible Protein Receptor and Its Inhibitor.

In order to predict the accurate binding configuration as well as the binding affinity for a flexible protein receptor and its inhibitor drug, enhanced sampling with multicanonical molecular dynamics (McMD) simulation and thermodynamic integration (TI) were combined as a general drug docking method. CDK2, cyclin-dependent kinase 2, is involved in the cell cycle regulation. Malfunctions in CDK2 can cause tumorigenesis, and thus it is a potential drug target. Here, we performed a long McMD simulation for docking the inhibitor CS3 to CDK2 starting from the unbound structure. Subsequently, a potential binding/unbinding pathway was given from the multicanonical ensemble, and the binding free energy was readily computed by TI along the pathway. Using this combination, the correct binding configuration of CS3 to CDK2 was obtained, and its affinity coincided well with the experimental value.

Molecular mechanisms of cooperative binding of transcription factors Runx1-CBFß-Ets1 on the TCRα gene enhancer.

Ets1 is an essential transcription factor (TF) for several important physiological processes, including cell proliferation and differentiation. Its recognition of the enhancer region of the TCRα gene is enhanced by the cooperative binding of the Runx1-CBFß heterodimer, with the cancelation of phosphorylation-dependent autoinhibition. The detailed mechanism of this interesting cooperativity between Ets1 and the Runx1-CBFß heterodimer is still largely unclear. Here, we investigated the molecular mechanisms of this cooperativity, by using molecular dynamics simulations. Consequently, we detected high flexibility of the loop region between the HI2 and H1 helices of Ets1. Upon Runx1-CBFß heterodimer binding, this loop transiently adopts various sub-stable conformations in its interactions with the DNA. In addition, a network analysis suggested an allosteric pathway in the molecular assembly and identified some key residues that coincide with previous experimental studies. Our simulations suggest that the cooperative binding of Ets1 and the Runx1-CBFß heterodimer alters the DNA conformation and induces sub-stable conformations of the HI2-H1 loop of Ets1. This phenomenon increases the flexibility of the regulatory module, including the HI2 helix, and destabilizes the inhibitory form of this module. Thus, we hypothesize that this effect facilitates Ets1-DNA binding and prevents the phosphorylation-dependent DNA binding autoinhibition.

Variation of free-energy landscape of the p53 C-terminal domain induced by acetylation: Enhanced conformational sampling.

The C-terminal domain (CTD) of tumor suppressor protein p53 is an intrinsically disordered region that binds to various partner proteins, where lysine of CTD is acetylated/nonacetylated and histidine neutralized/non-neutralized. Because of the flexibility of the unbound CTD, a free-energy landscape (FEL) is a useful quantity for determining its statistical properties. We conducted enhanced conformational sampling of CTD in the unbound state via virtual system coupled multicanonical molecular dynamics, in which the lysine was acetylated or nonacetylated and histidine was charged or neutralized. The fragments were expressed by an all-atom model and were immersed in an explicit solvent. The acetylation and charge-neutralization varied FEL greatly, which might be convenient to exert a hub property. The acetylation slightly enhanced alpha-helix structures that are more compact than sheet/loop conformations. The charge-neutralization produced hairpins. Additionally, circular dichroism experiments confirmed the computational results. We propose possible binding mechanisms of CTD to partners by investigating FEL. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Elastic properties of dynein motor domain obtained from all-atom molecular dynamics simulations.

Dyneins are large microtubule motor proteins that convert ATP energy to mechanical power. High-resolution crystal structures of ADP-bound cytoplasmic dynein have revealed the organization of the motor domain, comprising the AAA(+) ring, the linker, the stalk/strut and the C sequence. Recently, the ADP.vanadate-bound structure, which is similar to the ATP hydrolysis transition state, revealed how the structure of dynein changes upon ATP binding. Although both the ADP- and ATP-bound state structures have been resolved, the dynamic properties at the atomic level remain unclear. In this work, we built two models named 'the ADP model' and 'the ATP model', where ADP and ATP are bound to AAA1 in the AAA(+) ring, respectively, to observe the initial procedure of the structural change from the unprimed to the primed state. We performed 200-ns molecular dynamics simulations for both models and compared their structures and dynamics. The motions of the stalk, consisting of a long coiled coil with a microtubule-binding domain, significantly differed between the two models. The elastic properties of the stalk were analyzed and compared with the experimental results.

Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro. We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF.

A novel approach of dynamic cross correlation analysis on molecular dynamics simulations and its application to Ets1 dimer-DNA complex.

The dynamic cross correlation (DCC) analysis is a popular method for analyzing the trajectories of molecular dynamics (MD) simulations. However, it is difficult to detect correlative motions that appear transiently in only a part of the trajectory, such as atomic contacts between the side-chains of amino acids, which may rapidly flip. In order to capture these multi-modal behaviors of atoms, which often play essential roles, particularly at the interfaces of macromolecules, we have developed the "multi-modal DCC (mDCC)" analysis. The mDCC is an extension of the DCC and it takes advantage of a Bayesian-based pattern recognition technique. We performed MD simulations for molecular systems modeled from the (Ets1)2-DNA complex and analyzed their results with the mDCC method. Ets1 is an essential transcription factor for a variety of physiological processes, such as immunity and cancer development. Although many structural and biochemical studies have so far been performed, its DNA binding properties are still not well characterized. In particular, it is not straightforward to understand the molecular mechanisms how the cooperative binding of two Ets1 molecules facilitates their recognition of Stromelysin-1 gene regulatory elements. A correlation network was constructed among the essential atomic contacts, and the two major pathways by which the two Ets1 molecules communicate were identified. One is a pathway via direct protein-protein interactions and the other is that via the bound DNA intervening two recognition helices. These two pathways intersected at the particular cytosine bases (C110/C11), interacting with the H1, H2, and H3 helices. Furthermore, the mDCC analysis showed that both pathways included the transient interactions at their intermolecular interfaces of Tyr396-C11 and Ala327-Asn380 in multi-modal motions of the amino acid side chains and the nucleotide backbone. Thus, the current mDCC approach is a powerful tool to reveal these complicated behaviors and scrutinize intermolecular communications in a molecular system.

Theoretical investigation on nearsightedness of finite model and molecular systems based on linear response function analysis.

We examined nearsightedness of electronic matter (NEM) of finite systems on the basis of linear response function (LRF). From the computational results of a square-well model system, the behavior of responses obviously depends on the number of electrons (N): as N increases, LRF, δρ(r)/δv(r'), decays rapidly for the distance, |r-r'|. This exemplifies that the principle suggested by Kohn and Prodan holds even for finite systems: the cause of NEM is destructive interference among electron density amplitudes. In addition, we examined double-well model systems, which have low-lying degenerate levels. In this case, there are two types of LRF: the cases of the half-filled and of full-filled in low-lying degenerate levels. The response for the former is delocalized, while that of the later is localized. These behaviors of model systems are discussed in relation to the molecular systems' counterparts, H2, He22+, and He2 systems. We also see that NEM holds for the dissociated limit of H2, of which the mechanism is similar to that of the insulating state of solids as suggested by Kohn. We also examined LRF of alanine tripeptide system as well as butane and butadiene molecules, showing that NEM of the polypeptide system is caused by sp3 junctions at Cα atoms that prevent propagation of amplitudes of LRF, which is critically different from that of NEM for finite and infinite homogeneous systems.

Structure of the entire stalk region of the Dynein motor domain.

Dyneins are large microtubule-based motor complexes that power a range of cellular processes including the transport of organelles, as well as the beating of cilia and flagella. The motor domain is located within the dynein heavy chain and comprises an N-terminal mechanical linker element, a central ring of six AAA+ modules of which four bind or hydrolyze ATP, and a long stalk extending from the AAA+ring with a microtubule-binding domain (MTBD) at its tip. A crucial mechanism underlying the motile activity of cytoskeletal motor proteins is precise coupling between the ATPase and track-binding activities. In dynein, a stalk region consisting of a long (~15nm) antiparallel coiled coil separates these two activities, which must facilitate communication between them. This communication is mediated by a small degree of helix sliding in the coiled coil. However, no high-resolution structure is available of the entire stalk region including the MTBD. Here, we have reported the structure of the entire stalk region of mouse cytoplasmic dynein in a weak microtubule-binding state, which was determined using X-ray crystallography, and have compared it with the dynein motor domain from Dictyostelium discoideum in a strong microtubule-binding state and with a mouse MTBD with its distal portion of the coiled coil fused to seryl-tRNA synthetase from Thermus thermophilus. Our results strongly support the helix-sliding model based on the complete structure of the dynein stalk with a different form of coiled-coil packing. We also propose a plausible mechanism of helix sliding together with further analysis using molecular dynamics simulations. Our results present the importance of conserved proline residues for an elastic motion of stalk coiled coil and imply the manner of change between high-affinity state and low-affinity state of MTBD.

Understanding the role of epigenomic, genomic and genetic alterations in the development of endometriosis (review).

Endometriosis is a complex disease influenced by genetic, epigenetic and environmental factors. The aim of the present study was to describe genomic instability, genetic polymorphisms and their haplotype, epigenetic alterations associated with predisposition to endometriosis, and the key factors associated with endometriosis-related ovarian neoplasms. Focus has been given on the developing paradigm that epigenetic alterations or genetic mutations in endometriosis may start in utero or in adolescent and young adults. A search was conducted between 1966 and 2010 through the English language literature (online Medline PubMed database) using the keywords endometriosis combined with epigenetic, genetic and environment. Genetic/epigenetic alterations include single­nucleotide polymorphisms (SNPs), copy number variation, loss of heterozygosity (LOH), and promoter methylation. Several genes with genetic polymorphisms analyzed in the present study tended to overlap previously reported endometriosis susceptibility genes. Retrograde menstruation leads to iron overload, which facilitates the accumulation of somatic mutations through Fenton reaction-mediated oxidative stress. The epigenetic disruption of gene expression plays an important role in the development of endometriosis through interaction with environmental changes. There seems to be at least three spatiotemporally distinct phases of the development of endometriosis: the initial phase of genetic background inherited from parents; followed by epigenetic modifications in the female offspring; and iron overload, which is subject to dynamic modulation later in life. In conclusion, the marked regulation of endometriosis susceptibility genes may stem from a mechanism responsible for epigenetic and genetic mutations based on the microenvironmental changes.

LigandBox: A database for 3D structures of chemical compounds.

A database for the 3D structures of available compounds is essential for the virtual screening by molecular docking. We have developed the LigandBox database (http://ligandbox.protein.osaka-u.ac.jp/ligandbox/) containing four million available compounds, collected from the catalogues of 37 commercial suppliers, and approved drugs and biochemical compounds taken from KEGG_DRUG, KEGG_COMPOUND and PDB databases. Each chemical compound in the database has several 3D conformers with hydrogen atoms and atomic charges, which are ready to be docked into receptors using docking programs. The 3D conformations were generated using our molecular simulation program package, myPresto. Various physical properties, such as aqueous solubility (LogS) and carcinogenicity have also been calculated to characterize the ADME-Tox properties of the compounds. The Web database provides two services for compound searches: a property/chemical ID search and a chemical structure search. The chemical structure search is performed by a descriptor search and a maximum common substructure (MCS) search combination, using our program kcombu. By specifying a query chemical structure, users can find similar compounds among the millions of compounds in the database within a few minutes. Our database is expected to assist a wide range of researchers, in the fields of medical science, chemical biology, and biochemistry, who are seeking to discover active chemical compounds by the virtual screening.

Comparison of Neoadjuvant Intraarterial Chemotherapy Versus Concurrent Chemoradiotherapy in Patients With Stage IIIB Uterine Cervical Cancer.

BACKGROUND: The purpose of this study was to compare the long-term survival of patients with stage IIIB squamous cell carcinoma of the cervix treated with neoadjuvant intraarterial chemotherapy (IA-NAC) versus those treated with concurrent chemoradiotherapy (CCRT). METHODS: We retrospectively reviewed the clinical records of 38 patients with stage IIIB squamous cell carcinoma of the cervix admitted between January 1994 and December 1999 who received IA-NAC followed by abdominal radical hysterectomy (ARH) or radiotherapy (RT). IA-NAC consisted of bilateral infusion via the internal iliac artery of cisplatin, bleomycin and pirarubicin for 2-3 courses. A historical control group of 64 patients who underwent primary CCRT from January 2000 to September 2007 was used for comparison. RESULTS: In the IA-NAC group, 12 patients (31.6%) with operable tumors underwent ARH, and the remaining 26 patients (68.4%) received RT. The response rates were 86.8% (12 complete response + 21 partial response) for IA-NAC and 98.4% (26 complete response + 37 partial response) for CCRT (P = 0.077), respectively. The 5-year overall survival and disease-free survival rates were 62.4 and 44.5% for IA-NAC and 51.1 and 46.9% for CCRT (P = 0.247 and 0.776), respectively. The 5-year overall survival and disease-free survival rates were 75.0 and 58.3% for the patients receiving IA-NAC followed by ARH, and 55.3 and 37.6% for the patients receiving IA-NAC followed by RT (P = 0.368 and 0.262), respectively. CONCLUSIONS: In the present study, IA-NAC followed by ARH or RT and primary CCRT showed similar survival rates for stage IIIB squamous cell carcinoma of the cervix.

Computer-aided antibody design.

Recent clinical trials using antibodies with low toxicity and high efficiency have raised expectations for the development of next-generation protein therapeutics. However, the process of obtaining therapeutic antibodies remains time consuming and empirical. This review summarizes recent progresses in the field of computer-aided antibody development mainly focusing on antibody modeling, which is divided essentially into two parts: (i) modeling the antigen-binding site, also called the complementarity determining regions (CDRs), and (ii) predicting the relative orientations of the variable heavy (V(H)) and light (V(L)) chains. Among the six CDR loops, the greatest challenge is predicting the conformation of CDR-H3, which is the most important in antigen recognition. Further computational methods could be used in drug development based on crystal structures or homology models, including antibody-antigen dockings and energy calculations with approximate potential functions. These methods should guide experimental studies to improve the affinities and physicochemical properties of antibodies. Finally, several successful examples of in silico structure-based antibody designs are reviewed. We also briefly review structure-based antigen or immunogen design, with application to rational vaccine development.

Selection of in silico drug screening results for G-protein-coupled receptors by using universal active probes.

We developed a new protocol for in silico drug screening for G-protein-coupled receptors (GPCRs) using a set of "universal active probes" (UAPs) with an ensemble docking procedure. UAPs are drug-like compounds, which are actual active compounds of a variety of known proteins. The current targets were nine human GPCRs whose three-dimensional (3D) structures are unknown, plus three GPCRs, namely ß(2)-adrenergic receptor (ADRB2), A(2A) adenosine receptor (A(2A)), and dopamine D3 receptor (D(3)), whose 3D structures are known. Homology-based models of the GPCRs were constructed based on the crystal structures with careful sequence inspection. After subsequent molecular dynamics (MD) simulation taking into account the explicit lipid membrane molecules with periodic boundary conditions, we obtained multiple model structures of the GPCRs. For each target structure, docking-screening calculations were carried out via the ensemble docking procedure, using both true active compounds of the target proteins and the UAPs with the multiple target screening (MTS) method. Consequently, the multiple model structures showed various screening results with both poor and high hit ratios, the latter of which could be identified as promising for use in in silico screening to find candidate compounds to interact with the proteins. We found that the hit ratio of true active compounds showed a positive correlation to that of the UAPs. Thus, we could retrieve appropriate target structures from the GPCR models by applying the UAPs, even if no active compound is known for the GPCRs. Namely, the screening result that showed a high hit ratio for the UAPs could be used to identify actual hit compounds for the target GPCRs.