Potential Pocket Research Articles

Identification of Novel Cyclin A2 Binding Site and Nanomolar Inhibitors

It has been shown that dysfunction in tight regulation of DNA repair pathways can induce various syndromes, increase the risk of cancer, and trigger neurological defect. However, there are cases where inhibition of DNA repair process is necessary. For example, after treating cancer cells with radiotherapy, it is essential to inhibit DNA repair function of the radiation-induced cancer cells for successful apoptosis. Therefore, the key is to target a protein that is responsible for controlling the rapid and proper resolution of DNA strand breaks. The recent discovery of CCNA2 (cyclin A2), the main mammalian S-phase cyclin, as a regulator of neuronal genomic stability and DNA repair positioned cyclin A2 as a potential therapeutic target for DNA repair dysfunction and as a radiation sensitizer. Thus, identifying small molecule regulators of cyclin A2 activity carries significant potential to regulate diverse cellular processes in both ageing/neurodegeneration and in cancer. No functional modulators of cyclin A2 are known to date. Here, we identified a potential allosteric cyclin A2 ligand binding pocket based on high-resolution structural data. Molecular dynamics simulations were used to generate diverse binding pocket conformations for application of the relaxed complex scheme. We then used structure-based virtual screening to find potential cyclin A2 inhibitors. Based on a consensus score of docked poses from Glide and AutoDock Vina, we identified about 60 promising hit compounds, where all PAINS scaffolds were removed from consideration. A biochemical luminescence assay of cyclin A2 function was used for experimental verification and identified three nanomolar inhibitors of cyclin A2. These novel cyclin A2 inhibitors are the first reported cyclin A2 inhibitors that do not target the cyclin A2 - CDK 2 protein-protein interface.

Formation of Carbon-enhanced Metal-poor Stars As a Consequence of Inhomogeneous Metal Mixing

Abstract We present a novel scenario for the formation of carbon-enhanced metal-poor (CEMP) stars. Carbon enhancement at low stellar metallicities is usually considered a consequence of faint or other exotic supernovae. An analytical estimate of cooling times in low-metallicity gas demonstrates a natural bias, which favors the formation of CEMP stars as a consequence of inhomogeneous metal mixing: carbon-rich gas has a shorter cooling time and can form stars prior to a potential nearby pocket of carbon-normal gas, in which star formation is then suppressed due to energetic photons from the carbon-enhanced protostars. We demonstrate that this scenario provides a natural formation mechanism for CEMP stars from carbon-normal supernovae, if inhomogeneous metal mixing provides carbonicity differences of at least one order of magnitude separated by >10 pc. In our fiducial (optimistic) model, 8% (83%) of observed CEMP-no stars ([Ba/Fe] < 0) can be explained by this formation channel. This new scenario may change our understanding of the first supernovae and thereby our concept of the first stars. Future 3D simulations are required to assess the likelihood of this mechanism to occur in typical high-redshift galaxies.

Preference for Glucose over Inositol Headgroup during Lysolipid Activation of G Protein-Coupled Receptor 55.

G protein-coupled receptor 55 (GPR55) is highly expressed in brain and peripheral nervous system. Originally deorphanized as a cannabinoid receptor, recently GPR55 has been described as a lysophospholipid-responsive receptor, specifically toward lysophosphatidylinositol and lysophosphatidyl-β-d-glucoside (LysoPtdGlc). To characterize lysolipid-GPR55 interaction, synthetic access to LysoPtdGlc and selected analogues was established utilizing a phosphorus(III)-based chemical approach. The biological activity of each synthetic lipid was assessed using a GPR55-dependent chemotropism assay in primary sensory neurons. Combined with molecular dynamics simulations the potential ligand entry port and binding pocket specifics are discussed. These results highlight the preference for gluco- over inositol- and galacto-configured headgroups.

Discovery of small molecule inhibitors of adenovirus by disrupting E3-19K/HLA-A2 interactions

The binding of the adenovirus (Ad) protein E3-19K with the human leukocyte antigen (HLA) plays an important role in Ad infections, which is the causative agent of a series of gastrointestinal, respiratory and ocular diseases. The objective of this research is to evaluate the essential interactions between E3-19K and HLA-A2 using the X-ray crystal structure of the E3-19K/HLA-A2 complex, and to identify small molecules that could potentially disrupt their binding. Computational methods, including molecular dynamic simulations, MM/GBSA calculations, and computational solvent mapping, were implemented to determine potential binding site(s) for small molecules. The previous experimentally determined hot spot residues, Q54 and E177 in HLA-A2, were also predicted to be the dominant residues for binding to E3-19K by our theoretical calculations. Several other residues were also found to play pivotal roles for the binding of E3-19K with HLA-A2. Residues adjacent to E177, including Q54 and several other residues theoretically predicted to be crucial in HLA-A2 were selected as a potential binding pocket to perform virtual screening with 1200 compounds from the Prestwick library. Seven hits were validated by surface plasmon resonance (SPR) as binders to HLA-A2 as a first step in identifying molecules that can perturb its association with the Ad E3-19K protein.

Catalytic Efficiency of Basidiomycete Laccases: Redox Potential versus Substrate-Binding Pocket Structure

Laccases are copper-containing oxidases that catalyze a one-electron abstraction from various phenolic and non-phenolic compounds with concomitant reduction of molecular oxygen to water. It is well-known that laccases from various sources have different substrate specificities, but it is not completely clear what exactly provides these differences. The purpose of this work was to study the features of the substrate specificity of four laccases from basidiomycete fungi Trametes hirsuta, Coriolopsis caperata, Antrodiella faginea, and Steccherinum murashkinskyi, which have different redox potentials of the T1 copper center and a different structure of substrate-binding pockets. Enzyme activity toward 20 monophenolic substances and 4 phenolic dyes was measured spectrophotometrically. The kinetic parameters of oxidation of four lignans and lignan-like substrates were determined by monitoring of the oxygen consumption. For the oxidation of the high redox potential (>700 mV) monophenolic substrates and almost all large substrates, such as phenolic dyes and lignans, the redox potential difference between the enzyme and the substrate (ΔE) played the defining role. For the low redox potential monophenolic substrates, ΔE did not directly influence the laccase activity. Also, in the special cases, the structure of the large substrates, such as dyes and lignans, as well as some structural features of the laccases (flexibility of the substrate-binding pocket loops and some amino acid residues in the key positions) affected the resulting catalytic efficiency.

Rational Engineering of Multiple Active Sites in an Ester Hydrolase.

Effects of altering the properties of an active site in an enzymatic homogeneous catalyst have been extensively reported. However, the possibility of increasing the number of such sites, as commonly done in heterogeneous catalytic materials, remains unexplored, particularly because those have to accommodate appropriate residues in specific configurations. This possibility was investigated by using a serine ester hydrolase as the target enzyme. By using the Protein Energy Landscape Exploration software, which maps ligand diffusion and binding, we found a potential binding pocket capable of holding an extra catalytic triad and oxyanion hole contacts. By introducing two mutations, this binding pocket became a catalytic site. Its substrate specificity, substrate preference, and catalytic activity were different from those of the native site of the wild type ester hydrolase and other hydrolases, due to the differences in the active site architecture. Converting the binding pocket into an extra catalytic active site was proven to be a successful approach to create a serine ester hydrolase with two functional reactive groups. Our results illustrate the accuracy and predictive nature of modern modeling techniques, opening novel catalytic opportunities coming from the presence of different catalytic environments in single enzymes.

Crystal structures of the extracellular domains of the CrRLK1L receptor-like kinases ANXUR1 and ANXUR2.

Catharanthus roseus Receptor-Like Kinase 1-like (CrRLK1L) proteins contain two tandem malectin-like modules in their extracellular domains (ECDs) and function in diverse signaling pathways in plants. Malectin is a carbohydrate-binding protein in animals and recognizes a number of diglucosides; however, it remains unclear how the two malectin-like domains in the CrRLK1L proteins sense the ligand molecule. In this study, we reveal the crystal structures of the ECDs of ANXUR1 and ANXUR2, two CrRLK1L members in Arabidopsis thaliana that have critical functions in controlling pollen tube rupture during the fertilization process. We show that the two malectin-like domains in these proteins pack together to form a rigid architecture. Unlike animal malectin, these malectin-like domains lack residues involved in binding to the diglucosides, suggesting that they have a distinct ligand-binding mechanism. A cleft is observed between the two malectin-like domains, which might function as a potential ligand-binding pocket.

A potential substrate binding pocket of BdcA plays a critical role in NADPH recognition and biofilm dispersal

Biofilm dispersal is characterized by the cell detachment from biofilms and expected to provide novel “anti-biofilm” approaches of prevention and treatment of biofilms in clinical and industrial settings. The E.coli protein BdcA has been identified as a biofilm dispersal factor and designed to be an important component in engineered applications to control biofilm formation. It belongs to short-chain dehydrogenase/reductase (SDR) family with the specific affinity to NADPH. Here, we show the structure of BdcA in complex with NADPH and confirm that NADPH binding is requisite for BdcA facilitating cell motility and increasing biofilm dispersal. Especially, we observe a potential substrate binding pocket surrounded by hydrophobic residues upon NADPH binding and present evidences that this pocket is essential for BdcA binding NADPH and exerting its biological functions. Our study provides the clues for illuminating the molecular mechanism of BdcA regulating biofilm dispersal and better utilizing BdcA to eliminate the biofilms.

Total-Ionizing Dose Effects on Charge Transfer Efficiency and Image Lag in Pinned Photodiode CMOS Image Sensors

The total-ionizing dose (TID) effects on image lag in pinned photodiode CMOS image sensors are investigated thanks to various device variants in order to isolate the major radiation-induced effects on the charge transfer. It is shown that the main cause of the charge transfer degradation is the radiation-induced defects generation in the premetal dielectric (PMD) and in the transfer gate (TG) spacer vicinity which modifies the potential diagram at the photodiode/TG interface by the creation of a potential pocket retaining the electrons that are not transferred. For 0.1 kGy(SiO2) 5 kGy(SiO2), the defects generated in the PMD influence the whole photodiode potential inducing a pinning voltage increase and degrading the charge transfer by enlarging the potential pocket effect which becomes the main image lag source. The reported results clarify the impact of ionizing radiation on the charge transfer, suggesting radiation hardened by design solutions for future space or nuclear applications.

The orientation distribution of tunneling-related quantities

In the nuclear tunneling processes involving deformed nuclei, most of the tunneling-related quantities depend on the relative orientations of the participating nuclei. In the presence of different multipole deformations, we study the variation of a few relevant quantities for the α-decay and the sub-barrier fusion processes, in an orientation degree of freedom. The knocking frequency and the penetration probability are evaluated within the Wentzel–Kramers–Brillouin approximation. The interaction potential is calculated with Skyrme-type nucleon–nucleon interaction. We found that the width of the potential pocket, the Coulomb barrier radius, the penetration probability, the α-decay width, and the fusion cross-section follow consistently the orientation-angle variation of the radius of the deformed nucleus. The orientation distribution patterns of the pocket width, the barrier radius, the logarithms of the penetrability, the decay width, and the fusion cross-section are found to be highly analogous to pattern of the deformed-nucleus radius. The curve patterns of the orientation angle distributions of the internal pocket depth, the Coulomb barrier height and width, as well as the knocking frequency simulate inversely the variation of the deformed nucleus radius. The predicted orientation behaviors will be of a special interest in predicting the optimum orientations for the tunneling processes.

Abstract 5741: Evaluating the role of recurrent ESR1-CCDC170 fusion in breast cancer endocrine resistance

Abstract Background: Recurrent gene fusions resulting from chromosome rearrangements are central to the formation of cancer. Previously, our lab identified recurrent rearrangements between the estrogen receptor α (ERα) gene ESR1 and its neighbor gene CCDC170, in ~6-8% of luminal B tumors, a more aggressive form of ER+ breast cancer that has worse clinical outcome after endocrine therapy. ESR1-CCDC170 rearrangements enable the expression of different-sized N-terminally truncated CCDC170 (ΔCCDC170) under ESR1 promoter, such as the E2-E7 or E2-E10 variants in which exon 2 of ESR1 fused with exon 7 or exon 10 of CCDC170. Consistent with the behavior of luminal B tumors, ectopic ΔCCDC170 expression in ER+ breast cancer cells transformed cells to a more aggressive form and led to reduced endocrine sensitivity in vitro, as well as enhanced xenograft growth in vivo. In the present study, we examined the role of ESR1-CCDC170 in breast cancer endocrine resistance in vivo and further elucidated the potential engaged mechanisms of its actions. Results: Our in vivo endocrine sensitivity study showed that, while tamoxifen (Tam) treatment rendered tumor regression in the vector-expressing T47D xenograft tumors, it made the E2-E7 overexpressing T47D xenografts static and less likely to regress, whereas the E2-E10 overexpressing xenografts, though grew initially upon Tam treatment, became cytostatic and showed significant reduction in tumor regression. Kaplan-Meier analysis revealed a significantly worse progression-free survival for E2-E7 (p<0.01) and E2-E10 (p<0.001) overexpressing tumors treated with Tam compared to the control tumors. These data suggest that ESR1-CCDC170 variants differ in level of reduced responsivity to Tam, and may render the T47D xenografts less sensitive or resistant to Tam in vivo. Further studies suggest that ΔCCDC170 protein preferentially localizes to cytoplasm (different from wild-type CCDC170 protein enriched in nucleus), physically interacts with ERα and HER2, and forms homodimers. RPPA analysis showed that silencing of ESR1-CCDC170 repressed ERα and BCL2 protein levels, as well as total/phospho-HER2 levels, consistent with the findings of ΔCCDC170 interactions with ERα and HER2. Importantly, analysis of ΔCCDC170 protein sequence revealed a potential high-affinity ATP-binding pocket at its C-terminus, suggesting that ΔCCDC170 may be directly druggable. Conclusion: These data suggest a potential role of ESR1-CCDC170 in mediating breast cancer endocrine responsivity, which may possibly through cytoplasmic mislocalization of ΔCCDC170 and interactions with ERα and HER2. Further studies will affirm and elucidate the role of overexpressed or endogenous ESR1-CCDC170 in breast cancer endocrine resistance, and pinpoint the precise mechanisms of its oncogenic function, in order to develop a novel target therapy against it for the fusion-carrying patient community around the world. Citation Format: Yiheng Hu, Jamunarani Veeraraghavan, Xian Wang, Ying Tan, Jin-Ah Kim, Rachel Schiff, Xiao-Song Wang. Evaluating the role of recurrent ESR1-CCDC170 fusion in breast cancer endocrine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5741. doi:10.1158/1538-7445.AM2017-5741

Thermal decay rate of a metastable state with two degrees of freedom: Dynamical modelling versus approximate analytical formula

Accuracy of the Kramers approximate formula for the thermal decay rate of the metastable state is studied for the two-dimensional potential pocket. This is done by the comparison with the quasistationary rate resulting from the dynamical modeling. It is shown that the Kramers rate is in agreement with the quasistationary rate within the statistical errors provided the absorptive border is far enough from the potential ridge restricting the metastable state. As the absorptive border (or its part) gets closer to the ridge the Kramers formula underestimate the quasistationary rate. The difference reaches approximately the factor of 2 when the absorptive border coincides with the ridge.

Identification of a putative binding site critical for general anesthetic activation of TRPA1

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

Simulation and measurement of total ionizing dose radiation induced image lag increase in pinned photodiode CMOS image sensors

This paper presents an investigation of total ionizing dose (TID) induced image lag sources in pinned photodiodes (PPD) CMOS image sensors based on radiation experiments and TCAD simulation. The radiation experiments have been carried out at the Cobalt −60gamma-ray source. The experimental results show the image lag degradation is more and more serious with increasing TID. Combining with the TCAD simulation results, we can confirm that the junction of PPD and transfer gate (TG) is an important region forming image lag during irradiation. These simulations demonstrate that TID can generate a potential pocket leading to incomplete transfer.

Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket

The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A2AR-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson's disease.

Creating an Artificial Tail Anchor as a Novel Strategy To Enhance the Potency of Peptide-Based HIV Fusion Inhibitors.

The hydrophobic groove of the human immunodeficiency virus type 1 (HIV-1) gp41 NHR trimer has been known as the classic drug target to develop fusion inhibitors derived from the gp41 CHR. Here, we developed a novel and simple strategy to improve the existing peptide-based HIV fusion inhibitors. We identified a shallow pocket adjacent to the groove in the NHR trimer and added a short artificial peptide consisting of three amino acids (IDL) to the C terminus of a fusion inhibitor to fit this new target. The inhibition activity of this new conjugated peptide was significantly enhanced, by 77-fold, making it much more potent than T20 (enfuvirtide) and suggesting that the IDL tail can be adopted for optimizing existing HIV-1 CHR peptide fusion inhibitors. This new approach of identifying a potential binding pocket outside the traditional target and creating an artificial tail anchor can be widely applied to design novel fusion inhibitors against other class I enveloped viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV).

Exploration of the conformational landscape in pregnane X receptor reveals a new binding pocket.

Ligand-regulated pregnane X receptor (PXR), a member of the nuclear receptor superfamily, plays a central role in xenobiotic metabolism. Despite its critical role in drug metabolism, PXR activation can lead to adverse drug-drug interactions and early stage metabolism of drugs. Activated PXR can induce cancer drug resistance and enhance the onset of malignancy. Since promiscuity in ligand binding makes it difficult to develop competitive inhibitors targeting PXR ligand binding pocket (LBP), it is essential to identify allosteric sites for effective PXR antagonism. Here, molecular dynamics (MD) simulation studies unravelled the existence of two different conformational states, namely "expanded" and "contracted", in apo PXR ligand binding domain (LBD). Ligand binding events shifted this conformational equilibrium and locked the LBD in a single "ligand-adaptable" conformational state. Ensemble-based computational solvent mapping identified a transiently open potential small molecule binding pocket between α5 and α8 helices, named "α8 pocket", whose opening-closing mechanism directly correlated with the conformational shift in LBD. A virtual hit identified through structure-based virtual screening against α8 pocket locks the pocket in its open conformation. MD simulations further revealed that the presence of small molecule at allosteric site disrupts the LBD dynamics and locks the LBD in a "tightly-contracted" conformation. The molecular details provided here could guide new structural studies to understand PXR activation and antagonism.

A new piperidinol derivative targeting mycolic acid transport in Mycobacterium abscessus.

The natural resistance of Mycobacterium abscessus to most commonly available antibiotics seriously limits chemotherapeutic treatment options, which is particularly challenging for cystic fibrosis patients infected with this rapid-growing mycobacterium. New drugs with novel molecular targets are urgently needed against this emerging pathogen. However, the discovery of such new chemotypes has not been appropriately performed. Here, we demonstrate the utility of a phenotypic screen for bactericidal compounds against M. abscessus using a library of compounds previously validated for activity against M. tuberculosis. We identified a new piperidinol-based molecule, PIPD1, exhibiting potent activity against clinical M. abscessus strains in vitro and in infected macrophages. Treatment of infected zebrafish with PIPD1 correlated with increased embryo survival and decreased bacterial burden. Whole genome analysis of M. abscessus strains resistant to PIPD1 identified several mutations in MAB_4508, encoding a protein homologous to MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis was unaffected, PIPD1 strongly inhibited the transport of trehalose monomycolate, thereby abrogating mycolylation of arabinogalactan. Mapping the mutations conferring resistance to PIPD1 on a MAB_4508 tridimensional homology model defined a potential PIPD1-binding pocket. Our data emphasize a yet unexploited chemical structure class against M. abscessus infections with promising translational development possibilities.

Structural insights into the benzophenanthridines binding to human glycine transporter GlyT1

We previously identified cysteine 475 as a key residue for the inhibitory action of sanguinarine on the human glycine transporter GlyT1c. To define potential benzophenanthridine binding pocket more closely, we created a structural homology model of GlyT1 and also mutated several amino acids in the vicinity of cysteine 475. Even though this area contains the molecular determinants of the glycine and sodium permeation pathways, and several mutations resulted in an inactive transporter, we found that the mutation of a polar aromatic tyrosine 370 to purely aromatic phenylalanine, but not to an aliphatic leucine, significantly increased the sensitivity of GlyT1 to both sanguinarine and chelerythrine. The reduction of sanguinarine to dihydrosanguinarine completely eliminated the alkaloid’s inhibitory potency. Both these results suggest that aromaticity is important in the interaction of benzophenanthridines with GlyT1. Even though tyrosine 370 is part of the conformationaly highly flexible glycine binding site, and is accesible during the transport process from both intra and extracellular sites, the cytoplasmic location of the second alkaloid sensitive residue, cysteine 475, suggests that the benzophenanthridines might attack the area of the GlyT1 intracellular gates.

Modified fusion probability by reflection boundary**Supported by National Natural Science Foundation of China (11175021) and Specialized Research Fund for the Doctoral Program of Higher Education (20120003110025)

We investigate the time-dependent probability for a Brownian particle passing over the barrier to stay at a metastable potential pocket against escaping over the barrier. This is related to the whole fusion-fission dynamical process and can be called the reverse Kramers problem. By the passing probability over the saddle point of an inverse harmonic potential multiplying the exponential decay factor of a particle in the metastable potential, we present an approximate expression for the modified passing probability over the barrier, in which the effect of the reflection boundary of the potential is taken into account. Our analytical result and Langevin Monte-Carlo simulation show that the probability of passing and against escaping over the barrier is a non-monotonous function of time and its maximal value is less than the stationary result of the passing probability over the saddle point of an inverse harmonic potential.