Performing an accurate canalith repositioning procedure (CRP) is important for treating benign paroxysmal positional vertigo, because inadequate rotational head angles can result in ineffective otolith mobilization and consequent treatment failure. Specialists-guided Epley maneuver reportedly had mean errors of 13.7°–24.4° while they were significantly larger (40.0°–51.5°) when self-administered. Similar results were obtained for the Barbeque maneuver: mean errors were 9.2°–13.0° by the specialists while they were significantly larger (22.9°–28.6°) when self-administered. Our study aimed to validate the feasibility of an inertial measurement unit sensor-based CRP (IMU-CRP) by analyzing the differences in accuracy in the rotational angles, comparing them with education-based conventional CRP (EDU-CRP). A pilot validation was also performed by analyzing the treatment success rate of IMU-CRP in patients with BPPV. This single-institution prospective, comparative effectiveness study examined 19 participants without active vertigo or prior knowledge of benign paroxysmal positional vertigo and CRP. Participants conducted the Epley and Barbeque roll maneuvers without and with auditory guidance  twice, and head rotation accuracies were compared. Differences in target angles based on the American Academy of Otolaryngology-Head and Neck Surgery guidelines were considered errors. For BPPV participants, treatment success was assessed based on the presence or absence of nystagmus, vertigo, and dizziness. For all the Epley and Barbeque roll maneuvers steps, the absolute errors were smaller for IMU- than for EDU-CRPs, with significant differences in steps 2–4 and 3–6 of the Epley and Barbeque roll maneuvers, respectively. A learning effect was found in steps 4 and 5 of the Barbeque roll maneuver but not in the Epley maneuver. The treatment success rates after 1 h were 71.4% and 100% for the Epley and Barbeque roll maneuvers, respectively. Real-time feedback on head rotation angles induced more appropriate movements in the Epley and Barbeque roll maneuvers. A guiding device based on head monitoring providing real-time auditory feedback may increase the self-administered CRP success rates in treating benign paroxysmal positional vertigo. However, only 58% of patients with BPPV were discharged following the first CRP, whereas 94% needed up to three follow-up appointments5. Repeated CRPs at home can improve treatment outcomes. A home CRP program including Brandt–Daroff, Semont, 360° rotation, and deep head hanging maneuvers following conventional office-based CRP was 8.3% more effective than office-based CRP alone6. Home CRP also reduced BPPV recurrence by 9%7. Therefore, repeated or self-administered CRP can support effective BPPV care.Benign paroxysmal positional vertigo (BPPV) can be treated by a series of rotational head movements geometrically aligned with the affected semicircular canal. A symptom reduction in patients with BPPV with posterior canal involvement is 3.3–107.7 times more likely following a canalith repositioning procedure (CRP) than under control conditions9. Without specialist assistance, patients have difficulties performing precise CRP maneuvers due to the complexity of sequential 3-dimensional (3-D) head movements. In the Epley maneuver, the absolute mean error was as large as 39.8° in step 4, even after detailed explanations. The comparatively simpler Barbeque (BBQ) roll maneuver still caused substantial rotation errors, especially in elderly participants, the population mostly affected by BPPV8. Patients can also perform CRP using only audiovisual media such as YouTube. However, their reliability can be as low as 64%10, and watching a well-made video clip does not guarantee well-performed CRPs. One of the reasons is that video clips cannot provide feedback on the accuracy of head movements or how to correct them in real-time.Self-administered CRPs showed significantly higher errors in rotational head angles compared to specialists-guided CRPs adhering to American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) guidelinesThe present study aimed to validate the feasibility of an inertial measurement unit (IMU) sensor-based CRP guiding system that provides step-by-step instructions and real-time feedback while participants perform CRP independently. Epley and BBQ roll maneuvers were performed, and the 3-D rotational head movements were quantitatively tracked with head-mounted IMU sensors. The rotational angle errors were compared between education-based conventional (EDU)-CRP and IMU-CRP. Learning effects were also investigated with test–retest sessions. Finally, we analyzed the treatment success rate after conducting a preliminary validation of IMU-CRP for patients with BPPV.4. All the participants performed Epley and BBQ roll maneuvers for the right ear. For the Epley maneuver .Between July 2019 and January 2021, we enrolled 19 adults aged ≥ 60 years . Based on a systematic interview or physical examination, it was confirmed that they had no disease or surgery that could induce BPPV and no prior knowledge of BPPV or CRP. In this study we focused on older adults, because this age group has the greatest difficulty in understanding and preforming an accurate CRPThe Institutional Review Board of Seoul National University Hospital approved this prospective, single-institution study with normal participants (IRB No. H-1412-012-630). Prospective, single-institution study for a pilot validation with BPPV patients was approved by the Institutional Review Board of Myung-ji Hospital (IRB No. MJH-2019-09-021-002). Both studies were conducted in accordance with the ethical standards of the Helsinki Declaration. All participants provided written informed consent.To measure head motion in real-time, we developed a small (36 × 39 × 14 mm) and lightweight (16 g) HMTD Fig. a. An att11. The attitude heading reference system sensor calculated the Euler angles with reference to the earth's center. The rotation matrix calculated the rotation angle relative to an arbitrary axis using the Euler angle. A rotation between two points can be represented by A rotation matrix was applied to calculate the 3-D rotation angle for each maneuver step from Euler anglesentclass1pt{minima8. In brief, we used rotation angle errors (in degrees) to evaluate EDU- and IMU-CRP performances. Errors were defined as differences between actual rotation angles and corresponding target angles specified in the AAO-HNS guidelines9. Positive and negative values represent excessive and insufficient head rotation, respectively. The absolute mean error was averaged across all participants.The details of rotation vector and angle analyses have been reported previously8, with reference to the AAO-HNS-specified rotation angles. Half the SD was used to represent the minimum difference between groups13. Thus, target ranges were 26.1°, 31.0°, and 18.9° for steps 2–4, respectively, of the Epley maneuver and 17.2°, 14.6°, 20.1°, and 12.8° for steps 2–5, respectively, of the BBQ roll maneuver.CRP was considered reliable when the absolute mean error was within the target range defined as the absolute mean error of specialist + half the SD in a previous studyt tests of errors and absolute errors between EDU- and IMU-CRPs were performed using SPSS . Paired t tests between the first and second sessions per treatment were conducted to evaluate the effects of repetitive trials. Effect size (Glass’s delta) for all the performed paired t tests was calculated as well14. In a pilot validation with BPPV patients, the treatment success rates of the three outcome measures were calculated for each maneuver.Due to data loss during the Bluetooth transfer, data of seven participants  were excluded from further analyses. Data are presented as the mean ± SD. Paired The Institutional Review Board of Seoul National University Hospital approved this prospective, single-institution study (IRB No. H-1412-012-630), and the first registration took place on 19/06/2019. Prospective, single-institution study for a pilot validation with BPPV patients was approved by the Institutional Review Board of Myung-ji Hospital (IRB No. MJH-2019-09-021-002). Both studies were conducted in accordance with the ethical standards of the Helsinki Declaration. All participants provided written informed consent.All participants provided written informed consent for participation and publication.8. There are no target range data in step 1 of the Epley maneuver and step 6 of the BBQ roll maneuver due to different start and end points in the specialist CRPs. For the Epley maneuver, the mean errors of steps 1–4 during the EDU-CRP were 5.51 ± 20.43, − 8.98 ± 15.97, 10.89 ± 26.36, and − 24.80 ± 21.84, respectively. During the IMU-CRP, the respective mean errors were 1.87 ± 8.21, 6.25 ± 5.42, 1.62 ± 6.81, and 0.34 ± 9.32. Effect size concerning the mean errors for each Epley maneuver step between EDU-CRP and IMU-CRP were 0.18, 0.95, 0.35, and 1.15, respectively. The mean errors during the IMU-CRP were significantly smaller in steps 2 (P = 0.004) and 4 (P < 0.001). For the BBQ roll maneuver, the mean errors of steps 2–6 during the EDU-CRP were − 5.03 ± 12.01, − 5.97 ± 18.75, 3.48 ± 21.23, 15.26 ± 14.55, and − 5.05 ± 17.52, respectively. During the IMU-based treatment, the corresponding mean errors were − 2.65 ± 6.46, − 4.07 ± 9.57, 3.25 ± 5.99, 8.25 ± 3.73, and − 0.31 ± 6.48. Effect size concerning the mean errors for each BBQ maneuver step between EDU-CRP and IMU-CRP were 0.20, 0.10, 0.01, 0.48, and 0.27, respectively. The mean error during the IMU-CRP was significantly smaller in step 5 (P = 0.049).Figure 8. For the Epley maneuver, the absolute mean errors of steps 1–4 during EDU-treatment were 14.24 ± 15.28, 18.04 ± 15.71, 24.85 ± 18.12, and 33.98 ± 20.25, respectively. The overall absolute mean error was 22.78 ± 22.14. During the IMU-CRP, the absolute mean errors of steps 1–4 were 8.23 ± 4.52, 6.52 ± 5.18, 7.10 ± 2.96, and 8.67 ± 3.26, respectively. The overall absolute mean error was 7.63 ± 5.43. Effect size concerning the absolute mean errors for each Epley maneuver step between the EDU-CRP and IMU-CRP were 0.39, 0.73, 0.98, and 1.25, respectively. The absolute mean errors during the IMU-CRP were significantly smaller in steps 2 (P = 0.012), 3 (P = 0.002), and 4 (P < 0.001). The percentages of data with absolute mean errors within the target range were 73.3%, 66.7%, and 40.0% during the EDU-CRP and 96.7%, 100.0%, and 100.0% during the IMU-CRP for steps 2–4, respectively.Figure P = 0.002), 5 (P = 0.002), and 6 (P = 0.002). The percentages of data with absolute mean errors within the target range were 84.4%, 53.1%, 65.6%, 40.6% during the EDU-CRP and 100%, 84.4%, 100%, and 90.6% during the IMU-CRP for steps 2–5, respectively. All steps considered, the percentages of data with absolute mean errors within the target range were 60.6% and 95.9% for EDU- and IMU-CRPs, respectively.For the BBQ roll maneuver, the absolute mean errors of steps 2–6 during the EDU-CRP were 10.91 ± 9.43, 16.00 ± 14.21, 18.28 ± 12.19, 18.82 ± 11.41, and 18.55 ± 14.30, respectively. The overall absolute mean error was 16.65 ± 17.13. During the IMU-CRP, the corresponding absolute mean errors were 6.28 ± 2.68, 8.84 ± 6.62, 6.92 ± 3.33, 8.43 ± 3.48, and 5.52 ± 2.62. The overall absolute mean error was 7.22 ± 4.94. Effect size concerning the absolute mean errors for each BBQ maneuver step between the EDU-CRP and IMU-CRP were 0.49, 0.50, 0.93, 0.91, and 0.91, respectively. The absolute mean errors during the IMU-CRP were significantly smaller in steps 4  and 5 (P = 0.026). Effect sizes concerning the absolute mean errors for each BBQ maneuver step between the first and second session of EDU-CRP were 0.44, 0.27, 0.64, 0.61, and 0.25, respectively. The absolute mean error differences between the first two sessions (1st–2nd) were 8.11 ± 19.45, 5.08 ± 19.90, 15.56 ± 27.52, 10.23 ± 16.37, and 4.87 ± 25.88; thus, repeated education improved the performance of the BBQ roll maneuver by 15.56° in step 4 and 10.23° in step 5.For the BBQ roll maneuver, the absolute mean errors of steps 2–6 during the first EDU-CRP were 15.11 ± 18.23, 18.73 ± 19.13, 26.40 ± 24.33, 24.26 ± 16.85, and 20.65 ± 19.40, respectively. The corresponding mean errors during the second EDU-CRP were 7.00 ± 5.68, 13.65 ± 15.34, 10.85 ± 9.45, 14.04 ± 10.18, and 15.77 ± 18.91, respectively. The absolute mean error of the second EDU-CRP was significantly smaller in steps 4 (P = 0.022). Effect sizes concerning the absolute mean errors for each BBQ maneuver step between the first and second session of the IMU-CRP were 0.04, 0.22, 0.92, 0.08, and 0.03, respectively. The absolute mean error differences between the two sessions were 0.19 ± 5.79, − 1.48 ± 6.29, − 3.18 ± 4.97, 0.33 ± 3.51, and 0.11 ± 5.25; thus, the performance of the BBQ roll maneuver worsened after repetition of IMU-CRP by 3.18° in step 4.During the IMU-CRP, the absolute mean errors of steps 2–6 were during the first trial, 6.62 ± 4.33, 7.83 ± 6.73, 5.07 ± 3.46, 8.73 ± 4.01, and 5.82 ± 4.10, respectively, and during the second trial 6.44 ± 3.76, 9.31 ± 8.00, 8.25 ± 4.74, 8.40 ± 3.61, and 5.71 ± 3.98, respectively. The absolute mean error of the second IMU-CRP was significantly larger in step 4  mean errors were substantial despite repeated education, and the angular head rotation was within the target range of only 60.6%. Seemingly, participants can learn the concept of the Epley maneuver, but precise head rotation is difficult to achieve through education. Real-time feedback from the proposed IMU-based system seems key to enabling participants to perform Epley and BBQ roll maneuvers on their own accurately.The present study aimed to validate the feasibility of self-administered CRPs assisted by an IMU sensor-based guiding system. This study found a significant benefit of this guiding system. The (absolute) mean errors in IMU-CRPs were significantly smaller than those of conventional EDU-CRPs in both Epley and BBQ roll maneuvers. Moreover, the IMU-based system can assist patients in performing CRP at home as accurately as professionals in the clinic. In 95.9% of patients, the absolute mean errors of IMU-CRPs were within the target range derived from vestibular specialists with > 10 years of clinical experience. Repeated CRPs improve treatment outcomes in BPPV and prevent symptom recurrenceThe feedback system seems to be the most important difference between EDU- and IMU-CRPs. Education and feedback are both instructions on how to perform CRP correctly. Education describes the general approach, whereas feedback enables correction of the current head position. Even if participants perfectly understood each step, they might have difficulties executing the learned CRP. Since the IMU system instructs the participant to move the head until it is within the target range, the participant will eventually achieve the correct position without fully understanding the entire procedure. Therefore, IMU sensor-based guiding system can be an effective adjunct to CRP treatment.15. This cap-attached device guides the patient to navigate a ball within the device through head movements. These authors also developed a mobile app for an Epley maneuver guiding system16. A virtual reality-based system has also been proposed17. These systems were only evaluated by physicians subjectively rating CRP performances. Moreover, these systems do not provide real-time quantitative feedback on head motions. Recently, another research group developed an Epley maneuver visual assistive device18. In this study, the author performed a quantitative analysis of rotation error when performing the Epley maneuver with and without the assistive device. The result was consistent with our research finding. However, this visual assistive device helps the clinician to treat the patient more easily and does not help the patient perform the Epley maneuver by themselves more accurately. Our IMU-based wearable system computes the rotation angle in real-time and provides audio guidance to achieve AAO-HNS targets. It can be applied to Epley and BBQ roll maneuvers, the latter of which has not been addressed previously. Horizontal canal BPPV may be more common than posterior canal BPPV, depending on the evaluation time22. Moreover, the adaptability of our guiding system allows the addition of any treatable BPPV subtype.Several studies described in-home systems for BPPV treatment. A device called DizzyFix™ has been approved by the US Food and Drug AdministrationAs seen in Fig. 23. Our IMU-based guiding system has potential as a BPPV treatment device, as it showed a treatment success rate of 80% after 1 h in a pilot validation for 10 BPPV patients.According to Song et al., the resolution rate of positional nystagmus and vertigo in patients with posterior canal BPPV was 67.6% at post-1 h25. (2) Younger participants have less difficulty learning geometrically complex 3-D head movements8. (3) Recurrence and residual symptoms requiring additional management are more frequent in elderly participants28. Although clinically irrelevant, the study results may change if young participants were exclusively recruited.The present study has several limitations. First, effects of inaccurate head rotations on treatment outcomes were evaluated only with a small number of BPPV patients for preliminary validation. Further studies are required to validate the treatment outcomes of this IMU-based system for vertigo and nystagmus in more patients with BPPV. It may be difficult for some patients with BPPV to properly carry out the instructions when nystagmus and symptoms occur. Thus, we recommend using this system after the diagnosis and initial treatment by a medical personnel at the hospital  to avoid misdiagnosis, provide initial CRP education to the patient, and reduce symptoms to enable the patient to perform CRP alone. Second, age and sex effects on guiding effectiveness were not considered because of the limited number of study participants. We recruited only participants aged ≥ 60 years for three reasons: (1) BPPV is more prevalent in elderly participantsReal-time feedback on head rotation angles by an IMU sensor-based guiding system enabled the participants to perform Epley and BBQ roll maneuvers on their own accurately. IMU-CRP accuracy was comparable to that of vestibular specialists. Repeated self-administered CRPs have been reported to improve treatment outcomes and prevent BPPV recurrence; however, according to the present study results, education-based conventional self-administered CRP is unreliable. The IMU-based system can be useful in elderly patients with BPPV with recurring or residual symptoms by helping them repeat accurate CRPs independently, even when a vestibular specialist is unavailable.


ZDHHC genes in the human genome. These enzymes catalyze S-acylation in a two-step process involving “autoacylation” of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. Here, we developed and implemented a novel FRET-based high-throughput assay for the discovery of compounds that interfere with autoacylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. Our screen of >350,000 compounds identified two related tetrazole-containing compounds (TTZ-1 and TTZ-2) that inhibited both zDHHC2 autoacylation and substrate S-acylation in cell-free systems. These compounds were also active in human embryonic kidney 293T cells, where they inhibited the S-acylation of two substrates (SNAP25 and PSD95 [postsynaptic density protein 95]) mediated by different zDHHC enzymes, with some apparent isoform selectivity. Furthermore, we confirmed activity of the hit compounds through resynthesis, which provided sufficient quantities of material for further investigations. The assays developed provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating cellular activities of zDHHC enzymes in S-acylation.Protein S-acylation is a reversible post-translational modification that modulates the localization and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC (Asp-His-His-Cys) domain–containing (zDHHC) proteins encoded by 23 distinct  The ami studies .ZDHHC2 as a candidate gene for schizophrenia  and of many zDHHC enzymes in cells with a low level of toxicity.A major obstacle to progress of the S-acylation field is a lack of selective chemical inhibitors or modulators of zDHHC enzymes , 32, 33.n as 2BP . This prbis-cyclic piperazine backbone. Interestingly, the identified compounds were recently shown to inhibit zDHHC9-mediated S-acylation of the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 pandemic, and to reduce virus infectivity . The assay monitors time-resolved FRET (TR-FRET) between the energy donor terbium cryptate (attached to an antibody recognizing hemagglutinin [HA]-tagged zDHHC2) and the acceptor fluorophore nitrobenzoxadiazole (NBD) (attached to the lipid group). The excitation of the energy donor terbium cryptate (360 nm) followed by its emission at 480 nm results in energy transfer to the acceptor NBD and emission at 539 nm. Enzyme autoacylation by NBD-conjugated palmitoyl-CoA increases the proximity of the terbium cryptate and NBD, leading to an increase in FRET signal. The resulting interaction is quantified as the ratiometric measurement of NBD (539 nm) over terbium (480 nm) emission and constitutes the TR-FRET signal. Human zDHHC2 enzyme for use in this assay was produced with the Expi293 MembranePro expression system (Invitrogen), which enables membrane proteins to be displayed in a native context on highly enriched lipoparticles that are released in the supernatant of the transfected mammalian cells.S-acylation is a two-step process involving enzyme “autoacylation” (at the cysteine of the DHHC motif) followed by transfer of the acyl chain to a substrate protein. To identify potential inhibitors of S-acylation, we developed an assay measuring enzyme autoacylation and focused on zDHHC2 given its prominent role in synaptic function in the nervous system A. The asB. Autoacylation of zDHHC2, detected by TR-FRET signal, was shown to increase with increasing concentration of NBD-palmitoyl-CoA. Importantly, the TR-FRET signal was substantially reduced when a catalytically dead form of zDHHC2 with a substitution of the cysteine in the DHHC motif (C157S) was used, demonstrating that the FRET signal faithfully reports the autoacylation of the enzyme.An example of the TR-FRET measured using this assay system is shown in Fig. 1C outlines the final conditions chosen for the screen, which were then used to screen a high-throughput screen (HTS) library of >350,000 compounds at a concentration of 4.2 μM . About 1600 compounds that presented an inhibition equal to the median – 3 SD or less were then examined in duplicate and at two concentrations. Following this, we selected the 162 most potent compounds and again assessed their inhibitory activity by TR-FRET using freshly dissolved powder and at two different concentrations (12.5 and 4.2 μM). Their ability to affect substrate S-acylation was also assessed using the in vitro ELISA described later at two different concentrations (3 and 10 μM) and in duplicate experiments. Although several compounds were confirmed to have an activity in vitro, the results presented focus on two of these compounds (tetrazole [TTZ] -1 and -2) because they were subsequently found to have the highest inhibitory activity on S-acylation in cell-based assays (see later). The chemical structure of these two compounds is shown in Following optimization, the assay was miniaturized to a 1536-well plate format. A pilot screen was then performed at two concentrations (12.5 and 4.2 μM) using ONO pilot library containing a total of 15,792 compounds. RZ′ score was calculated to be between 0.72 and 0.80 for each plate showing that the assay design was robust. Fig. 1in vitro. For this, we designed an ELISA in a 96-well plate format. His6-tagged human zDHHC2 enzyme was purified from insect cells on Ni2+–nitrilotriacetic acid affinity resin according to methods developed previously (A). Glutathione-S-transferase (GST)-tagged SNAP25, a reported substrate of zDHHC2  for 2 h to block free cysteines, the azide-tagged palmitate that had been incorporated into GST-SNAP25 and zDHHC2 was reacted with DBCO-PEG4-Biotin in a copper-free cycloaddition reaction. Samples were then analyzed by immunoblotting with NeutrAvidin-800 to detect biotin (and hence S-acylation) and an anti-GST antibody (IR680) to detect GST and GST-SNAP25. Fig. 3B shows that this assay detects the S-acylation of GST-SNAP25 (band above 47 kDa) but not GST. The NeutrAvidin-800 signal also revealed the autoacylated enzyme (band above 36 kDa).We next assessed the ability of TTZ-1 and TTZ-2 to inhibit zDHHC2-mediated substrate S-acylation eviously A. Glutatf zDHHC2 , was theC). In vitro S-acylation and click chemistry labeling were carried out in uncoated 96-well plates. The processed samples were then diluted with PBS and bound for 1 h to glutathione plates before being incubated with NeutrAvidin-peroxidase (horseradish peroxidase [HRP]). Chemiluminescence signal was then measured following a 10 min incubation with a peroxidase substrate. Compounds tested in this ELISA were preincubated with the purified zDHHC2 enzyme for 15 min at room temperature prior to the addition of substrates. Fig. 3D shows the normalized chemiluminescent signal detected with the ELISA. Background levels of signal were detected with GST or GST-SNAP25 alone (with no added zDHHC2)  or with GST + zDHHC2 (6.1 ± 1.1), whereas there was a strong signal for samples incubated with GST-SNAP25 and the purified enzyme (100 ± 3.4). The ELISA was therefore suitable for testing the activity of the identified compounds on the S-acylation of SNAP-25 by purified zDHHC2.This assay was further developed into an ELISA format to more precisely quantify SNAP25 S-acylation C. In vitin vitro was then determined by calculating IC50 values (with concentrations ranging from 0.3 to 30 μM). 50 below 5 μM (C). Importantly, this confirms that the decreased signal detected with these compounds in the ELISA reliably reports on reduced S-acylation rather than any nonspecific effects.The ability of TTZ-1 and TTZ-2 to inhibit SNAP25 S-acylation by purified zDHHC2 low 5 μM . Selectelow 5 μM C. ImportA and B shows that both compounds inhibited SNAP25 S-acylation catalyzed by zDHHC2, with IC50 values of approximately 15 μM for TTZ-1 and 19 μM for TTZ-2.The inhibitory activity of TTZ-1 and TTZ-2 on cells expressing SNAP25 and zDHHC2 was also examined. Human embryonic kidney 293T (HEK293T) cells were transfected with plasmids encoding GFP-tagged SNAP25b and HA-tagged zDHHC2. Cells were then preincubated for 4 h with either dimethyl sulfoxide (DMSO) as a vehicle control or increasing concentrations of TTZ-1/TTZ-2 (diluted in serum-free medium) before being metabolically labeled for a further 4 h with C16:0-azide. Samples were then lysed and processed by copper-catalyzed click chemistry to reveal the extent of S-acylation. A shows complete inhibition of zDHHC2-mediated SNAP25 S-acylation by TTZ-1 (113 ± 5%) and a very strong reduction (84 ± 6%) by TTZ-2. Importantly, the level of enzyme expression was unaffected by the compounds. Interestingly, the inhibitory effect of the compounds on zDHHC15-mediated S-acylation of SNAP25 was similar to that seen with zDHHC2 . In contrast, S-acylation of SNAP25 by zDHHC3 and zDHHC7 was less inhibited: TTZ-1 had no significant effect on either enzyme in this assay, and TTZ-2 caused a 19 ± 3% reduction of SNAP25 S-acylation by zDHHC3 and had no significant effect on S-acylation by zDHHC7 (B and C). Finally, both compounds showed an intermediate effect on zDHHC17-mediated S-acylation of SNAP25, with a ∼35 to 40% reduction . In addition, there was a slight but significant reduction in the level of zDHHC17 expressed in cells treated with TTZ-2. As any major differences in the expression levels of the different enzymes could in theory contribute to the apparent differential effects of TTZ-1 and TTZ-2, we directly compared the expression of zDHHC2, zDHHC3, zDHHC7, zDHHC15, and zDHHC17 when transfected in HEK293T cells together with GFP-SNAP25. As shown in Fig. 6F, none of the zDHHC enzymes was expressed to a higher level than HA-zDHHC2, suggesting that the observed differential effects of TTZ-1 and TTZ-2 are not because of increased expression levels of a particular enzyme.We next investigated whether the identified compounds were effective at inhibiting zDHHC enzyme isoforms other than zDHHC2 . In addiFig. 6bis-nitration of chlorobenzene 3 led to the sulfonate salt 4. SNAr reaction with aqueous ammonia followed by protodesulfonation gave 2,6-dinotroaniline 6 in 36% overall yield for the initial sequence. Sandmeyer reaction of 6 under standard conditions gave 2,6-dinitrochlorobenzene 7 (57%), which was subjected to an efficient SNAr reaction with hydroxide followed by partial reduction and then reacted with 2-chloroacrylonitrile 10 to give benzoxazine 11 in a poor, but acceptable, yield of 15% after purification by silica gel chromatography. Reduction of the second nitro functionality led to the central scaffold of the target ligand 12 (70%) from which diversity could be introduced. Amide bond coupling followed by acid-catalyzed TTZ formation gave TTZ-1 and TTZ-2. Compounds TTZ-1 and TTZ-2 were prepared in 12 synthetic steps and an overall yield of 1.9% and 1.5%, respectively.To confirm the identity of the hit compounds and to allow further investigations to take place, we prepared samples of TTZ-1 and TTZ-2 through the multistep synthetic sequence outlined in The newly synthesized TTZ-1 and TTZ-2 compounds were then tested against GFP-SNAP25 S-acylation in cell-based assays with results comparable to the original hit compounds . We alsoPSD95 (postsynaptic density protein 95) is a major S-acylated protein that has a critical role in the maturation of excitatory neurons and in synaptic plasticity , 47. PSDA shows that SNAP23 S-acylation can indeed be detected by mPEG-click during the 4 h labeling period, as evidenced by the appearance of high molecular weight bands when cells are incubated with C16:0-azide but not with palmitate. The pretreatment of the cells with either TTZ-1 or TTZ-2 caused a significant reduction in SNAP23 S-acylation. Similarly, TTZ-1 and TTZ-2 decreased the S-acylation of calnexin, another well-characterized S-acylated protein .Finally, we tested whether TTZ-1 and TTZ-2 were able to block the S-acylation of endogenous proteins in the absence of zDHHC enzyme overexpression. In order to detect the S-acylation of endogenous proteins, we used mPEG-click, a technique that we developed recently  that is Fig. 10 protein , 53  that follows the post-translational attachment of lipid moieties and their subsequent binding to detergent micelles  developing new assay platforms for inhibitor discovery and (ii) using these platforms to define novel chemical inhibitors of zDHHC enzymes. We showed that the developed high-throughput autoacylation assay was indeed a robust platform as it led to the identification of hits from a screen of >350,000 individual compounds that also inhibited enzyme autoacylation and substrate S-acylation in established cell-based assays. The compounds that emerged from this screen, TTZ-1 and TTZ-2, both possess a 2,8-disubstituted 3,4-dihydro-2H-benzo[b] oxazine core. Common to both structures is a TTZ head group, a well-established bioisostere for carboxylic acids. In addition, both compounds contain a hydrophobic chain at the 8-position of the heterocycle linked through an amide bond. The resemblance of the polar and nonpolar components of these synthetic compounds to the natural fatty acid substrates for the zDHHC enzymes suggests that they may have a common binding site on the enzyme.B). We also attempted to develop a TR-FRET assay for substrate acylation. However, with the materials we used in this publication , we could not observe any TR-FRET signal on SNAP25 protein.An important strength of the study is that the identified inhibitors displayed activity in multiple cell-free and cell-based assays, undertaken at different research sites. The compounds were indeed first identified for their inhibitory activity toward the autoacylation of zDHHC2, as measured by TR-FRET. TR-FRET is a popular choice for HTS campaigns because of its relative simplicity and has been widely used for the screening of modulators of molecular interactions (protein–protein or protein–DNA). TR-FRET has also been used to identify inhibitors of several enzymes, such as kinases, heparanases, and ubiquitin ligases . This isFig. 1Following the identification of TTZ-1 and TTZ-2 in the HTS, the next step in the selection process was to screen their inhibitory activity toward substrate S-acylation in cell-free assays that used click chemistry for detection by either a newly developed ELISA or by direct SDS-PAGE analysis. A click-ELISA was developed previously for the detection of Hhat activity  with theversus zDHHC3 and zDHHC7, with zDHHC17 displaying intermediate levels of inhibition. Similarly, the inhibitors were more active against the S-acylation of PSD95 by zDHHC2 and zDHHC15 than by zDHHC3 and zDHHC7. We cautiously interpret these observations as indicating that the development of isoform-selective zDHHC inhibitors is a realistic possibility, perhaps because of subtle differences in the acyl-CoA binding pocket of zDHHC enzymes that affects lipid selectivity or differences in the turnover of the autoacylated enzyme intermediate; these possibilities will be explored in future work in this area. Finally, the compounds were tested for their ability to inhibit substrate S-acylation in cells overexpressing different zDHHC isoforms. Both inhibitors were active against the zDHHC2-mediated S-acylation of two different substrates, SNAP25 and PSD95. It is interesting to note that the inhibition profile of TTZ-1 and TTZ-2 on SNAP-25 S-acylation appeared to display some zDHHC isoform selectivity and was more potent against zDHHC2 and zDHHC15 The selective inhibition of zDHHC2-mediated S-acylation of PSD95  in neurons could be facilitated by the use of TTZ-1 or TTZ-2 and might provide a therapeutic tool for the modulation of postsynaptic domain organization and of synaptic plasticity , 61, 62.2+ is known to bind to the DHHC-CRD of zDHHC enzymes and plays an important structural role.There may also be potential for employing selective inhibitors of zDHHC enzymes for the treatment of some infectious diseases. Many viral proteins undergo S-acylation, and this is often key for efficient viral replication , 68. Thuin vitro and tumor growth in an in vivo model of KRAS-mutant lung adenocarcinoma , whereas TTZ-1 and TTZ-2 contain a TTZ unit that is acidic and would be expected to be deprotonated at physiological pH . We therefore expect ketoconazole and TTZ-1/TTZ-2 to have very different properties and hence biological targets/interactions.Based on the results of the TR-FRET assays partners , 31, 32.partners , 32, 78.partners , 80, 81.partners , 83, sugpartners . There ipartners , 88, 89.partners . A substpartners . Ketoconpartners , which iThe observation that TTZ-1/TTZ-2 inhibited the S-acylation of endogenous substrates mediated by endogenous enzymes is encouraging. Although the concentrations tested here were relatively high (25 and 50 μM), it has to be noted that the identity of the endogenous zDHHCs mediating the S-acylation of these substrates in HEK293T is unclear. Since these cells express the 23 members of the zDHHC family , a combie.g., using RNAi or CRISPR technology). These tools are significantly more drug like in character and offer an important advance over nonselective inhibitors such as 2BP. In addition, the apparent selectivity of these compounds toward zDHHC2 and zDHHC15 offers the potential to refine and develop their selectivity further through a detailed structure–activity relationship study. Truly selective inhibitors of the zDHHC enzyme family would represent a major development for the field.The identification of small-molecule inhibitors of zDHHC enzymes described herein provides valuable and much-needed tools to investigate S-acylation-dependent cellular pathways. Our data also suggest that they are capable of acting on native enzymes and substrates, highlighting their potential usefulness as tool compounds. It is envisaged that they will allow the acute effects of S-acylation disruption to be investigated without the need for long-term zDHHC depletion (2 in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum . Insect cells Sf9  and Sf9 TriEx  were grown at 28 °C in a dry incubator without CO2, in ESF21 media  and TriEx medium , respectively. All the components of the Expi293 MembranePro system (including the cells) were from Invitrogen.HEK293T cells  were grown at 37 °C in a humidified atmosphere containing 5% COGoat GST antibody , NeutrAvidin-DyLight 800 , and High-Sensitivity NeutrAvidin-HRP  were obtained from Thermo Fisher Scientific. Rat HA antibody  was from Sigma, mouse GFP antibody  was obtained from Takara, rabbit SNAP23 from Synaptic Systems (used at 1:1000 dilution), and mouse actin  was from Sigma. Sheep anti–red fluorescent protein antibody was a gift from Ian Prior (University of Liverpool). IR dye–conjugated secondary antibodies were used at a dilution of 1:20,000 and purchased from LI-COR Biosciences. Terbium cryptate–conjugated anti-HA antibody was from CisBio.Human complementary DNA (cDNA) encoding zDHHC2 NM_016353.4), zDHHC3 (NM_001135179), zDHHC7 (NM_017740.2), zDHHC15 (NM_144969.2), and zDHHC17 (NM_015336.2) were synthesized by GeneArt Technologies (Thermo Fisher Scientific) and subcloned in pEF-BOS-HA 353.4, zD and was 6 and StrepII, respectively) in the baculovirus transfer vector pIEX/Bac3 .The human cDNA encoding zDHHC2 was codon optimized for protein expression in insect cells and synthesized by GeneArt Technology (Invitrogen). It was inserted with N- and C-terminal tags .C16:0-azide has been described previously . Palmitog for 30 min at 4 °C and resuspended in the buffer provided. The optimized protocol for the HTS was as follows: compounds were tested at a final concentration on 4.2 μM (2.5 nl) premixed for a few minutes with 1.5 μl of HA-zDHHC2 lipoparticles (at 42 μg/ml) before the addition of 4.5 μl of a mixture of anti-HA Tb cryptate (1:200 dilution) and NBD-palmitoyl-CoA (1 μM). Compounds were dispensed by Echo 555 (Beckman Coulter), and other reagents were dispensed by Multi Drop Combi (Thermo Fisher Scientific). The TR-FRET signal was read after a 15 min incubation on a Biotek Synergy Neo apparatus (Agilent). The dynamic range during the campaign was between 1.5 and 2.3.The Expi293 MembranePro system was used to generate HA-tagged zDHHC2 lipoparticles according to the manufacturer’s instructions. Briefly, 90 ml of Expi293 cells were cotransfected with 30 μg of pEF-BOS-HA-zDHHC2 plasmid and 90 μg of a plasmid encoding the lentiviral protein Gag. A transfection enhancer was added the next day. The supernatant of the cell culture (containing the zDHHC2 lipoparticles) was collected 72 h post-transfection and mixed with the precipitation reagent provided in the kit for 1 or 2 days at 4°C. The lipoparticles were then pelleted by centrifugation at 6000TR-FRET ratio was calculated by Biotek Gen5 software (Agilent) using the equation: acceptor signal/donor signal ∗ 10,000. The percent of inhibition was normalized by vehicle of WT enzyme  and C157S inactive mutant (inhibitor control). Calculation of percent of inhibition and quality control of whole campaign was made with Screener software (Genedata).One of the main strengths of this high-throughput assay is the absence of wash steps. Terbium homogenous TRF is indeed one of the only methods allowing for the detection of the interaction between tagged proteins and a probe without any wash. A potential limitation would be the distance between the FRET donor and the acceptor, which could be solved by testing other tags.g for 20 min at 4 °C to remove broken cells. The recombinant viruses were titrated by serial dilution on 6-well plates of Sf9 cells and plaque titration.The recombinant Baculovirus was obtained following the manufacturer’s instructions . Briefly, Sf9 cells  were plated in 6-well plates and cotransfected with 1.2 μl of baculoFectin II, 100 ng of flashback Ultra DNA, and 500 ng of transfer vector (pIEX-Bac3-hDHHC2 or the control vector expressing lacZ). Cells were incubated for 5 days at 28 °C before the supernatant (containing the recombinant baculovirus seed stock) was harvested and stored at 4 °C until required. Virus amplification was performed in Sf9 TriEX cells  grown in TriEx media . The media were removed from cells in T75 flasks (at ∼50% confluence). About 100 μl of the seed stock virus was diluted to 2 ml with medium and added to the cells. The virus was allowed to adsorb for 1 h, with periodic rocking, before being removed. About 15 ml of fresh medium was then added, and the cells were incubated for at least 5 days before the medium containing recombinant virus was harvested (this virus stock is the inoculum) and centrifuged at 1000g for 5 min. About 10 ml of PBS (pH 6.4) was added to the flask to collect the remaining cells, which were combined with the cell pellets and centrifuged again at 1000g for 5 min. The cell pellets were then washed two further times in PBS (pH 6.4) and resuspended in a small volume of PBS supplemented with protease inhibitors , pelleted, and frozen at −80 °C until required.Large-scale production of zDHHC2 was performed in Sf9 TriEX cells. Medium was removed from 15 T75 flasks of TriEX cells, and the recombinant virus was added to the cells in a total volume of 2 ml and at a multiplicity of infection of 2. The virus was allowed to adsorb at room temperature for 1 h, during which time the cells were occasionally gently rocked. The inoculum was then removed and replaced with 13 ml of fresh TriEX medium and incubated for 96 h at 28 °C. The cells were then detached by tapping the flask and collected by centrifugation at 1000n-dodecyl-β-d-maltoside , 1 mM Tris(2 carboxyethyl) phosphine hydrochloride , and protease inhibitors [as aforementioned]). About 1 ml of lysis buffer was added per cell pellet (from three T75 flasks), and the cells were disrupted further by 10 strokes of a Dounce homogenizer. The cell lysates were then rotated for 30 min at 4 °C on a wheel at a gentle speed and then clarified by ultracentrifugation at 100,000g for 30 min at 4 °C. The lysates were then supplemented with 5 to 10 mM imidazole , added to 1 ml of washed Ni2+–nitrilotriacetic acid agarose , and rotated on a low-speed wheel for at least 1 h at 4 °C. The agarose was then pelleted by centrifugation at 2000g for 5 min at 4 °C and washed five times with 10 ml of wash buffer  containing 15 mM imidazole. The washed agarose beads were resuspended in 500 μl of elution buffer 1  and rotated on a wheel for 10 min at 4 °C. The agarose was then pelleted by centrifugation at 2000g for 5 min at 4 °C. This elution step was repeated once. The agarose beads were then eluted four times with 500 μl of elution buffer 2 containing 500 mM imidazole . The eluted samples were pooled (∼3 ml) and dialyzed twice (overnight and for 4 h) against 500 ml of elution buffer without imidazole or TCEP. Purified proteins were analyzed by SDS-PAGE and Instant Blue  staining. The concentration of the enzyme was determined by comparison with bovine serum albumin (BSA) standards and found to be between 1.5 and 3 μM depending on the batch.Cell pellets were thawed and resuspended in lysis buffer pLysS cells  were transformed with either pGEX-KG (containing the GST cDNA) or pGEX-KG-rat SNAP25b  and selected with the appropriate antibiotics. Transformed cells were grown in a 1 l culture of supermedia  with shaking at 225 rpm (37 °C), and protein expression was induced by the addition of 1 mM IPTG for 5 to 6 h. Cells were pelleted and resuspended in PBS. The cells were lysed by subjecting to a freeze–thaw cycle at −80 °C, addition of 1 mg/ml lysozyme  and incubation for 30 min on ice. The cells were then sonicated, and cell debris and membranes removed by centrifugation at 20,000g for 60 min. The clarified lysates were incubated with glutathione sepharose , washed, and eluted by the addition of 2 × 1.5 ml of 10 mM reduced l-glutathione  in 50 mM Tris (pH 8). Eluted proteins were dialyzed overnight at 4 °C against 5 l of PBS in a 3.5 molecular weight cutoff slide-A-lyzer G2 cassette . Purified proteins were analyzed by SDS-PAGE and Instant Blue staining. Their concentration was estimated from the intensity of their corresponding bands as compared with the standard curve obtained with BSA standards that were run in parallel.The production and purification of GST and GST-SNAP25b has been described . BrieflyIn an uncoated 96-well plate, 0.075 μM of purified zDHHC2 enzyme was incubated with 1 μM GST-tagged substrate protein and 1 μM azide-C16:0-CoA at 25 °C for 45 min. The assay buffer consisted of 50 mM MES (pH 6.4), 100 mM NaCl, 0.1% DDM, and 1 mM TCEP. Following the reaction, NEM was added to a final concentration of 50 mM, and the plate was incubated for 2 h at room temperature with gentle rocking. Following this, 10 μl of click reagent  diluted in PBS was added  and incubated at room temperature for 1 h with rocking. About 100 μl of PBS was added to each well of the uncoated plate to dilute the samples. For assessment of ONO compounds using this assay, the compounds were preincubated with enzyme for 15 min at room temperature prior to the addition of substrates. The concentration of compounds used refers to their final concentration in the S-acylation reaction, and they were therefore added at a higher initial concentration (1.66×) for the 15 min preincubation period.Samples prepared as detailed previously were supplemented with 4 × SDS-PAGE loading buffer (containing 100 mM DTT). They were heated at 95 °C for 5 min and loaded on an SDS-PAGE gel before being transferred to a nitrocellulose membrane. The membrane was incubated with NeutrAvidin-800 and a goat anti-GST antibody and then an anti–goat-680 antibody before being imaged on a LI-COR Odyssey infrared scanner.versus normalized response (variable slope) equation (GraphPad Software).Glutathione-coated plates were activated  with two brief washes with 200 μl of PBS 0.05% v/v Tween-20. About 80 μl of PBS was then added to each well, and 20 μl of each sample (prepared as aforementioned) was added (in duplicate) and incubated at room temperature for 1 to 2 h on a rocking platform. The plate was then washed several times with 200 μl of PBS-0.05% Tween-20. About 100 μl of NeutrAvidin-HRP  diluted at 1:20,000 in PBS 0.025% Tween-20 was added to each well and incubated at room temperature for 30 min on a rocking platform. The plate was then washed several times with 250 μl of PBS-0.05% Tween-20 and incubated with 100 μl of SuperSignal ELISA Pico chemiluminescent substrate . The resulting signal was read 10 min post addition of the substrate. For quantification, the average value of GST-SNAP25 samples (without zDHHC2) was subtracted from all raw values. These values were then normalized to the value of the DMSO control sample containing GST-SNAP25 and zDHHC2, providing a percentage of activity remaining after treatment with inhibitors. The percentage inhibition was then calculated as (100 − percentage activity remaining). Data were analyzed and fitted to a nonlinear curve using the log(inhibitor) d-lysine–coated 24-well plates  and transfected with 2.4 μg of pEF-zDHHC2 plasmid (or the control empty plasmid pEF-BOS-HA) for the measurement of zDHHC2 autoacylation and 4.8 μl of Lipofectamine 2000 (Invitrogen). For substrate acylation, cells were transfected with 1.6 μg of pEF-zDHHC plasmid (or the control empty plasmid pEF-BOS-HA) together with 0.8 μg of EGFP-SNAP25b plasmid and 4.8 μl of Lipofectamine 2000. Twenty-four hours post-transfection, cells were washed once with PBS and preincubated with ONO or UoS compounds diluted in 250 μl of serum-free DMEM for 4 h. S-acylation labeling was then started by the addition of 50 μl of 600 μM of either unmodified palmitic acid C16:0 or C16:0-azide  diluted in serum-free DMEM supplemented with 1 mg/ml fatty acid–free BSA . Cells were labeled for 4 h, washed once with PBS, then lysed on ice in 100 μl of 50 mM Tris (pH 8) containing 0.5% SDS and protease inhibitors . Conjugation of IR800 alkyne dye or mPEG5K alkyne to C16:0-azide was carried out for 1 h at room temperature with end-over-end rotation by adding an equal volume of freshly prepared click chemistry reaction mixture containing the following: 5 μM IRDye 800CW alkyne  or 200 μM of mPEG5K-alkyne , 4 mM CuSO4 , 400 μM Tris[methyl]amine , and 8 mM ascorbic acid  in distilled water. About 67 μl of 4 × SDS-PAGE sample buffer containing 100 mM DTT was then added to the 200 μl sample. Samples were heated at 95 °C for 5 min, and 20 μl was resolved by SDS-PAGE and transferred to nitrocellulose membrane for immunoblot analysis. Membranes were first stained with Revert 700 total protein stain kit , imaged, destained, and then analyzed by immunoblotting. Signals were quantified with ImageStudio software (LI-COR). S-acylation was calculated as the ratio (in percent) between click signal (IR800) and GFP signal (IR680) and normalized to control conditions on the same membrane (cells expressing GFP-SNAP25 + zDHHC and treated with DMSO). For alkyne mPEG-treated samples, the efficiency of S-acylation was calculated as the ratio between the sum of the signals of the higher molecular weight bands (corresponding to modified cysteines) over the sum of all the signals. The expression of the various zDHHCs was also quantified as a ratio between the HA signal (IR680) and the Total Protein Stain (IR680) and normalized to control conditions on the same membrane (cells expressing GFP-SNAP25 + zDHHC and treated with DMSO). For the determination of the in cell IC50, the percent of S-acylation inhibition was calculated as (100 − percent of S-acylation). Data were analyzed and fitted to a nonlinear curve using the log(inhibitor) versus normalized response (variable slope) equation (GraphPad Software).HEK293T cells were plated on poly-www.phylogeny.fr.The following human ZDHHC protein sequences were retrieved from the National Center for Biotechnology Information database: ZDHHC1 (NP_001310556.1), ZDHHC2 (NP_057437.1), ZDHHC3 (NP_001128651.1), ZDHHC4 (NP_001358225.1), ZDHHC5 (NP_056272.2), ZDDHC6 (NP_071939.1), ZDHHC7 (NP_060210.2), ZDHHC8 (NP_037505.1), ZDHHC9 (NP_057116.2), ZDHHC11 (NP_079062.1), ZDHHC12 (NP_001304944.2), ZDHHC13 (NP_061901.2), ZDHHC14 (NP_078906.2), ZDHHC15 (NP_659406.1), ZDHHC16 (NP_115703.2), ZDHHC17 (NP_056151.2), ZDHHC18 (NP_115659.1), ZDHHC19 (NP_001034706.1), ZDHHC20 (NP_694983.2), ZDHHC21 (NP_001341047.1), ZDHHC22 (NP_777636.2), ZDHHC23 (NP_001307395.1), and ZDHHC24 (NP_997223.1). Sequences corresponding to the 51 amino-acid DHHC CRD were aligned with MUSCLE, phylogenic analysis was performed with PhyML, and the tree was rendered with TreeDyn. They were analyzed on 3  was added to concentrated sulfuric acid (150 ml), and the solution was heated at 120 °C for 4 h  was added in four portions while stirring. The reaction was then heated at 115 °C for 20 h while stirring. While still hot, the solution was poured onto 1 kg of ice, once all the ice was melted the suspension was filtered and pressed dry. The solid was then dissolved in hot distilled water (240 ml), and most of insoluble solids were removed by decantation and filtration of the solution while still hot. The product was then left to crystallize and filtered to give a pale-yellow solid that was dissolved in aqueous ammonia (180 ml) and water (180 ml). The mixture was left stirring at reflux for 1 h. The solution was cooled down to 4 °C for 12 h, and the solid was filtered under suction to give an orange solid that was dissolved in 100 ml of concentrated sulfuric acid and 100 ml of water. The solution was heated at reflux for 16 h. While hot, the solution was poured onto 1 kg of cracked ice. When the ice had melted, the solid was filtered under vacuum to give the title compound6 as a yellow solid . Melting point (mp): 138 °C   3462, 3345, 3107, 1636, 1513; 1H NMR  δ 8.49 , 8.39 , 6.84 ; 13C NMR  δ 140.8, 134.9, 134.0, 113.8.Chlorobenzene  for 4 h . After c–142 °C) ; low-res6  in hot acetic acid (27 ml) was added at such a rate that the temperature was kept under 40 °C . mp: 87 °C (Lit: 86–87 °C)  3103, 1701, 1528; 1H NMR  δ 8.33 , 7.93 , 6.01 ; 13C NMR  δ 150.5, 130.6, 129.3, 119.7.Sodium nitrite  was added to concentrated sulfuric acid (11 ml), and the resulting solution was heated at 40 °C for 20 min to ensure that all the solid was dissolved, then a solution of 2,6-dinitroaniline er 40 °C . After t6–87 °C) ; υmax  and potassium hydroxide  were added to water (30 ml), and the solution was heated to reflux for 1 h . mp: 63 °C (Lit: 63.5 °C)  3215, 3096, 1767, 1932, 1809, 1692, 1623, 1534; 1H NMR  δ 8.35 , 7.20 ; 13C NMR  δ 150.3, 139.8, 131.8, 118.8.2-Chloro-1,3-dinitrobenzene  for 1 h . The hot63.5 °C) ; LRMS  and 10% palladium on activated carbon  in methanol (20 ml) for 15 min (in vacuo. The crude was purified by flash chromatography (toluene:dichloromethane 9:1) to give the title compound9 as a red solid . mp: 110 °C; LRMS (ESI + APCI): m/z calculated 154.1, found 155.1 [M + H]+; υmax  3481, 3385, 3223, 3103, 2921, 2852, 1538; 1H NMR  δ 10.73 , 7.48 , 6.95 , 6.79 , 4.09 ; 13C NMR  δ 143.4, 137.9, 133.8, 120.5, 119.9, 113.6.Hydrogen was bubbled through a nitrogen purged solution of 2,6-dinitrophenol r 15 min . The rea9  and cesium carbonate  were dissolved in toluene (18 ml) and heated at 50 °C for 30 min. Then, 2-chloroacrylonitrile 10  was added, and the reaction was heated at 110 °C in a sealed vial for 4 h. After the reaction was completed, the solvent was removed under reduced pressure to give a brown solid, which was filtered through a Celite plug and eluted with acetone. The solvent was removed in vacuo to give a brown solid. The crude product was purified on silica gel (toluene:EtOAc 8:2) to give the title compound11 as a brown solid . LRMS (ESI + APCI): m/z calculated 205.2, found 204.0 [M − H]−; υmax  3392, 3111, 2927, 1780, 1688, 1616, 1532; 1H NMR  δ 7.17 , 7.05 , 6.99 , 6.15 , 5.63-5.60 , 3.89-3.75 ; 13C NMR  δ 141.2, 136.1, 134.3, 123.1, 120.0, 116.8, 114.1, 63.2, 43.0.2-Amino-6-nitrophenol H-benzo[b]oxazine-2-carbonitrile 11  and 10% palladium on activated carbon  were dissolved in methanol (5 ml), and the solution was purged with hydrogen for 15 min and left stirring under a hydrogen atmosphere for 45 min. The solution was filtered through Celite, eluted with acetone, and the solvent was removed under vacuum to give the title compound12 as a brown oil . LRMS (ESI + APCI): m/z calculated 175.2, found 176.1 [M + H]+; υmax  3370, 2966, 2863, 2251, 1623, 1498; 1H NMR  δ 6.68 , 6.22 , 6.12 , 5.15-5.10 , 3.85 , 3.74 , 3.65-3.60 ;13C NMR  δ 136.3, 132.0, 129.2, 123.1, 116.4, 107.2, 106.5, 62.7, 43.9.8-Nitro-3,4-dihydro-213  and HATU  were dissolved in DMF (1 ml) and N,N-diisopropylethylamine  was added, and the resulting solution was left stirring for 45 min. A solution of compound 12  in DMF (100 μl) was added to the acid solution, and it was left stirring at room temperature for 16 h. An aqueous solution of 5% lithium chloride (30 ml) was added, and the aqueous phase was extracted with EtOAc , the organic fractions were combined and dried over anhydrous MgSO4, the organic solution was filtered, and the solvent was removed by rotatory evaporation. The crude was purified by flash chromatography (PhMe:EtOAc 8:2) to give the title compound15 as a brown oil . LRMS (ESI + APCI): m/z calculated 393.2, found 394.2 [M + H]+; υmax  3381, 2956, 2932, 2859, 2552, 1662, 1610, 1506;1H NMR  δ 8.68 , 7.96-7.92 , 7.71 , 7.09-7.01 , 6.82 , 6.53 , 5.58-5.51 , 4.09 , 3.79-3.65 , 1.86-1.76 , 1.55-1.26 , 0.96-0.83 ; 13C NMR  δ 165.0, 163.0, 134.0, 131.6, 130.0, 129.2, 128.1, 123.0, 117.5, 115.1, 111.8, 68.9, 63.5, 43.9, 32.6, 26.7, 23.3, 14.3; three carbons missing.4-(Heptyloxy)benzoic acid 14  and HATU  were dissolved in DMF (1 ml) and N,N-diisopropylethylamine  was added, and the resulting solution was left stirring for 45 min. A solution of compound 12  in DMF (100 μl) was added to the acid solution, and it was left stirring at room temperature for 16 h. A solution of 5% lithium chloride (30 ml) was added, and the aqueous phase was extracted with EtOAc , the organic fractions were combined and dried over anhydrous MgSO4, the organic solution was filtered, and the solvent was removed by rotatory evaporation. The crude was purified by flash chromatography (PhMe:EtOAc 8:2) to give the title compound16 as a brown oil . LRMS (ESI + APCI): m/z calculated 427.2, found [M + H]+; υmax  3375, 3087, 3062, 3029, 2940, 2863, 2543, 1660, 1608; 1H NMR  δ 8.68 , 7.97-7.91 , 7.72 , 7.32-7.22 , 7.20-7.14 , 7.06-7.01 , 6.82 , 6.53 , 5.55-5.51 , 4.15-4.09 , 3.77-3.64 , 2.71 , 1.91-1.72 ; 13C NMR  δ 165.0, 162.9, 143.2, 134.0, 131.6, 130.0, 129.2, 129.1, 128.1, 126.6, 123.0, 117.5, 115.1, 111.9, 68.7, 63.5, 43.9, 36.1, 28.7; three carbons missing.4-(Benzyloxy)benzoic acid 15  was dissolved in DMF (100 μl) and then sodium azide  and triethylammonium chloride  were added and the vial was sealed and heated at 140 °C for 2 h. The contents of the vial were left to cool down to room temperature, and a solution of 5% lithium chloride in water (30 ml) was added, the aqueous phase was extracted with ether , the organic fractions were combined, and the solvent was removed in vacuo to give the title compound TTZ-1 (1) as a brown oil . LRMS (ESI + APCI): m/z calculated 436.2, found 435.1 [M − H]−; υmax  3325, 3271, 2951, 2928, 2874, 2859, 2735, 2617, 2470, 1640; 1H NMR  δ 9.49 , 8.11-8.04 , 7.12-7.03 , 6.84 , 6.75 , 6.52 , 5.89 , 5.43 , 4.11 , 4.03-3.78 , 1.90-1.74 , 1.53-1.45 .1.59-1.20 , 0.95-0.82 ; 13C NMR  δ 167.5, 163.4, 156.7, 136.1, 135.2, 130.7, 127.1, 126.7, 122.6, 115.7, 115.2, 114.3, 69.2, 69.0, 43.5, 32.5, 29.9, 26.7, 23.3, 14.3; 1 carbon missing.Compound 16  was dissolved in DMF (100 μl), and then sodium azide  and triethylammonium chloride  were added and the vial was sealed and heated at 140 °C for 2 h. The contents of the vial were left to cool down to room temperature, and a solution of 5% lithium chloride in water (30 ml) was added, the aqueous phase was extracted with ether , the organic fractions were combined, and the solvent was removed in vacuo to give the title compound TTZ-2 (2) as a brown oil . LRMS (ESI + APCI): m/z calculated 470.2, found 469.1 [M − H]−; υmax  3331, 3027, 2928, 2858, 1701, 1638, 1608; 1H NMR  δ 9.49 b, 8.11-8.04 , 7.32-7.14 , 7.11-7.04 , 6.84 , 6.75 , 6.52 , 5.89 , 5.43 , 4.21-4.07 , 4.03-3.74 , 2.75-2.68 , 1.89-1.77 ; 13C NMR  δ 168.1, 168.0, 167.5, 163.4, 151.8, 143.2, 130.7, 129.3, 129.2, 126.6, 122.5, 115.7, 115.2, 114.3, 69.2, 68.8, 55.5, 43.5, 36.1, 28.7; two carbons missing.Compound 1H NMR and 13C NMR spectra are available as supporting information.All data are contained within the article. Copies of This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Acanthamoeba is a free-living protozoan known to cause keratitis most commonly, especially among contact lens wearers. Treatment of Acanthamoeba keratitis is challenging as Acanthamoeba can encyst from the active form, a trophozoite, into a hibernating cyst that is refractory to antibiotics and difficult to kill; therefore, there is a need for more effective anti-amoebic strategies. In this study, we have evaluated the anti-amoebic activity of the antimicrobial peptide mimic RK-758 against Acanthamoeba castellanii. RK-758 peptidomimetic was subjected to biological assays to investigate its amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects on A. castellanii. The anti-amoebic activity of the peptide mimic RK-758 was compared with chlorhexidine against the Acanthamoeba castellanii ATCC30868 and Acanthamoeba castellanii 044  with the concentrations of both ranging from 125 µM down to 7.81 µM. All experiments were performed in duplicate with three independent replicates. The data were represented as mean ± SE and analysed using a two-sample t-test and two-tailed distributions. A p < 0.05 was considered statistically significant. The peptidomimetic RK-758 had anti-Acanthamoeba activity against both trophozoites and cysts in a dose-dependent manner. The RK-758 had amoebicidal and growth inhibitory activities of ≥50% at a concentration between 125 µM and 15.6 µM against the trophozoites of both Acanthamoeba strains. Inhibitory effects on the cyst formation and trophozoite re-emergence from cysts were noted at similar concentrations. Chlorhexidine had 50% activity at 7.81 µM and above against the trophozoites and cysts of both strains. In the haemolysis assay, the RK-758 lysed horse RBCs at concentrations greater than 50 µM whereas lysis occurred at concentrations greater than 125 µM for the chlorhexidine. The peptidomimetic RK-758, therefore, has activity against both the trophozoite and cyst forms of Acanthamoeba and has the potential to be further developed as an anti-microbial agent against Acanthamoeba. RK-758 may also have use as an anti-amoebic disinfectant in contact lens solutions. Acanthamoeba is a single-celled eukaryotic microorganism known to cause rare but sight-threatening keratitis, especially among contact lens (CL) wearers [Acanthamoeba exists in two forms, the trophozoite is the pathogenic form which is metabolically active and responsible for causing the corneal infection, whereas the cyst form is metabolically dormant or inactive but resistant to disinfectants and antimicrobials [Acanthamoeba keratitis (AK) remains a challenge and it can lead to corneal toxicity. The current treatment mainly involves topical medications such as chlorhexidine (0.02–0.2%) or polyhexamethylene biguanide (PHMB) alone or in combination with diamidines (propamidine isethionate or hexamidine (0.1% w/v), oral miltefosine, and penetrating keratoplasty in non-responding cases [Acanthamoeba can be resistant to these normal treatment modalities, allowing the disease to worsen [ wearers ,2. Acantcrobials . The phecrobials . Even ifng cases ,5,6,7. Fng cases . The treng cases ,9. TopicAntimicrobial peptides (AMPs) are naturally occurring molecules that have gained importance due to their broad-spectrum antimicrobial properties . AMPs haCandida sp., Cryptococcus sp., and Saccharomyces sp.) and protozoa (Leishmania sp.). For example, histatin 5 is active against Candida sp. and Aspergillus fumigatus [Leishmania major, Cryptococcus neoformans, and Plasmodium falciparum [Most AMPs are highly cationic and act by membrane disruption with selective binding to the negatively charged surfaces of pathogens. The most membrane-active AMPs contain both hydrophilic and hydrophobic groups which aggregate at the cell membranes after initial electrostatic interactions with negatively charged lipid groups in prokaryotic membranes, followed by insertion into the cell membrane using their hydrophobic meioties . Cationiumigatus , and derlciparum ; howeverlciparum ,18.Acanthamoeba [Acanthamoeba trophozoites and cysts [A. castellanii but these peptides were sensitive to the proteases released by trophozoites [A. castellanii by permeabilising the cell membrane at relatively higher concentrations. This AMP was also able to reduce the number of Legionella residing within the Acanthamoeba [Acanthamoeba trophozoites and cysts in a phosphate buffer saline or ex vivo corneas [A. castellanii and Naegleria floweri [A. castellanii clinical isolate [Lactococcus lactis, can also be active against A. castellanii trophozoites at the end of a 24 h incubation [A castellanii trophozoites, indicating a possible role in combating amoebic infection [The highly cationic peptide, protamine, its derivative, melimine, and magainin are active against thamoeba ,19,20,21nd cysts . Additiohozoites . Anotherthamoeba . Poly-ep corneas . AMPs de floweri . A synth isolate . Nisin, cubation . Moreovenfection .Candida albicans and Gram-negative bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Proteus vulgaris [Trypanosoma brucei brucei, which causes sleeping sickness in humans [Staphylococcus aureus, Escherichia coli [Pseudomonas aeruginosa [S. aureus [Peptidomimetics are synthetic compounds designed to mimic the biological function of peptides and overcome the limitations of AMPs, such as their relatively short half-lives in vivo that occurs due to proteolytic degradation, the high cost of their synthesis, and the toxicity of certain AMPs. To mimic the biological function of natural AMPs, peptidomimetic molecules contain amphiphilic, i.e., hydrophobic and hydrophilic backbones with a net positive charge . Studiesvulgaris . Benzodin humans . The curhia coli , and Pseruginosa . Similarruginosa . Peptide. aureus .Acanthamoeba must not cause the amoeba to encyst, as this may result in a re-activation/re-infection after the cessation of therapy. The importance of encystment was demonstrated in an Acanthamoeba keratitis worldwide outbreak. This outbreak occurred as the result of contact lens wearers using Complete MoisturePlus  as a disinfecting solution [Acanthamoeba keratitis outbreak was that the solution, unlike other disinfectants, caused Acanthamoeba to encyst [Acanthamoeba testing of contact lens solutions [Any potential therapeutics for solution ,39. Thersolution . It was o encyst . A case-olutions . Some ofolutions ,42. In tolutions ,44.Acanthamoeba trophozoites and cysts in comparison with chlorhexidine.This study evaluated the anti-amoebic activity of an antimicrobial peptidomimetic, RK-758, against Acanthamoeba castellanii strains ATCC30868 and 044  [2 tissue culture flasks at 32 °C for 5–7 days. Trophozoites were collected once a 90% confluency was achieved by washing and resuspending in 1× phosphate buffered saline  or PYG (depending upon the subsequent assay) by centrifugation at 500 g for 2 min. Cysts were obtained by seeding approximately 5 × 105 trophozoites/mL on non-nutrient agar (NNA) plates  and incubating the plates at 32 °C for 14 days. The cysts were scrapped and washed in PBS by centrifugation at 3500× g for 10 min and resuspended in PBS [The isolate)  were culisolate)  in 75 cmd in PBS . The cysThe test compound in this study, peptidomimetic RK-758, was synthesized according to the patents WO2018081869A1 and Australian Provisional Patent Application No. 2021902457, and its chemical structure has been published . The chl5 trophozoites/mL were incubated with the peptidomimetic RK-758 or chlorhexidine in 24-well plates at concentrations ranging from 125 µM to 7.81 µM in PBS [Briefly, 5 × 10M in PBS . The chlM in PBS . PBS aloM in PBS .5 trophozoites/mL were incubated with the peptidomimetic RK-758 or chlorhexidine in 24-well plates at concentrations ranging from 125 µM to 7.81 µM in PYG [Briefly, 2 × 10M in PYG . The PYG2 and 10% glucose to 1× PBS by filter sterilization using 0.22 µM membrane filters  [5 trophozoites/mL) were incubated with either peptidomimetic RK-758 or chlorhexidine in 24-well plates with concentrations ranging from 125 µM to 7.81 µM in the encystment media. The encystment medium alone was used as a control. The trophozoites were incubated at 30 °C for 72 h followed by adding 0.25% (w/v) sodium-dodecyl sulphate to each well to burst the trophozoites leaving the cysts intact [To measure the ability of the peptidomimetic RK-758 or chlorhexidine compared to the ability of the trophozoites to encyst, an encystment medium was prepared by adding 50 mM MgClIreland) . The tros intact . The num5 cysts/mL were incubated with the peptidomimetic RK-758 or chlorhexidine in 24-well plates at concentrations ranging from 125 µM to 7.81 µM in PYG. The PYG medium alone was used as a control. The plates were observed every day at a 10× and 40× magnification to assess the emergence of trophozoites during incubation at 30 °C for 72 h. At the end of the incubation period, the number of trophozoites that re-emerged was counted using a Neubauer haemocytometer.In this assay ,48, 5 × g for 5 min. Chlorhexidine and peptidomimetic RK-758 concentrations ranging from 200 µM to 12.5 µM were added to the washed HRBCs and incubated at 37 °C for 4 h. The PBS was used as a negative control to achieve a 0% lysis. HRBCs in distilled water were used as positive controls to achieve 100% lysis. At the end of the incubation, the cells were pelleted at 1057× g for 5 min, and the supernatant was removed to assess the release of haemoglobin by measuring the OD at 540 nm. The relative OD of the HRBCs treated with the compounds, chlorhexidine and peptidomimetic RK-758, was compared to that of those treated with distilled water and used to determine the relative percentage of haemolysis as follows:The haemolytic activities of both the chlorhexidine and peptidomimetic RK-758 were determined using horse red blood cells  as described previously ,50. Briet-test and two-tailed distributions. A p < 0.05 was considered statistically significant.The statistical analyses were performed using the GraphPad Prism 8.4.3 software . All the experiments were performed in duplicate with three independent replicates. The data were represented as mean ± SE and analysed using a two-sample A. castellanii ATCC30868 and 044 strains. The number of viable trophozoites was 1.73 × 105 for A. castellanii ATCC30868 and 2.69 × 105 for A. castellanii 044 following a 24 h incubation in PBS alone. The RK-758 caused statistically significant killing of trophozoites between 79%, 87%, and 76% against A. castellanii ATCC30868 (the number of viable trophozoites reduced to 3.58 × 104 (p = 0.03), 2.17 × 104 (p = 0.01), and 1.6 × 104 (p = 0.03), respectively) (castellanii 044 (the number of viable trophozoites reduced to 5 × 103 (p = 0.001), 3.3 × 104 (p = 0.003), and 1.1 × 105 (p = 0.01),respectively) (castellanii ATCC30868 (the number of viable trophozoites reduced to 6.67 × 103 (p = 0.01), 3.17 × 104 (p = 0.03), and 3.08 × 104 (p = 0.02), respectively) (castellanii 044 (the number of viable trophozoites reduced to 5.8 × 103 (p = 0.001), 2.5 × 104 (p = 0.002), and 7.1 × 104 (p = 0.004),respectively)  (p < 0.05).Amoebicidal assays were performed to determine the activity of the peptidomimetic RK-758 in comparison to the chlorhexidine on the viability of ctively) A, and 98ctively) B, at tesctively) A, and 98ctively) B, at thectively) B at 15.6A. castellanii ATCC30868 and 044 strains. The number of viable trophozoites was enumerated to be 3.08 × 105 for A. castellanii ATCC30868 and 2.17 × 105 for A. castellanii 044 following a 48 h incubation in PYG alone. The RK-758 showed the statistically significant growth inhibition of trophozoites between 97% and 63% against A. castellanii ATCC30868 (the number of viable trophozoites reduced to 9.17 × 103 at 62.5 µM (p = 0.0008) and 1.13 × 105 at 7.81 µM (p = 0.03) (castellanii 044 (the number of viable trophozoites reduced to 3.08 × 104 at 7.81 µM (p < 0.0001) (castellanii ATCC30868 (the number of viable trophozoites reduced to 1.67 × 103 at 125 µM (p = 0.0007) and 5.25 × 104 at 7.81 µM (p = 0.002)  (p < 0.05), except for the A. castellanii ATCC30868 at 125 µM, where the chlorhexidine had greater activity (p = 0.005).Growth inhibition assays were performed to determine the amoebistatic activity of the peptidomimetic RK-758 and chlorhexidine against the  = 0.03) A, and 10 0.0001) B, at tes= 0.002) A, and 10< 0.001) B, at simA. castellanii ATCC30868 and 044 strains. At the end of the 72 h incubation, 9.75 × 104 cysts of A. castellanii ATCC30868 and 8.75 × 104 cysts of A. castellanii 044 had formed in the encystment medium. The RK-758 inhibited cyst formation by 81% and 68% against A. castellanii ATCC30868   (Encystation assays were performed to determine the anti-encystation ability of the chlorhexidine and peptidomimetic RK-758 against ctively) A at 125 ctively) B, at tesA. castellanii ATCC30868   (A. castellanii ATCC30868 at 62.5 µM and 31.25 µM concentrations (p = 0.003 and p = 0.04), respectively. Similarly, a statistically significant difference was noted between the activities of the RK-758 and chlorhexidine against the A. castellanii 044 at 62.5 µM and 15.6 µM concentrations .Chlorhexidine inhibited cyst formation by between 94% and 54% for ctively) A, at tes =0.002) B at testA. castellanii ATCC30868 and 044 strains. At the end of the 72 h incubation, 2.56 × 105 trophozoites of A. castellanii ATCC30868 and 2.52 × 105 trophozoites of A. castellanii 044 had re-emerged in the PYG medium. The peptidomimetic RK-758 inhibited the trophozoites’ re-emergence between 100% and 98% at concentrations ranging from 125 µM to 7.81 µM against A. castellanii ATCC30868  and between the activities of the RK-758 and chlorhexidine against A. castellanii 044 at 15.6 µM and 7.81 µM concentrations .There was a statistically significant difference between the activities of the RK-758 and chlorhexidine at 7.81 µM against A. Chlorhexidine and peptidomimetic RK-758 caused haemolysis in a dose-dependent manner. Chlorhexidine showed a negligible haemolysis between 31.25 µM and 7. 81 µM. The therapeutic index  for the chlorhexidine ranged from 16 to 4. The peptidomimetic RK-758 showed haemolysis of < 50% between 31.25 µM and 7.81 µM. The therapeutic index of the RK-758 ranged from 8 to 1.Acanthamoeba castellanii, most probably due to its cationic charge [Acanthamoeba infections [Acanthamoeba infection.This study demonstrated that the peptidomimetic RK-758 was active against both the trophozoite and cyst forms of c charge ,20,25. Tc charge ,4,5. Thifections . A laborfections . Recentlfections . The curAcanthamoeba effects of peptidomimetics had not been evaluated.Previous studies have already established that peptidomimetics are highly effective against bacteria ,36,37, bAcanthamoeba trophozoites’ adhesion to contact lens at a concentration of 152 µg per lens [A. castellanii trophozoites to nisin, a bacteriocin AMP, resulted in reductions in cultivable numbers after a 24 h exposure but there was a recovery in the amoeba growth after a 72 h exposure. This may have been due to the lack of a nisin effect on the membrane integrity of the trophozoites [Previously, the naturally occurring antimicrobial peptide protamine had been shown to have amoebicidal activity at 228 µM, and its derivative melimine inhibited per lens ,19. Addiper lens ,21. The per lens . An AMP per lens . The exphozoites . The pephozoites , which mhozoites .Acanthamoeba clinical isolates at 3.1 to 25 µg/mL (6 to 49 µM) [The current study found that RK-758 performed almost equivalently to the currently used topical treatment, chlorhexidine. Chlorhexidine has previously been reported to have minimum cysticidal activity against nine o 49 µM)  and the o 49 µM) . Future 50 (a dose causing 50% death in mice) as a measure of toxicity, polyhexamethylene biguanide, another commonly used therapeutic for Acanthamoeba keratitis [50 are not directly comparable; therefore, experiments directly comparing the therapeutic index of currently used therapies with RK-758 are required. A previous study showed that chlorhexidine at 20 µg/mL (31.97 µM) gave a 20% lysis of rabbit red blood cells after 2 h of incubation [A haemolysis assay is often used as a primary measure of the toxicity of antimicrobial peptides . The RK-eratitis ,58, has eratitis . Howevercubation . SimilarA. castellanii. The authors suggested that the inclusion of 50 mM of MgCl2 and 10% glucose in the PBS formulation induced encystment [The current study used PBS as a negative control to evaluate the viability of trophozoites in an amoebicidal assay; however, a previous study reported that PBS alone induced 28% encystment after 24 h of incubation . A latercystment . Elsewhecystment . The curAcanthamoeba activity on the trophozoites and cysts of A. castellanii ATCC30868 and 044 strains. Its activity is very similar to chlorhexidine, which indicates that RK-758 has the potential to be developed as a new therapeutic agent for the treatment of Acanthamoeba keratitis or a new disinfectant for contact lenses with good activity against Acanthamoeba.In summary, the peptidomimetic RK-758 has excellent anti-


Objective: Numerous pathogenic variants in KCNB1, which encodes the voltage-gated potassium channel, KV2.1, are linked to developmental and epileptic encephalopathies and associated with loss-of-function, -regulation, and -expression of the channel. Here we describe a novel de novo variant (P17T) occurring in the KV2.1 channel that is associated with a gain-of-function (GoF), with altered steady-state inactivation and reduced sensitivity to the selective toxin, guanxitoxin-1E and is clinically associated with neurodevelopmental disorders, without seizures.Methods: The autosomal dominant variant was identified using whole exome sequencing (WES). The functional effects of the KCNB1 variant on the encoded KV2.1 channel were investigated using whole-cell patch-clamp recordings.Results: We identified a de novo missense variant in the coding region of the KCNB1 gene, c.49C>A which encodes a p.P17T mutation in the N-terminus of the voltage-gated, KV2.1 potassium channel. Electrophysiological studies measuring the impact of the variant on the functional properties of the channel, identified a gain of current, rightward shifts in the steady-state inactivation curve and reduced sensitivity to the blocker, guanxitoxin-1E.Interpretation: The clinical evaluation of this KCNB1 mutation describes a novel variant that is associated with global developmental delays, mild hypotonia and joint laxity, but without seizures. Most of the phenotypic features described are reported for other variants of the KCNB1 gene. However, the absence of early-onset epileptic disorders is a much less common occurrence. This lack of seizure activity may be because other variants reported have resulted in loss-of-function of the encoded KV2.1 potassium channel, whereas this variant causes a gain-of-function. V2.1  is a voltage-gated potassium channel protein which, in humans, is encoded by the KCNB1 gene (V2.1 channels give rise to a predominant delayed rectifier current (IK) that regulates action potential duration, firing frequency and neuronal excitability , with a pore (P) loop between S5 and S6, (the S5-S6 linker) which incorporates the selectivity filter of the channel, while the voltage sensor is found on S4 . Proline 17 is located in the N-terminal region of the KV2.1 channel, proximal to the inactivation domain, which is distinct from all but one of the mutations described above , was used to assist in interpretation. Additional sequencing technology and the variant interpretation protocol has been previously described (http://www.ncbi.nlm.nih.gov/clinvar/submitters/26957/).Exome sequencing was carried out by GeneDx. Using genomic DNA from the proband and parents, the exonic regions and flanking splice junctions of the genome were captured using the SureSelect Human All Exon V4 (50 Mb), the Clinical Research Exome kit  or the IDT xGen Exome Research Panel v1.0 . Massively parallel (NextGen) sequencing was done on an Illumina system with 100 bp or greater paired-end reads. Reads were aligned to human genome build GRCh37/UCSC hg19 and analyzed for sequence variants using a custom-developed analysis tool. A prediction tool, PROVEAN,  and human voltage-gated channel modifier subfamily S member 3 , Genbank™ NM_002252 incorporated into pCMV6-XL5  was utilised. The reporter plasmid, Green Fluorescent Protein (GFP) incorporated in the pcDNA3.1 vector, was a kind gift of Helen Meadows, GlaxoSmithKline, United Kingdom. The clinically identified KV2.1 mutation, proline (P) to threonine (T) at position 17  was introduced using the QuikChange kit  as previously described (Human full-length wild-type (WT) voltage-gated channel, escribed . All conV2.1 or KV2.1_P17T was co-expressed with the KV9.3 subunit a ratio of 0.5:0.5 µg was transfected.All individual expression plasmids were transiently co-expressed along with the green fluorescent reporter gene GFP at a concentration of 0.5 µg per well , using a modified calcium-phosphate protocol, as previously described  in a mod2, 1 CaCl2 and 10 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and an intracellular solution comprising 150 KCl, 3 MgCl2, 5 EGTA and 10 HEPES, adjusted to pH 7.4 for both solutions. For extracellular solutions, containing 10 mM tetraethylammonium (TEA) an equimolar amount of NaCl was substituted. Guangxitoxin 1E (GxTx-1E) was purchased from Tocris  and prepared in distilled water to a concentration of 10 µM and stored at −20°C until required. GxTX-1E was diluted directly into the extracellular solution prior to experimentation at a concentration of either 10 or 100 nM and cells were incubated in toxin for 20 min prior to recording.Electrophysiological experiments were conducted using an extracellular solution comprising (in mM): 145 NaCl, 2.5 KCl, 2 MgCloC) using an Axopatch 1D patch clamp amplifier (molecular Devices), low-pass filtered at 5 kHz before sampling (2–10 kHz).Currents passing through WT or mutant channels transiently expressed in tsA201 cells were identified by their green fluorescence  and were recorded using whole-cell patch clamp electrophysiology using three different protocols (A–C). Protocol A see  consisten represents the number of individual cells for each experimental condition. Where current amplitudes were used for comparisons, control currents  were recorded on the same experimental days. Statistical comparisons were carried out using a one-way analysis of variance (ANOVA) with a post-hoc Dunnett’s multiple comparisons test. Data was considered statistically different p < .05 (*), p < .01 (**), p < 0.001 (***) and p < 0.0001 (****).A measurement of current  for WT or the mutant channel was taken at the +50 mV step following the −80 mV prepulse of Protocol B and normalized to the cells membrane capacitance . Data acquisition was performed using pClamp 10.2 software (Axon Instruments). Data acquired was analysed using GraphPad Prism six software and Microsoft Excel and expressed as the mean ±95% Confidence Intervals (CI) and This patient was the full-term product of a normal pregnancy, labor and delivery born to a 30- year-old mother and a 39-year-old father. At 18.5 months old, he presented to our clinic due to global developmental delays. He had no intelligible speech, pulled to stand, took a few steps with support, army crawled, and scooted on his bottom. At 5 years 2 months, his height was 115 cm (85th percentile), with weight 18.5 kg (45th percentile), and head circumference 52.5 cm (81st percentile). His facial features were normal with no dysmorphic features. His speech articulation was compromised by a flaccid tongue that did not articulate speech sounds well. He had mild pectus excavatum, prominent fetal finger tip pads on his middle 3 fingers, and malalignment of his toes with incurving of his fourth and fifth toes, as well as incurving of his hallux and first toe. He had everted flat feet with a wide-based gait, mild joint laxity, good strength, and mild hypotonia. He had good social skills, a very short attention span, and a high level of activity. Developmental evaluation at age 34 months showed receptive language skills at 27–30 months, expressive language skills at 15–18 months, play skills at 30–33 months, interaction/attachment at 15–18 months, and social-emotional skills at 31 months. EKG and EEG were normal, and he has never had any seizures.de novo variant (p.Pro17Thr (CCG>ACG): c.49 C>A) in exon 1 in the KCNB1 gene of this patient (Gene Dx #1859197). This individual’s mother (GeneDx #1859680) and father (GeneDx #1860837) do not harbour this P17T variant and sequence analysis and deletion testing of the mitochondrial genome in this patient was negative.Exome sequencing revealed a KCNB1 gene has not been reported previously. This variant is a non-conservative amino acid substitution, which is likely to impact secondary protein structure as these residues differ in polarity, charge, size and/or other properties. In-silico analyses (see Methods), suggested that P17T is a likely pathogenic variant, consistent with the global developmental delay and hypotonia seen in this individual. These findings led us to investigate the functional properties of the P17T variant directly in this study.The P17T variant in the V2.1-WT channels transiently transfected into human tsA-201 cells, were elicited by 10 mV steps in voltage from −80 to +60 mV  95% CI = 221 to 367 ] to cells expressing 500 ng GFP alone, (−1 (n = 13) 95% CI = 28 to 37  the endogenous current to 7.4 pA pF−1 (n = 4) 95% CI = 0.4 to 14  95% CI = 466 to 775  than both background and KV2.1-WT currents, respectively.Similar experiments with homozygous human K6 to 775  that werV2.1-P17T with KV2.1-WT in a 1:1 ratio. Co-expression of KV2.1-WT with KV2.1-P17T resulted in large outward currents of 707 pA pF−1 (n = 3) 95% CI = 76 to 1,338 that were not significantly different  from the homozygous mutant channel (KV2.1-P17T) but were significantly different  from homozygous KV2.1-WT  do cause changes in reversal potential, indicative of effects on ion selectivity with a loss of selectivity for K+ over Na+ seen in these mutated channels  with KV2.1-WT (500 ng) gave a current density of 95 pA pF−1 (n = 8) 95% CI = −3 to 193 (see V2.1-P17T when co-expressed with KV9.3 was 473 pA pF−1 (n = 6) 95% CI = 365 to 581 (see V2.1-P17T/KV9.3 current density is significantly larger than KV2.1-WT/KV9.3 [p < 0.0001 (95% CI = −665 to −90), see V2.1-P17T also acts as a dominant positive in the presence of Kv9.3 channels. Using protocol B, it is clear that there is no difference in the reversal potential of currents evoked in cells transfected with KV2.1-WT/KV9.3  occurs at −32.5 mV  compared to KV2.1 channels alone (8.8 mV).The P17T mutation shifts the steady state inactivation curve for KV2.1 channels whether alone or co-expressed with KV9.3.These shifts in the steady state inactivation curves can explain the increased currents seen for the mutated channels, when steps are made from a voltage close to the resting membrane potential , 3, sincV2.1 channels (V2.1 channel (V2.1 channels was not seen for the mutated channels. Guanxitoxin-1E (10 nM) reduced KV2.1-WT current from 199 ± 30 pA/pF (n = 12) to 122 ± 39 pA/pF (n = 6), (p < .05) but had no effect on KV2.1-P17T current . However, the toxin was effective on both WT and mutated KV2.1 channels at a 10-fold higher concentration (100 nM) showing that whilst the toxin has a reduced potency against the mutated channels, it can still block these channels at higher concentrations.The tarantula toxin, guanxitoxin-1E, is a known blocking agent of Kchannels , whose a channel , being l channel . Given tKCNB1  resulting in neurodevelopmental delay, speech apraxia, normal growth, normal EKG, and normal EEG. However, unlike 85% of patients identified with mutations in KCNB1, the patient has not developed seizures . As suchKCNB1 .A receptor GABRA5 which may underlie many if not all of the clinical symptoms observed  where mutations of the channel lead to paroxysmal non-kinesigenic dyskinesia (PNKD3) with a broad spectrum of developmental and neurological phenotypes and seizures seen in a proportion of the patients (It is perhaps surprising that although the novel GoF mutation described here does not lead to epileptic seizures it does lead to several symptoms such as developmental delay and speech apraxia. This may suggest that alteration in channel function in either direction away from the baseline activity seen in the absence of mutations can lead to the same clinical outcome, as seen for the K2P potassium channel TASK-3 in syndrome . A similpatients . Some ofpatients .per se that leads to at least some of the common phenotypes observed across all mutations whether GOF or LOF, but rather, some other property of the mutated KV2.1 channels. In this respect it is of interest that KV2.1 channels have a non-conducting role in forming stable junctions between endoplasmic reticulum and plasma membranes and that this function produces effects that are independent of the channels roles in regulating membrane excitability (Alternatively, it may not the level of channel activity tability .V2.1 channel toxin, GxTx-1, is less effective at blocking P17T mutated channels compared to KV2.1-WT channels (V2.1 channels may also have a different potency when used to block mutated channels, compared to WT channels, because of the altered functional properties of the channel. This has implications for any therapeutic approaches designed to reduce channel activity to resting levels, where it will be important that these approaches are tested against the mutated KV2.1 channels and not solely KV2.1-WT channels.Interestingly, we have shown that the Kchannels , becausechannels . Other e