There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials. Parkinson's disease is a common and complex neurodegenerative disorder affecting ∼1% of people over the age of 65 –4. The lMounting evidence indicates that all pathogenic mutations, including G2019S, exert their effects by increasing LRRK2 kinase activity. As such there is considerable interest in targeting LRRK2 for the prevention and possibly treatment of Parkinson's . AccordiRecent work has defined a subset of Rab GTPase proteins as the first validated physiological substrates of LRRK2 . LRRK2 din vivo efficacy of LRRK2 inhibitors in cell line and animal models  to a final total protein concentration of 1 µg/µl and heated at 70°C for 10 min. Ten microgram of samples were loaded onto NuPAGE 4–12% Bis–Tris Midi Gel  and electrophoresed at 130 V for 2 h with the NuPAGE MOPS SDS running buffer . At the end of electrophoresis, proteins were electrophoretically transferred onto the nitrocellulose membrane  at 100 V for 90 min on ice in the transfer buffer (48 mM Tris–HCl and 39 mM glycine). Transferred membrane was blocked with 5% (w/v) skim milk powder dissolved in TBS-T  at room temperature for 1 h. The membrane was then cropped into three pieces, namely the ‘top piece’ (from the top of the membrane to 75 kDa), the ‘middle piece’ (between 75 and 30 kDa) and the ‘bottom piece’ (from 30 kDa to the bottom of the membrane). The top piece was incubated with rabbit anti-LRRK2 pS935 UDD2 antibody multiplexed with mouse anti-LRRK2 C-terminus total antibody diluted in 5% (w/v) skim milk powder in TBS-T to a final concentration of 1 µg/ml for each of the antibody. The middle piece was incubated with mouse anti-GAPDH antibody diluted in 5% (w/v) skim milk powder in TBS-T to a final concentration of 50 ng/ml. The bottom pieces were incubated with rabbit MJFF-pRAB102 to a final concentration of 10 mM before loading gels. Gels for Phos-tag SDS–PAGE consisted of a stacking gel  and a separating gel . After centrifugation at 20 800 g for 1 min, 10 µg of samples were loaded and electrophoresed at 70 V for the stacking part and at 120 V for the separating part with the running buffer . For immunoblot analysis, gels were washed for 10 min in the transfer buffer  containing 10 mM EDTA and 0.05% (w/v) SDS three times, followed by one wash in the transfer buffer containing 0.05% SDS for 10 min. Proteins were electrophoretically transferred onto nitrocellulose membranes  at 100 V for 180 min on ice in the transfer buffer (48 mM Tris–HCl and 39 mM glycine). Transferred membranes were blocked with 5% (w/v) skim milk powder dissolved in TBS-T  at room temperature for 1 h. Membranes were then incubated with primary antibodies diluted in 5% (w/v) skim milk powder or 2% (w/v) bovine serum albumin in TBS-T overnight at 4°C. After washing membranes in TBS-T, membranes were incubated with secondary antibody (horseradish peroxidase-labelled secondary antibodies diluted in 5% skim milk powder in TBS-T) for Phos-tag immunoblot at room temperature for 1 h. After washing membranes in TBS-T, protein bands were detected by exposing films [Amersham Hyperfilm ECL ] to the membranes using an ECL solution [SuperSignal West Dura Extended Duration (Thermo Fisher Scientific)].Phos-tag acrylamide was stored at 5 mM aqueous solution (3.43 mg of compound in 1 ml of solution) at −80°C in black tubes that block out light as Phos-tag acrylamide is light-sensitive. HPLC analysis of stock Phos-tag acrylamide was undertaken every 4–5 weeks to ensure stock had not started to polymerise. Phos-tag analysis including sample preparation was undertaken as described recently . Brieflyhttp://www.immprot.org/) [5 copies per cells) as well as monocytes (∼1 × 105 copies per cells), but is virtually undetectable in B and T lymphocytes as well as natural killer and dendritic cells that constitute most of the PBMCs (6 copies per cells) and is up to 10-fold less abundant in all other PBMCs (2–4 × 105 copies per cell)  . This suhe PBMCs . Rab10 ier cell) . Blood cTo our knowledge, the LRRK2 pathway activity has not been assessed in human neutrophils before. We therefore isolated neutrophils from peripheral blood from 12 healthy volunteers exploiting an immunomagnetic negative isolation approach, in which all non-neutrophils are targeted for removal with antibody complexes recognizing unwanted cells, including red blood cells and platelets, leaving only neutrophils in the supernatant. Flow cytometry analysis with the CD66b− Fluorescein isothiocyanate neutrophil marker revealed that the purity of neutrophils isolated from each volunteer was 97–99% and viability of cells was ∼99% assessed with DAPI staining , sufficiTo ensure that observed LRRK2 Ser935 and Rab10 phosphorylation was mediated by LRRK2, we incubated each batch of neutrophils with or without 100 nM MLi-2, a very selective and potent LRRK2 inhibitor ,27. NeutImmunoblotting using the MJFF-pRab10 monoclonal antibody revealed a robust signal in all neutrophil samples, which was markedly suppressed by MLi-2 LRRK2 inhibitor treatment . Rab10 pWe next explored the feasibility of analysing LRRK2 pathway activity in neutrophils isolated from a small number of Parkinson's patients with and without the G2019S LRRK2 mutation. The purpose of the present study was to test the practicability of assessing LRRK2-mediated Rab10 phosphorylation in a patient cohort in a clinical setting and not to address whether Rab10 phosphorylation is elevated in patients with Parkinson's or G2019S carriers. To answer this question, further studies with much larger numbers of patients and controls will be required.Neutrophils were isolated from 13 healthy controls, 7 patients with sporadic Parkinson's and 6 individuals with a heterozygous G2019S LRRK2 mutation — 5 with Parkinson's and 1 non-manifesting carrier –5. DemogWe quantified Rab10 phosphorylation in four different ways, by normalising pRab10 signal obtained with no MLi-2 treatment with either GAPDH  or totalTo compare LRRK2-mediated phosphorylation of Rab10 in neutrophils and PBMCs, we isolated neutrophils and PBMCs from the same healthy donors . Flow cyImmunoblot analysis of PBMCs revealed that full-length LRRK2 was the major species, with only low levels of the ∼170 kDa form observed. Full-length LRRK2 levels were 20- to 30-fold higher in PBMCs compared with neutrophils . Levels To assess LRRK2 inhibitor dose response, we treated human neutrophils with increasing concentrations of two structurally diverse inhibitors namely MLi-2  and PF-0We also explored the kinetics of the MLi-2 inhibitor mediated Rab10 Thr73 and LRRK2 Ser935 dephosphorylation in human neutrophils, which revealed that Rab10 was fully dephosphorylated within 10 min following treatment with 100 nM MLi-2 . DephospAs the DIFP protease inhibitor is a potent organophosphorus neurotoxin, we investigated whether it was essential to add this to the neutrophil lysis buffer. We therefore lysed human neutrophils with or without DIFP and, in the first instance, explored whether DIFP could be replaced with 1% (w/v) SDS, which has been used to lyse neutrophils in other studies . This stWe next explored whether a delay in purifying neutrophils from human blood would affect on LRRK2-dependent Rab10 phosphorylation. Blood was drawn from three healthy donors and left unprocessed at room temperature for up to 24 h before neutrophils were isolated and with or without MLi-2 inhibitor treatment. Immunoblot analysis of these samples reveals that storing blood for up to 24 h does not compromise LRRK2-dependent phosphorylation of Rab10 .Figure Finally, we investigated whether Rab10 phosphorylation in neutrophils could be monitored by employing the Phos-tag assay, in which the retardation of LRRK2-phosphorylated Rab10 is visualised following Phos-tag polyacrylamide gel electrophoresis . NeutropThere is compelling evidence for the role of LRRK2 and in particular its kinase function in Parkinson's disease. This has sparked considerable interest in exploiting LRRK2 as a drug and biomarker target. Here, we describe a facile and robust assay for monitoring LRRK2 kinase pathway activity by measuring its effect on phosphorylation of its physiological substrate Rab10 in (a) the homogenous pool of human peripheral blood neutrophils and (b) with the use of a highly selective phospho-specific Rab10 antibody .The rational for using human peripheral blood neutrophils is that they represent a homogenous subset of cells that make up the dominant leukocyte population in humans –41. MoreWhen looking at the total LRRK2 levels in neutrophil lysates, we noticed two bands: one at the expected size for full-length LRRK2 at ∼286 kDa and a shorter one at ∼170 kDa. We suspect that the truncated ∼170 kDa band corresponds to a proteolytic species lacking the N-terminal domain resulting from the high protease activity present in neutrophils, but cannot exclude an alternative splice variant. To our knowledge, no other LRRK2 isoform of that size has been reported ,42. The For assessing LRRK2 kinase pathway activity, we have utilised the newly developed phospho-specific MJFF-pRab10 monoclonal antibody that selectively recognises Rab10 phosphorylated by LRRK2 at Thr73 . Unlike The data obtained with the phospho-immunoblotting approach is vastly more robust and sensitive than the Phos-tag method compare . Compariin vivo assessment of LRRK2 activity; similar to the characterisation of the MJFF-pRab10 antibodies used in this study [Paramount for the phospho-immunoblot approach is the availability of an antibody that is fully selective against the Rab10 Thr73 phospho-epitope and does not cross-react with other phosphorylated Rab proteins. Another phospho-Rab10 polyclonal antibody has recently been published , but itsis study .We went on to explore the practicability of using our assay in the clinical setting to test how sample collection, processing, as well as any other uncontrollable and unforeseeable aspects that are intrinsic to clinical practise might affect the outcome. We recruited 13 healthy controls, 7 patients with sporadic Parkinson's, 5 patients with G2019 LRRK2 associated Parkinson's and 1 asymptomatic G2019S LRRK2 carrier (Supplementary Tables S1 and S2). By including participants with LRRK2 G2019S associated Parkinson's, we anticipated to gain more insights into the effect size of LRRK2 kinase activity by means of Rab10 phosphorylation. In a mouse model, a G2019S knock-in mutation in the homozygous state results in an ∼2-fold increase in Rab10 phosphorylation ,15,18. OWe speculate that G2019S LRRK2 mutation carriers would have the highest levels of Rab10 phosphorylation followed by those with sporadic Parkinson's, while healthy controls would have the lowest levels. This trend is observed when LRRK2-mediated Rab10 phosphorylation is normalised to GAPDH . In the in vivo LRRK2 kinase pathway activity with Rab10 phosphorylation as a readout. We would also recommend that Parkinson's bio-repositories consider including neutrophils as an additional bioresource.As many other groups have studied LRRK2 in PBMCs ,45,46 an50 for MLi-2 on Rab10 Thr73 dephosphorylation in neutrophils (30 nM) is higher than what has previously been observed in mouse embryonic fibroblasts (3–10 nM) [50 value could be explained by the high levels of LRRK2 activity in neutrophils   (Figure trophils . The IC5roblasts , suggestIn summary, we have developed an assay to robustly measure LRRK2 kinase pathway activity by monitoring Rab10 phosphorylation in human peripheral blood neutrophils. Neuroprotective and disease-modifying trials in neurodegenerative conditions have largely failed in the past. There is reasonable hope that this may be different in LRRK2 associated Parkinson's. Orally bioavailable, brain penetrant LRRK2 inhibitors have been generated and individuals who have not yet developed or are early on in their disease course can be genetically stratified based on their LRRK2 genotype. Measuring LRRK2-mediated phosphorylation of Rab10 in neutrophils or other tissues might represent a biomarker that allows for additional patient stratification. We acknowledge that this may be challenging due to the moderate changes of LRRK2 kinase activity associated with pathogenic mutations. Also, our assay captures a peripheral rather than a central readout of LRRK2 kinase function, but to date no non-invasive central measure of LRRK2 activity exists. In the present study, we have highlighted key considerations why neutrophils are a useful biomaterial for analysing LRRK2 pathway activity and the importance of using highly specific phospho-Rab protein antibodies. We also demonstrate the relative ease and feasibility of performing our assay in the clinical setting. We envision that our LRRK2 kinase pathway assay may find wider use for pharmacodynamic and target engagement studies for future LRRK2 inhibitor clinical trials. In future work, it would also be exciting to explore the biological role that the LRRK2 pathway plays in neutrophils and whether it is involved in controlling migration to the sites of the infection and/or phagocytosis and destruction of pathogens.


Tibial aperture fixation with a bioabsorbable interference screw is a popular fixation method in anterior cruciate ligament reconstruction (ACLR). An interference screw containing β-tricalcium phosphate (β-TCP) to improve bony integration and biocompatibility was recently introduced. This study aims to compare the clinical outcomes and radiological results of tunnel enlargement effect between the 2 bioabsorbable fixative devices of pure poly-L-lactic acid (PLLA) interference screws and β-TCP-containing screws, for tibial interference fixation in ACLR using hamstring autografts.Eighty consecutive patients who had undergone double-bundle ACLR between 2011 to 2012 were prospectively reviewed and randomly divided into two groups based on the type of tibial interference screw: 28 were assigned to the pure PLLA screw group (Group A), while the other 29 were assigned to the β-TCP-containing screw fixation group (Group B). Clinical evaluations and radiological analyses were conducted in both groups with a minimum 2- year follow-up.There was no significant difference in subjective or objective clinical outcome between the 2 groups. In radiological analyses, the use of a β-TCP-containing screw reduced tunnel widening in the portion of the tunnel with screw engagement compared to the pure PLLA screw, while the use of a β-TCP-containing screw resulted in greater tunnel enlargement in the proximal portion of the tunnel without screw engagement than use of a pure PLLA screw.Use of a β-TCP-containing interference screw in tibial aperture fixation reduced tunnel enlargement in the vicinity of the screw, whereas greater enlargement occurred proximal to the screw end relative to use of a pure PLLA interference screw. These paradoxical enlargements in use of β-TCP containing screws suggest that for reducing tunnel enlargement, the length of the interference screw should be as fit as possible with tunnel length in terms of using soft grafts.Level of Evidence: II, Prospectively comparative study.(NCT02754674), Date of trial registration: February 10, 2016.Retrospectively registered with ClinicalTrials.gov. The online version of this article (10.1186/s12891-017-1757-0) contains supplementary material, which is available to authorized users. Interference screws are commonly used for fixation in orthopedic surgery, especially in tibial aperture fixation for ACL reconstruction. Biodegradable cannulated interference fixation screws made of poly-L-lactic acid (PLLA) were introduced in the early 1990s to improve postoperative imaging, decrease stress shielding, avoid graft laceration, and make revision easier . These sHowever, there are concerns that explanted pure PLLA biodegradable interference screws may not degrade, and that replacement with bone may be incomplete , 6–8, anThe Matryx®  screw, made of 96 L/4D poly lactic acid (PLA) and 30% β-tricalcium phosphate (β-TCP), was introduced to enhance osteoconduction and to be more biocompatible with bone than existing screw materials. However, to the best of our knowledge, the effect of this screw against tunnel widening effect by osteoconduction and bony integration around tunnels has not been investigated in a clinical setting. Therefore, we sought to investigate the clinical advantages of β-TCP containing biodegradable screws under their theoretical advantages.We hypothesized that use of β-TCP-containing interference screws would improve coaptation of the graft to the tunnel with replacement of the degraded screw by new bone formation, and result in better tunnel remodeling by preventing tunnel widening. The purpose of this study was to evaluate whether use of a β-TCP-containing interference screw resulted in superior clinical and radiological outcomes  in ACL reconstruction using autologous hamstring grafts compared to a commercially available pure PLLA biodegradable screw.From February 2011 to June 2012, a total of 119 patients with ACL injury were enrolled in this study. Inclusion criteria were patients who underwent a primary anatomical double bundle ACL reconstruction with tibial aperture fixation by Bioscrew® , which is made of 100% poly-L-lactic acid (PLLA) or Matryx® (Linvatec Corp), which is made of 70% 96 L/4D poly lactic acid (PLA) & 30% β-TCP, and who were at least 20 years of age; patients younger than 20 years of age were treated on the basis of adolescent ACL and single-bundle reconstruction with no interference screw placed in the tibial tunnel if open physis. Of these 119 patients, 39 were excluded for the following reasons: single-bundle reconstruction (4), revisional ACL reconstruction (7), combined surgeries such as meniscal allograft transplantation (2), any previous ligament surgery or bony procedure in the area of graft attachment (10), degenerative arthritic knee (1), pregnancy (1), and other reasons (14) .To assess differences in tibial tunnel widening according to fixation device, the cross sectional areas at four locations were compared between the two groups on the immediate postoperative and 1- year follow-up CT images. All operations were performed by a single surgeon (J.H.W.) experienced in ACLR using both the transportal and outside-in techniques. All patients underwent CT examination twice on the same CT device using the same protocol within 3 days after the initial surgery and at the postoperative 1-year follow-up. The current study received Institutional Review Board approval from our institution  before study onset, and our protocol was also approved. Informed consent was obtained from all participants  to ensure maximal contact between the graft and the tunnel walls on the femoral side, and with bioabsorbable interference screws with a post tie on the tibial side for all cases.Graft was normally trimmed to retain its original size with a length of 26 to 30 cm. Then, it was folded and sutured together with the use of no. 1 absorbable sutures to form two triple-strand grafts. A 6-stranded graft, composed of triple semitendinosus (6.5 to 9.5 mm) for the anteromedial (AM) bundle and triple gracilis (4.5 to 7 mm) for the posterolateral (PL) bundle, was created for each group.o of knee flexion and applying a 30–35 N tension to the graft using the dedicated tie tensioner [Anatomic tibial insertion sites of both bundles were marked with an ArthroCare device , and the tip of the guide was aimed at the centers of the AM and PL bundle remnant tibial insertion sites. A 3.2-mm guide pin was inserted into the bases of the AM and PL tibial insertion sites. The AM and PL tibial tunnels were then drilled with a cannulated drill. Bioscrew® or Matryx® (length 30 mm) with a diameter 1 mm larger than the tunnel diameter was used for tibial aperture fixation at 0ensioner , and an All patients began active quadriceps isometric exercise and active range-of-motion exercise immediately after surgery. Four to 5 days after surgery, an ACL limited-motion brace was applied, and joint motion exercise was carried out at 15° increments per week. At 4 and 6 weeks after surgery, 90° and 135° of motion, respectively, were allowed. A pair of crutches was used to allow partial weight bearing from 3 days to 6 weeks after surgery. Patients were educated on performing proprioceptive balancing exercise at 3 months after surgery. Return to competitive sports involving jumping, pivoting, or sidestepping was prohibited until 6 months after the reconstruction .A CT scanner Light Speed VCT  was used for all examinations. All patients were placed in a supine position with knee full extension. The collimation was 16 × 0.625 mm, the tube parameters were 120 kVp and 200 mA, and the acquisition matrix was 512 × 512 pixels. Images were processed for multiplane reconstruction, and cross-sectional area (CSA) was measured using Osirix . We measured CSAs in the plane perpendicular to the long axis of the tibial tunnel using Osirix at the following four cutting levels . The preoperative evaluation was conducted by the Lysholm knee score, Hospital for Special Surgery (HSS) score, Tegner score, the International Knee Documentation Committee Score (IKDC) –17 and aP < 0.05 was considered significant . The 2-sample t-test was applied to compare the cross-sectional areas of “mid-tunnel” and “mid-screw”. The difference of clinical outcome scales between two groups was evaluated with a 2-sample t test or Mann-Whitney test. Statistical analyses were executed using SAS version 9.3 .Two orthopedic surgeons (independent observers) developed and agreed to the measurement methods together; however, they were blinded to each other’s measurements and their previous measurements. They measured the cross-sectional areas at the four cutting levels for all knees twice with an interval of 2 weeks. Reliability of the measurements was assessed by an independent statistician who examined interobserver reliability using the intraclass correlation coefficient. A priori power analysis was performed to determine the sample size using the two-sided hypothesis test at an α level of 0.05 and a power of 0.8. A post hoc power analysis was performed to determine whether the results of our study of 25 cases indicated adequate power, and the power was 85.21%, which was adequate to detect a difference of 1 mm widening , 19. The2 . The mean duration of postoperative follow-up was 33 months . There were no significant differences between groups in age, gender, BMI, femoral tunneling technique, and tunnel diameter, values of which are shown in Table The mean age was 34.7 years  and the mean body mass index (BMI) was 24.6 kg/mNo cases of complete absorption and replacement of the interference screw with new bone were observed at the 1-year follow-up CT in either group. No clinical evidence of any adverse events related to these interference fixation screws was observed, and degenerative radiographic changes were not noted.p = 0.024). At the mid-tunnel level, the mean increase in CSA after 1 year was significantly larger in group 2 (β-TCP screw) than in group 1 (PLLA screw)   the use of a β-TCP-containing screw reduced tunnel widening in the portion of the tunnel with screw engagement compared to the pure PLLA screw, while (2) the use of a β-TCP-containing screw resulted in greater tunnel enlargement in the proximal portion of the tunnel without screw engagement than use of a pure PLLA screw. These findings might be explained that in the screw-bone contact area, particles during degradation of β-TCP led to less local reactivity or inflammatory responses under their neutralizing effects, whereas they activated osteoclasts and resulted in osteolysis with mechanical stress or synovial fluid influx in proximal portion without screw engagement.The use of biodegradable interference screws is widely accepted because of their ease of handling and effective fixation. Ideally, a biodegradable interference screw should degrade with minimal or no host-bone reaction. It is reasonable to assume that the faster a material degrades, the earlier the osseous replacement takes place , 21. HowIn terms of bone-patellar tendon-bone (BPTB) grafts, interference screws have the benefit of reducing the potential space created in bone by the compaction method, which forms the walls of the container . SeveralHowever, our finding of a smaller increase in tunnel widening in the portion of the tunnel with β-TCP screw engagement has demonstrated a better behavior in clinical settings. Our hypothesis was that early degradation and the osteoconduction-promoting effect of β-TCP screws would enhance bony integration and reduce tunnel enlargement. As hypothesized, the tunnel was less enlarged in the section with screw engagement in the β-TCP-containing screw group than the PLLA screw group. The addition of β-TCP to a degradable polymer such as PLLA creates an inorganic osteoconductive scaffold and changes the properties of the scaffold. During degradation, β-TCP breaks down into calcium ions and phosphates, which maintain an elevated pH around the implant –28. ThisMeanwhile, abruptly increased tunnel enlargement was observed in the proximal portion to β-TCP containing screw relative to a PLLA screw Fig. . This coAlmost studies of explanted biodegradable interference screws have been overstated regarding to concerns about whether claims of bio-absorbability because of limited evidences of degradation , 33, 34.Several studies have reported that use of β-TCP-containing screws reduces tunnel enlargement . HoweverThe strength of this study is that to evaluate bone tunnel widening, immediate and postoperative 1- year CT scans were used instead of digital plan radiograph. Tunnel enlargement is believed to occur both intraoperatively and postoperatively. Intraoperative enlargement may be due to eccentric reaming, graft and reamer diameter mismatch, or use of the press-fit technique, so a difference in CSA between the reamer and actual tunnel is inevitable. In the current study, immediate postoperative CT images were used to obtain baselines values, so postoperative enlargement could be evaluated without intraoperative change bias. Furthermore, the tunnel was segmented into four different sections for more precise analysis; this enabled us to observe paradoxical enlargement in the area of the tunnel without screw engagement.Nevertheless, the present study had some limitations. First, no histologic data were obtained to confirm complete degradation of the PLLA screws. Second, the follow-up period was relatively short. However, several studies have shown that most tunnel widening occurs during the first 3 months –39. AccoUse of a β-TCP-containing interference screw in tibial aperture fixation reduced tunnel enlargement in the vicinity of the screw, whereas greater enlargement occurred proximal to the screw end relative to use of a pure PLLP interference screw. These paradoxical enlargements in use of β-TCP containing screws suggest that for reducing tunnel enlargement, the length of the interference screw should be as fit as possible with tunnel length in terms of using soft grafts.


Eight candidates demonstrated RdRp inhibition (>25% inhibition at 10 µM), which was confirmed using a gel-shift RdRp assay for two of them. The two molecules were identified as initial hits and selected for structure-activity relationship studies, which resulted in the synthesis of novel compounds that were examined for inhibitory activity. Five compounds inhibited human norovirus RdRp activity (>50% at 10 µM), with the best candidate, 54, demonstrating an IC50 of 5.6 µM against the RdRp and a CC50 of 62.8 µM. Combinational treatment of 54 and the known RdRp site-B inhibitor PPNDS revealed antagonism, indicating that 54 binds in the same binding pocket. Two RdRps with mutations (Q414A and R419A) previously shown to be critical for the binding of site-B compounds had no effect on inhibition, suggesting 54 interacts with distinct site-B residues. This study revealed the novel scaffold 54 for further development as a norovirus antiviral.Human norovirus causes approximately 219,000 deaths annually, yet there are currently no antivirals available. A virtual screening of commercially available drug-like compounds  was performed on the suramin and PPNDS binding-sites of the norovirus RNA-dependent RNA polymerase (RdRp). Selected compounds (n = 62) were examined for inhibition of norovirus RdRp activity using an  While norovirus-induced illness is usually self-limiting in healthy individuals, the disease can be severe or life threatening in immunocompromised and immunosuppressed patients3, neonates4, young children6 and elderly populations8. As such, a significant research effort has been invested to identify antiviral candidates for the treatment of norovirus infections and for prophylactic use in outbreak settings.Human norovirus is responsible for over 200,000 global deaths, USD $60 billion in societal costs, and USD $4.2 billion in health expenditure each year10. However, the recently developed human norovirus culture systems are not readily amenable to antiviral screening due to low replication levels, with only 100- to 1000-fold increases in viral titres10, and the systems’ complexities and costs. Most norovirus antiviral research currently relies on the closely related murine norovirus (MNV), for screening potential human antivirals due to its robust and reliable culture, and available mouse models11.Several factors have hampered norovirus antiviral research, including the inability to successfully propagate human norovirus in cell culture until recently12  and non-nucleoside inhibitors (NNIs)23. NA prodrugs can be phosphorylated after cellular uptake, then incorporated into the growing viral nucleic acid chain by the RdRp24. These antivirals work by competing with incoming nucleoside triphosphates (NTPs) to cause either chain termination25, or lethal mutagenesis of the progeny virus26. NAs have proven successful in the treatment of several viral infections including hepatitis B virus27, hepatitis C virus (HCV)28, herpesviruses29 and human immunodeficiency virus (HIV)30. However, due to their mode of action, they frequently result in off-target side effects32, and no NAs have yet reached clinical trials for norovirus treatment. One NA, 2′-C-methylcytidine (2CMC)  of 2.5 µM15, and a half maximal effective concentration (EC50) of 8.2 µM in cell culture against the human norovirus replicon15. More recently, 2CMC was also found to be effective against human norovirus cultured in the B-cell culture system, with a reported EC50 of 0.3 µM33. However, despite these promising preclinical results, development of the oral 2CMC prodrug  34. NNIs have been clinically approved for treating HIV35 and HCV36 infections, however, while several potential norovirus NNIs have been identified in pre-clinical studies39, none are in clinical trials for the treatment of human norovirus infections. Amongst these pre-clinical candidates is the anthelmintic drug suramin  tetrasodium salt (PPNDS)  Fig. , has als44. To-date, three NNI binding sites have been identified on the norovirus RdRp, including the NTP pathway, site-A in the fingers domain and site-B in the thumb domain37. Co-crystallisation studies of the norovirus RdRp and suramin identified its binding pocket in the NTP access pathway, between the fingers and thumb domains37. Site-A lies within a positively charged cleft for NTP traversal, but due to highly flexible amino acid side-chains it is less suitable as an NNI target, due to challenges for structure-based antiviral design17. However, site-B is a highly conserved binding site within the thumb region and can be exploited for both structure-activity and targeted de-novo antiviral design of novel compounds against human norovirus16.The human norovirus polymerase forms the canonical ‘right-hand’ RdRp structure, with fingers, palm and thumb domainsin silico screening identified 62 compounds that were examined for inhibition of norovirus RdRp activity using polymerase assays. Two molecules were found to have an interesting RdRp inhibitory activity and were selected for a structure-activity relationship study synthesising new chemical derivatives. Among the newly prepared compounds, we identified a lead candidate site-B binder, which inhibited the human norovirus RdRp at a low micromolar concentration. This study revealed a novel NNI that presents a promising scaffold for further chemical enhancement, to improve potency and specificity, in the pursuit of effective norovirus antivirals.This study aimed to identify promising chemical scaffolds for antiviral development targeting the human norovirus RdRp. To achieve this aim, a series of structure-based and ligand-based virtual screenings were carried out on approximately 300,000 commercially available compounds with favourable drug-like properties, to identify binding capabilities focused primarily on the suramin and PPNDS binding-sites of the norovirus RdRp. This 45. The molecules selected with the two methods were then subjected to different molecular docking evaluations described below.Two different virtual screening approaches have been used for the initial selection of potential hit compounds: a structure-based virtual screening and a ligand-based virtual screening. While the first approach takes into consideration the protein structure and the ligand spatial disposition in relation to the active site (pharmacophore features), the ligand-based methodology compares the shapes of known ligands with unknown ones, evaluating two-dimensional similarities  or three-dimensional similarities  without considering the protein structure46. In particular, two screens were conducted using two different RdRp crystal structures: the MNV RdRp co-crystallised with suramin 37 and the human RdRp co-crystallised with PPNDS (PDB ID: 4LQ3)17. Suramin inhibits human norovirus and MNV RdRp activity through a mixed inhibition mode (nucleic acid and GTP) and occupies site-A. Site-A encompasses the access route for the incoming NTP that is linked to the nascent RNA chain. PPNDS inhibits human norovirus RdRp activity by blocking the access of both the ssRNA template and the NTPs, and occupies the inner pocket of site-B, which is located in the RdRp thumb domain17. The two binding sites are very close to each other and overlap in the central part of the protein structure.The norovirus RdRp was used to perform a structure-based virtual screening of the SPECS library, a database of ~300,000 commercially available compounds47 on the suramin and on the PPNDS binding sites, respectively. The docking results (docking poses) obtained for each binding site were subsequently re-scored using other two different docking programs: LeadIT-FlexX and Glide XP49. Applying an internal consensus score procedure, a methodology validated by our research group in the past50, the values of the three different scoring functions for each docking pose were analysed together. Only the docking poses falling in the top 25% of the score value range in all the three scoring functions were considered as hits. 799 compounds were selected for the suramin pocket, 755 for the PPNDS. A final selection was made by visually inspecting the docking poses of the different molecules and their potential binding to the target sites . 19 derivatives were selected for the suramin binding site and 20 for the PPNDS site. The molecules were purchased and screened for inhibition of norovirus RdRp activity using a transcription enzyme assay.The SPECS database of compounds was pre-filtered using a pharmacophore model based on the suramin binding site in one case and on the PPNDS binding site in the second case Fig. . The req51. Two different queries were generated: one based on the partial structure of suramin, and a second prepared on the PPNDS structure , three compounds were selected for suramin and 20 for PPNDS. The new derivatives were purchased and biologically evaluated.A shape-based screening of the SPECS library, previously prepared with the Omega conformations generator program, was performed using ROCS 3Dure Fig. . In bothin silico were screened for inhibition of norovirus RdRp activity using an in vitro fluorescent, de novo RdRp assay52. Compounds were tested at fixed concentrations of 10 µM and 100 µM, and compared to mock treated samples containing the vehicle only (0.5% DMSO [vol/vol])  Fig. . Of the l]) Fig.  exhibitel]) Fig.  and morel]) Fig. . For cerl]) Fig.  or enhanIn addition, some compounds (i.e. AJ-077/33270018 and AP-970/12851847) exhibited insolubility, innate fluorescence and even apparent RdRp enhancement at 100 µM  reaching over 25% inhibition at 10 µM and more than 50% inhibition at 100 µM. Three additional compounds  that reached more than 25% inhibition at both 10 and 100 µM were selected for counter-screening. The three compounds (green bars) were examined using the gel-based assay due to the unusual behaviour of demonstrating higher inhibition at 10 µM compared to 100 µM, potentially due to solubility issues. Two additional compounds (AE-562/12222591 and AK-968/41925073) demonstrated more than 25% inhibition at 10 and 100 µM but were not pursued further as they both precipitated heavily in 100% DMSO and therefore lacked the desirable drug-like property of solubility.To confirm RdRp inhibition and to exclude any potential false positives from the first fluorescence RdRp assay, the eight selected compounds Fig.  were the16. The NA 3′-deoxyguanosine-5′-triphosphate (3′dGTP) was used as a positive control and exhibited complete inhibition of transcription at 10 µM. Inhibition of primed elongation of the PE44-NoV template was examined at a fixed concentration of 100 µM  only demonstrated partial inhibition of primed elongation activity, and were excluded from further development. Based on these results, we decided to focus our attention on these two molecules.In this assay, a 32 nucleotide RNA template (PE44-NoV) is extended to 44 nucleotides in the presence of an active RdRp µM Fig. . In the  µM Fig. , evidenc µM Fig. , while t11 and 13 were chosen as starting point for the preparation of a series of analogues, with which to explore the structure-activity relationships associated with the two scaffolds.Due to their confirmed inhibitory activity against the RdRp polymerases, the structures of 13 is characterized by a methylbenzoic acid portion and a morpholine ring part linked by a central furan-2-yl-methylene-thiazolidine-2,4-dione core. We focused our initial synthetic efforts on the two lateral portions of the molecule, without modifying the central core, in order to investigate the role of these two parts in the RdRp inhibitory activity.16a–d were prepared by reacting the corresponding phenylboronic acids (14a–d) with 5-bromofuran-2-carbaldehyde (15) in a mix of tetrahydrofuran/water, through a Suzuki coupling reaction using sodium carbonate (Na2CO3) as base and tetrakis (Pd[Ph]3]4) as catalyst modifying a reported procedure53. The desired products were obtained in a very good yield range (62–98%) after flash column chromatography purification. A Knoevenagel condensation was exploited for the preparation of derivatives 18a–d by reacting compounds 16a–d with thiazolidine-2,4-dione (17), using β-alanine as catalyst and acetic acid as solvent53. In the final step, compounds 18a–d were converted into the corresponding final products 23a–c, 24a–d, 25a–d by reacting them with 20–22 in dimethylformamide (DMF) at 60 °C for 72 h using potassium carbonate (K2CO3) as base54. The final compounds were obtained in a good yield range (42–76%) after either re-crystallization or flash column chromatography purification. Compounds 20–22 were previously prepared by reacting 2-bromoacetyl chloride (19) with different secondary amines in dichloromethane (DCM) and N,N-diisopropylethylamine (DIPEA) as base55. Synthetic efforts were made to achieve good yields for this step by monitoring the reaction temperature and evaluating different bases. Following this synthetic route, 11 derivatives of 13 were prepared.The synthetic pathway reported in Fig. 13 presents a free acid group, therefore a basic hydrolysis of 23b with LiOH in water and methanol at room temperature was attempted. Unfortunately, NMR analysis revealed complete degradation of the starting material with the opening of the thiazolidinedione ring, revealing instability for these compounds in basic environments. A second attempt was made by performing an acid hydrolysis in HCl 6 M increasing the reaction temperature to 100 °C over 3 days. In this case, the desired hydrolysis did not take place and the starting ester was recovered.As reported above, 16b as second step after the Suzuki coupling, obtaining 26. Hydrolysed 26 was then condensed with 17 to give 27. In the last step, the reaction between 27 and 20 did not yield the desired product 28, the unreacted staring materials were recovered, along with small amounts of a minor by-product, 29, in which the substitution of the bromide compound occurred on the carboxylic acid portion of the molecule instead of the thiazolidinedione ring, as also reported by Zhang et al.56. The free acid seems to interfere with the SN2 reaction and a different approach was therefore investigated.In the attempt to find a strategy to prepare the free acid derivatives, the previously used synthetic pathway was slightly modified, as reported in Fig. 28. In this case, the Knoevenagel condensation was performed as a first step giving 30. Nucleophilic substitution with 20 gave the central furan-2-yl-methylene-thiazolidine-2,4-dione core with the attached morpholine ring portion (31). For the last step, the Suzuki coupling between the 4-boronobenzoic acid 32, previously prepared starting from its ester derivative (14b), and 31 was attempted, but no desired product was obtained, recovering the unreacted boronic acid and observing degradation of the thiazolidinedione ring.Figure 28 was developed, considering the target molecule as an assembly of two portions: the 4-(5-formylfuran-2-yl)benzoic acid (26), prepared by Suzuki coupling , obtained by the SN2 reaction between 17 and 20. The two portions were then linked together in the last step through a Knoevenagel condensation affording the desired product 28 with the free acid , the reaction conditions needed to be changed according to the substituted amine present. In the case of piperidine and dimethylamine derivatives , the use of sodium hydride (NaH) as base and THF as solvent gave a less impure reaction and a higher yield if compared to the method used for the other derivatives.A final and alternative method for the preparation of the desired free acid ic acid 2, prepareion Fig. . Also fo11 has a central furan-2-ylmethylene-pyrazolidine-3,5-dione core substituted at position 1 of the pyrazolidine with a methyl benzene and at position 5 of the furane ring with a thiazol-2-yl-benzenesulfonamide. A five-step synthetic pathway was developed and optimised and a small family of two new derivatives was prepared in order to initially understand the role of the methyl group and the thiazole ring.46–47 were prepared by reacting the corresponding phenylhydrazines (43–44) with ethyl-3-chloro-3-oxopropanoate (45) in THF and triethylamine (Et3N), following a reported methodology57. 46–47 were then converted into the corresponding 1-phenylpyrazolidine-3,5-dione 48–49 through an ester displacement reaction in the presence of sodium hydroxide (NaOH) and ethanol (EtOH)57. 4-Bromobenzene-1-sulfonyl chloride (50) and aniline 51 in pyridine gave sulfonamide 52, which was then converted in 53 by a Suzuki coupling. In particular, synthetic efforts were made to find the best conditions for this coupling, since the procedure previously described did not work and only the starting materials were recovered after each attempt, potentially due to an interference of the sulfonamide bond with the catalyst used. After changing solvent systems, reaction temperature, base and phosphine ligands, the best reaction conditions were found using potassium phosphate (K3PO4) as base, Pd(dppf) as catalyst, water/DMF as solvent and heating in a microwave reactor at 130 °C, which gave the desired product in a good yield (61%).Hydrazides 48–49 and 53 according to a Knoevenagel condensation, giving the desired products 54 and 55.In the last step Fig. , the two13 and some synthesis intermediates , further supporting the reproducibility of the assay and more importantly confirming the RdRp inhibitory activity associated with this particular molecule. The shorter phenylfuran-2-yl-(methylene)thiazolidine-2,4-dione derivatives , regardless of the substituent on the aromatic ring, and the furan-2-yl(methylene)-3-(morpholine-4-carbonyl)thiazolidine-2,4-dione (31) lacked RdRp inhibitory activity, indicating that RdRp inhibition was associated with the whole scaffold of 13 and not only with the methylbenzoic acid portion or the morpholine ring part. These derivatives are likely too small to entirely occupy the RdRp binding site area to block polymerase activity. The overall RdRp inhibitory activity of the derivatives of 13 seems to be the result of the combination of the two portions forming the molecule. In the morpholine derivatives, the most important influence for the activity was given by the free acid in position para of the aromatic ring. In fact, its removal  or its conversion to a methyl ester  lead to a complete loss of activity, whereas the derivative bearing only the free acid (28) showed improved activity when compared with the initial hit, inhibiting the RdRp activity by almost 50%. The methyl group at position ortho seems irrelevant for inhibitory activity, as demonstrated by the lack of activity of 23c, and its removal, together with the presence of the free acid, lead to a significant increment of inhibitory activity. In the piperidine compounds, an unsubstituted aromatic ring (24a) and an ortho methyl substituent (24c) are related with the loss of inhibitory activity, giving the same result obtained for the morpholine series. Replacement of the morpholine ring with piperidine (40e) in the original structure of 13 abolished the activity, whereas esterification of the same derivative (24d) resulted in 25% inhibition, similar to the initial hit. Removal of the ortho methyl group of 24d (24b) increased the degree of RdRp inhibitory activity up to 60%, compared with 13, confirming the detrimental effect of the ortho methyl group. Replacing the morpholine ring with a non-cyclic secondary amine, such as the dimethyl amine, slightly improved the activity compared to the initial hit, but a maximum of 45% inhibition at 10 µM was reached. Also in this case, the presence of the acid or the ester seems fundamental for activity retention . Insertion of an aniline moiety, in replacement of the morpholine, had a positive impact on inhibitory activity, with derivative 42a, characterised by the unsubstituted aromatic ring on one side and the aniline on the other side, reaching 75% RdRp inhibition at 10 µM.Results of the assay are reported in Fig. 11 derivatives, in which the original thiazole ring has been replaced by an unsubstituted phenyl ring, showed 75% RdRp inhibition, with no substantial differences between the methyl benzene and the unsubstituted phenyl derivative (55 vs. 54), and an activity comparable with the inhibition found in the initial screening for 11.Hit 24b, 42a, 42b, 54, 55) demonstrated more than 50% inhibition of the human norovirus Sydney 2012 RdRp at 10 µM , whilst 55 only displayed minimal inhibition and 24b had no appreciable effect.Among the tested compounds, five , and the three newly synthesised derivatives, which exhibited near complete abolishment of norovirus RdRp activity in the gel-based assay , were further assessed using a fluorescent RdRp assay to ascertain IC50 values (concentration range examined: 0.1–100 µM). Dose-dependent inhibitory response curves were used to establish IC50 values for the five test compounds  were examined for inhibition of RNA polymerases from other viruses spanning three genera of the Caliciviridae family, including Norovirus (human norovirus and MNV), Lagovirus (rabbit haemorrhagic disease virus [RHDV]) and Sapovirus (human sapovirus). All four compounds examined exhibited broad-spectrum inhibitory activity of the calicivirus RdRps at a fixed concentration of 100 µM, ranging between 44.4% inhibition (42a with MNV RdRp) through to 99.0% inhibition; (54 with RHDV RdRp)  Fig. . The proTo ensure the four hit compounds selectively inhibited calicivirus polymerase activity, they were further counter screened against a viral-like RdRp (bacteriophage Φ6) and an RNA-dependent DNA polymerase Moloney murine leukaemia virus reverse transcriptase (MMLV RT). None of the lead compounds reached 50% inhibition of the Φ6 and MMLV polymerases examined up to 100 μM  displayed an IC50 of 12.1 µM against MNV RdRp  exhibited a mean half maximal cytotoxic concentration (CC50) of 62.8 µM  were examined against MNV plaque formation in cell culture. There was no appreciable effect on plaque numbers up to 25 µM, with RAW264.7 cells thinning at 50 µM, due to cytotoxicity . This could be due to poor cell membrane permeability of 54, as observed with PPNDS16 and suramin40, which, despite inhibiting the RdRp in an isolated enzyme activity assay, demonstrated a lack of antiviral activity in the cell-based assay. Compound solubility and its cell-membrane permeability properties could be further improved with structure-activity relationship enhancement of the 54 scaffold to facilitate cellular uptake, and therefore to increase antiviral efficacy. Work on this aspect is ongoing, and it will be reported in due course.As mentioned above, the only robust cellular assay available for the evaluation of potential norovirus inhibitors is the closely related MNV assay. Out of the novel compounds examined  or site-B (PPNDS). 54 and PPNDS were examined in a combinational study to test for antagonism  were examined alone and together in a 1:4 ratio respectively , dose-response curves for 54 (0.1–100 µM) were produced to compare the IC50 values between the wild-type RdRp and the two mutant RdRps, Q414A and R419A  and R419A RdRp (10.2 µM). The lack of effect of these two mutations on the inhibitory activity of 54, together with the antagonism found with PPNDS, suggest that 54 may bind the site-B pocket of the polymerase with no specific interactions with Q414 and R419.Given the evidence of 19A Fig. . The mea54 on the PPNDS binding site of human norovirus RdRp were performed using Glide SP. The proposed binding mode is shown in Fig. 54 is predicted to occupy site-B in a different orientation compared to PPNDS, spanning an area that includes the central core of site-B and the initial part of site-A, where suramin places its sulphate head , interacting through its sulphonamide group with Glu407 and Arg413, and through an arene-cation interaction between Arg392 and the benzene ring. No interactions are present between the phenyl ring and the protein, therefore its replacement by a thiazole, as in the initial hit 11, does not seem to influence binding, in accordance with the similar inhibitory activity found for 54 and 11 in the biological assay. The phenyl-pyrazolidine portion is occupying the area where PPNDS places is naphthylazo part  on the inhibitory activity of 54 , which was confirmed using a gel-shift RdRp assay. Among them, two compounds, 11 and 13, were selected for an initial structure-activity relationship study. Synthetic efforts were made to develop the best synthetic pathway for the preparation of different derivatives of both hits. Among the newly prepared compounds, two showed interesting RdRp inhibition profiles. The two initial hits 11 and 13, together with the two newly prepared derivatives 42a and 54, displayed IC50 values against human norovirus RdRp activity in the micromolar range (5.0–27.4 µM), with the best results obtained for 11 and its close derivative 54. These four compounds also displayed broad-spectrum inhibitory activity of RdRps across three Caliciviridae genera, including Norovirus, Lagovirus and Sapovirus. There is little protein sequence identity between the examined calicivirus polymerases (30.2–61.4%), and therefore the broad-spectrum activity could be attributed to a common, highly-conserved binding pocket across the RdRps, as has been previously reported with broad-spectrum activity of other site-B binding NNIs16. While 54 demonstrated an IC50 of 12.1 µM against the MNV RdRp in vitro, it had little effect against MNV in cell culture (EC50 > 50 µM). This could be due to poor cell membrane permeability, as previously observed with PPNDS16 and suramin40.A virtual screening evaluation of commercially available, drug-like compounds  was performed on the suramin and PPNDS binding-sites of the human norovirus RdRp. These two RdRp inhibitors occupy two different sites, A and B, situated in the NTP access pathway located between the fingers and thumb domains, and overlap each other in the central part of the protein. Selected compounds (n = 62) were examined for inhibition of human norovirus RdRp activity using an 54 is predicted to bind to the central core of site-B of the human norovirus RdRp, which has previously been shown to be an ideal antiviral target due to its highly conserved structure16. The proposed 54 binding mode was supported by combinational studies with PPNDS, the previously reported site-B binding norovirus NNI17. PPNDS and 54 were antagonistic in combination against norovirus RdRp activity, indicating that both compounds bind in the site-B pocket.Molecular docking studies revealed that the novel compound 54 may form distinct interactions with other residues within site-B, as mutations of previously reported site-B residues (Q414A and R419A) had no appreciable effect on the inhibitory activity of 54 compared to wild-type RdRp activity.17. It is possible that 54 works in the same fashion, and the actual binding site of 54 could be resolved through further crystallography and mutational studies.The mode of inhibitory action of PPNDS was reported to be through fixation of the C-terminus of the polymerase within the active site, blocking incoming NTPs and the RNA template54, was found to be an effective and specific inhibitor of RdRps across three genera of the Caliciviridae, although there was little effect against MNV replication in cell culture, indicating that cell permeability could be further optimised. In silico and combinational studies suggest that 54 binds the highly conserved site-B in the RdRp, and therefore this compound represents a promising starting point for further structure-activity relationship enhancement, to improve potency, solubility and specificity profiles in the quest for an effective antiviral against human norovirus.In conclusion, a novel NNI scaffold, 61. RdRp resistance mutants were generated using the QuikChange site-directed mutagenesis kit  and confirmed by Sanger sequencing.Recombinant calicivirus RdRps (GenBank accession numbers) norovirus GII.Pe/GII.4 Sydney 2012 (KT239579), MNV GV CW1 (DQ285629), RHDV Czech V351 (KF594473.1), and SaV GI.1 Mc114 (AY237422) were expressed and purified as described previously62. Briefly, RdRp activity was quantified by monitoring the formation of double-stranded RNA (dsRNA) from a single stranded homopolymeric template, poly(C) , using the fluorescent dye PicoGreen . RdRp assays were performed in 384-well plates, and each reaction mixture contained 400 ng enzyme, 45 µM GTP, 10 ng/µL poly(C) RNA, 2.5 mM MnCl2, 5 mM dithiothreitol, (DTT), 0.01% bovine serum albumin (BSA), and 0.005% Tween 20 in 20 mM Tris-HCl, pH 7.5, with a final reaction volume of 25 µL. The previous published norovirus NNI NIC02 was used as a positive control14. RdRps were incubated for 10 mins at 30 °C in the presence of the test compounds or the compound vehicle DMSO (0.5% vol/vol) before addition into the reaction mixture, which was then allowed to run 15 min at 30 °C then terminated with 10 mM EDTA, followed by PicoGreen staining and dsRNA quantitation. GraphPad Prism V6.05  was used to plot the IC50 values. In cases where fluorescent enhancement was recorded for a compound instead of inhibition, a secondary gel-based polymerase activity assay was used as a counter-screen as described below, to exclude the possibility of RdRp activity enhancement  were tested alone and together against norovirus Sydney 2012 RdRp.Fluorescent RdRp activity assays were carried out as previously describedPseudomonas Φ6 bacteriophage RdRp (NEB) reactions were set up as described above (Fluorescent RdRp activity assays), using 400 ng of RdRp per reaction.The 14. Replication was examined using plaque assays65. Cytotoxicity was examined using a CellTitre Blue  viability assay as described previously14.MNV-1 (CW1) culture assays were performed as previously described15 primer, 1 × MMLV NTP buffer (NEB) and 0.1 mM dTTP in a final volume of 25 µL. The RT was incubated with the compound or the compound vehicle (0.5% DMSO) for 10 min, and then reactions were initiated by the addition of the RNA and primer mix. The reaction was incubated for 1 h at 42 °C then the RT was deactivated by incubation at 65° for 20 min. Picogreen stain was added and the relative fluorescence was measured as described above in the ‘Quantitative RdRp activity and gel-based assays’ section.Lead hits were examined for inhibition of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) (kit M02535 (NEB)). MMLV RT activity reactions were carried out using 200 U MMLV RT (NEB) per reaction, with 250 ng homopolymeric poly(A) RNA template , 50 nM oligo dTTM i7-3770 vPro CPU@ 3.40 GHz × 8 running Ubuntu 14.04. Molecular Operating Environment (MOE) 2015.1066, Maestro (Schrödinger Release 2017–1), LeadIT (version 2.1.8), OpenEye Scientific Software and PLANTS were used as molecular modelling software. Pharmacophoric filters were created within MOE choosing the PCH (polar-charged-hydrophobic) scheme. Shape-comparison screening was performed with ROCS version 3.2.1.4. Both shape and color screen criteria were applied to the query. Output conformations were ranked according to the Tanimoto combo score and the shape Tanimoto score. A library of commercially available compounds was downloaded from SPECS website (www.specs.net) in sdf format and prepared using the Conformational Search tool in MOE for the pharmacophore studies and the Omega version 2.5.1.4 for the shape-based screening.All molecular modelling studies were performed on a Viglen Genie Intel®Corehttp://www.rcsb.org/); PDB code 3UR0 (suramin), 4LQ3 (PPNDS) and prepared with the MOE Protein Preparation tools for the pharmacophore studies and PLANTS docking. Docking PLANTS simulations for the virtual screening were performed using the following parameters: search algorithm: aco_ants 20, aco_evap 0.15, aco_sigma 2.0; binding site: bindingsite_center [−2.665 5.272 33.756] for suramine crystal [−19.229 −25.402 −4.634] for PPNDS crystal, bindingsite_radius 10; cluster algorithm: cluster_rmsd 2.0, cluster_strucures 5; scoring function: chemplp. The docking results obtained were then rescored using Glide XP and FlexX scoring functions.RdRp crystal structures were downloaded from Protein Data Bank (PDB)  was prepared using the Schrödinger Protein Preparation Wizard by assigning bond orders, adding hydrogens and performing a restrained energy minimization of the added hydrogens using the OPLS_2005 force field. An 11 Å docking grid (inner-box 11 Å and outer-box 220 Å) was prepared using as centroid the co-crystallised structure of PPNDS. Molecular docking of 54 was performed using Glide SP precision keeping the default parameters and setting 10 as number of output poses to include in the solution. The docking output database was saved as mol2 file and the docking poses visually inspected for their binding mode in MOE.Docking of consensus score procedure were evaluated considering the following criteria:ability of a compound to overall occupy the binding site (suramine or PPNDS binding site);number of interactions formed between the compound and the target protein ;coverage of different chemical scaffolds, discarding similar chemical entities;potential chemical instability;presence of potential toxic groups .The visual inspection process, conducted as last step of the structure-based and the ligand-based virtual screening, was performed using MOE 2015.10. The docking poses of the compounds obtained from the 1H and 13C NMR spectra were recorded with a Bruker Avance DPX500 spectrometer operating at 500 MHz for 1H and 125 MHz for 13C, with Me4Si as internal standard. Deuterated chloroform was used as the solvent for NMR experiments, unless otherwise stated. 1H chemical shifts values (δ) are referenced to the residual non-deuterated components of the NMR solvents . The 13C chemical shifts (δ) are referenced to CDCl3 . Mass spectra were determined with a Bruker microTOF spectrometer using electrospray ionization (ESI source). For mass spectra, solutions were made in HPLC grade methanol. TLC was performed on silica gel 60 F254 plastic sheets. Flash column chromatography was performed using silica an Isolera Biotage system. UPLC-MS analysis was used for purity/mass determination of the tested compounds using a Waters UPLC system with both Diode Array detection and Electrospray (+’ve and –‘ve ion) MS detection. The stationary phase was a Waters Acquity UPLC BEH C18 1.7 um 2.1 × 50 mm column. The mobile phase was H2O containing 0.1% Formic acid (A) and MeCN containing 0.1% Formic acid (B). Column temperature: 40 °C. Sample diluent: acetonitrile. Sample concentration 1 µg/mL. Injection volume 2 µL. A linear gradient standard method (A) was used: 90% A (0.1 min), 90–0% A (2.6 min), 0% A (0.3 min), 90% A (0.1 min); flow rate 0.5 mL/min. All compounds tested in biological assays were >95% pure. Purity of intermediates was >90%, unless otherwise stated.All solvents and reagents were used as obtained from commercial sources unless otherwise indicated. All solvents used for chromatography were HPLC grade from Fisher Scientific (UK). All reactions were performed under a nitrogen atmosphere. Supplementary Information


Pyridoxal demonstrated superior inhibition against maltase (0.38 mg/mL IC50) and glucoamylase (0.27 mg/mLIC50). In addition, pyridoxal showed significant higher α-amylase inhibitory activity (10.87 mg/mL of IC50) than that of pyridoxine (23.18 mg/mL of IC50). This indicates that pyridoxal can also inhibit starch hydrolyzing by pancreatic α-amylase in small intestine. Based on these in vitro results, the deeper evaluation of the anti-hyperglycemic potential of pyridoxine and its derivatives using Sprague-Dawley (SD) rat models, was initiated. The post-prandial blood glucose levels were tested two hours after sucrose/starch administration, with and without pyridoxine and its derivatives. In the animal trial, pyridoxal (p < 0.05) had a significantly reduction to the postprandial glucose levels, when compared to the control. The maximum blood glucose levels (Cmax) of pyridoxal administration group were decreased by about 18%  and 19%  in sucrose and starch loading tests, respectively, when compared to the control in pharmacodynamics study. The pyridoxal administration significantly decreased the minimum, maximum, and mean level of post-prandial blood glucose at 0.5 h after meals. These results indicate that water-soluble vitamin pyridoxine and its derivatives can decrease blood glucose level via the inhibition of carbohydrate-hydrolyzing and absorption-linked enzymes. Therefore, pyridoxal may have the potential to be used as a food ingredient for the prevention of prediabetes progression to type 2 diabetes. In the current study, we investigated the inhibitory activity of pyridoxine, pyridoxal, and pyridoxamine, against various digestive enzymes such as α-glucosidases, sucrase, maltase, and glucoamylase. Inhibition of these enzymes involved in the absorption of disaccharide can improve post-prandial hyperglycemia due to a carbohydrate-based diet. Pyridoxal (4.14 mg/mL of IC Type 2 Diabetes Mellitus (T2DM), a common disorder of glucose metabolism, is linked to insulin resistance and high calorie diets. T2DM is associated with changes in dietary pattern towards high calorie sweetened foods with disaccharides such as maltose and sucrose . T2DM ac® , Voglibose® , and Miglitol®  are generally prescribed either alone, or in combination with insulin sensitizers in T2DM patients, clinically.High blood glucose levels are typical in type 2 diabetes patients, and more specifically this is characterized by a postprandial increase of glucose levels following food consumption, due to digestion of carbohydrates by pancreatic α-amylase and small intestinal α-glucosidases, resulting in elevated liberation of glucose . A stratPyridoxine (Vitamin B6) has been used for managing normal cognitive function and in lowering the incidence of coronary heart disease (CHD) among the seniors ,9,10. InHowever, pyridoxine and its derivatives have not been thoroughly evaluated for their possible blood glucose lowering effect and there is no study suggesting the exact mechanism of vitamin B6 for this specific health benefit. Therefore, the aim of this study is to investigate mode of action and effect of pyridoxine and its derivatives on postprandial hyperglycemia. To determine the above, inhibitory activities of pyridoxine, pyridoxal, and pyridoxamine are investigated against the α-glucosidase and α-amylase . Anti-hyperglycemic effect of these compounds was also evaluated in Sprague-Dawley (SD) rat models. This work will contribute towards the understanding of the activity and the mechanism of action of vitamin B6 and its derivatives, specifically towards the management and prevention of type 2 diabetes. p-nitrophenyl-α-d-glucopyranoside (pNPG), porcine pancreatic α-amlyase enzyme powder, starch, sucrose, and maltose were purchased from Sigma-Aldrich . Unless noted, all chemicals were purchased from Sigma-Aldrich .Water-soluble pyridoxine and its derivatives was used from reagent companies in Sigma-Aldrich , Daejung , Duksan , and Junsei . Rat intestinal acetone powder, g, 30 min, 4 °C), the resulting supernatant was used for the assay. Sample solution (50 μL) and 0.1 phosphate buffer  containing α-glucosidase  solution (1.0 U/mL) was incubated at 37 °C for 10 min. After pre-incubation, 5 mM p-nitrophenyl-α-d-glucopyranoside solution (50 μL) in 0.1 M phosphate buffer (pH 6.9) was added to each well at timed intervals. The reaction mixtures were incubated at 37 °C for 30 min. Before and after incubation, absorbance was read at 405 nm and compared to a control, which had 50 μL of buffer solution in place of the extract by micro-plate reader . The α-glucosidase inhibitory activity was expressed as inhibition % and was calculated as follows:The rat intestinal α-glucosidase assay was administered as per the method of Kwon et al.  with sliThe porcine pancreatic α-amlyase inhibition assay was administered as per the method of Kwon et al.  Sample sg, 30 min, 4 °C), the resulting supernatant was used for the assay. Maltase, sucrose, and glucoamylase inhibitory activity were assay by modifying a method developed by Dahlqvist [The crude enzyme solution prepared from rat intestinal acetone powder Sigma-Aldrich Co.  was used as the small intestinal maltase, sucrose, and glucoamylase, showing specific activities of 0.70, 0.34, and 0.45 units/mL, respectively. Rat intestinal acetone powder (1.0 g) was suspended in 3 mL of 0.1 M sodium phosphate buffer, and the suspension was sonicated 12 times for 30 s at 4 °C. After centrifugation  rats was determined by an inhibitory action of water-soluble vitamins and Acarbose on postprandial hyperglycemia. All animal procedures were approved by Institutional Animal Care and Use Committee (IACUC) of the Hannam University . Four-week-old male SD rats were purchased from Raon Bio Co.  and fed a solid diet  for one week. The rats were housed in a ventilated room at 23 ± 2 °C with 50 ± 5% relative humidity, and under an alternating 12 h light/dark cycle. After 7 groups of 4 male SD rats (130~150 g) were fasted for 24 h, 2.0 g/kg of sucrose or starch were orally administrated concurrently with 0~100 mg/kg inhibitors (water-soluble vitamins and Acarbose). The blood samples were then taken from the tail after administration and blood glucose levels were measured at 0, 0.5, 1, 2, and 3 h. The glucose level in blood was determined by glucose oxidase method and compared with that of the control group, which had not taken the inhibitors.p < 0.05.All data are presented as mean ± Standard deviation (SD). Statistical analysis was carried out using statistical package SPSS 10 , and the significance of each group was verified with the analysis of one-way analysis of variance (ANOVA) followed by Duncan’s test of ® and Voglibose®, delay the digestion of oligosaccharide and disaccharide to monosaccharide by inhibiting α-glucosidases on the small intestinal brush-border, and reduce the rate of glucose absorption [α-Glucosidase inhibitors, such as Acarbosesorption . Inhibit50 values were calculated, we observed that pyridoxal had the lowest value (4.15 mg/mL), while pyridoxine had the highest (5.02 mg/mL)  .Our observations suggest that pyridoxal has the highest inhibitory potential against α-glucosidase while pyridoxine had the lowest .50 values were calculated, we observed that pyridoxal had the lowest value (12.92 mg/mL), while pyridoxine had the highest (23.17 mg/mL)  . Pyridox7 mg/mL) .Our observations are similar to our previous results, suggesting that pyridoxal has the highest inhibitory potential against α-amylase, while pyridoxine had the lowest .Since α-glucosidase inhibitory effects of the vitamin B6 group was shown, we examined whether vitamin B6 group could selectively inhibit enzymes in the small intestine, namely sucrase, maltase, and glucoamylase. The sucrase inhibitory effect of the tested compounds revealed that all of them had similar inhibitory effects , Table 1Similar to maltase inhibitory activity, all tested vitamin B6 structures resulted in dose-dependent glucoamylase inhibition , and pyr50) of samples in vitro system was shown in 50 value for maltase and glucoamylase , suggesting higher inhibition potential. Against sucrase all tested samples yielded similar and not significant different IC50 values  and starch group (216.92 ± 12.46 mg) , pyridoxAmong the three tested compounds, change of blood glucose level in the pyridoxal-treated group with sucrose was increased by 64.88 ± 12.06 mg/dL at 30 min after administration . This ismax, there is no significance between sucrose/starch and vitamin B6-treated groups. In contrast, vitamin B6-treated groups resulted in significantly decreased Cmax and AUCt values (both with sucrose and starch loading), but less effective than the Acarbose group. Pharmacodynamic parameters of the sucrose and starch loading test are shown in max and AUC  of the pyridoxal administration group were decreased by about 18%  and 19%  in the sucrose and starch loading tests, respectively, when compared to the control in the pharmacodynamics study.In sucrose loading, pyridoxal and pyridoxamine resulted to the highest reduction both in terms of C and AUC . In the C values . In termOur findings suggest that vitamin B6 and its derivatives can inhibit the breakdown of disaccharide to monomer in brush-border and suppress the entrance of blood glucose into the bloodstream.In this manuscript, we are presenting the first report of vitamin B6 and its derivatives, for the potential management of type 2 diabetes, via the inhibition of small-intestinal α-glucosidases. Our significant in vitro and in vivo findings indicate that pyridoxine (vitamin B6) has α-glucosidase inhibitory activity, resulting in reduced postprandial blood glucose levels following sucrose and starch ingestion. The purpose of this research was to evaluate the mode of action and type 2 diabetes-relevant effect of vitamin B6 and its derivatives. More specifically, we evaluated the effect on in vivo postprandial hyperglycemia via the in vitro inhibition of carbohydrate-hydrolyzing enzymes. Vitamin B6  forms are readily absorbed by passive diffusion in the jejunum and ileum , while a


The conformational influence of a single stereogenic centre in an otherwise achiral oligomer behaves as a signal that decays with distance. M and P helices that such oligomers typically adopt by the inclusion of an N or C terminal chiral inducer. We show that decay constants may be very low  in non-polar solvents, and we evaluate the increase in decay constant that results in polar solvents, at higher temperatures, and with more conformationally flexible residues such as Gly. Decay constants are independent of whether the signal originates from the N or the C terminus. By interpreting the decay constant in terms of the probability with which conformations containing a screw-sense reversal are populated, we quantify the populations of these alternative minor conformers within the overall ensemble of secondary structures adopted by the foldamer. We deduce helical persistence lengths for Aib polymers that allow us to show that in a non-polar solvent a peptide helix, even in the absence of chiral residues, may continue with the same screw sense for approximately 200 residues.Although foldamers, by definition, are extended molecular structures with a well-defined conformation, minor conformers must be populated at least to some extent in solution. We present a quantitative analysis of these minor conformers for a series of helical oligomers built from achiral but helicogenic α-amino acids. By measuring the chain length dependence or chain position dependence of NMR or CD quantities that measure screw-sense preference in a helical oligomer, we quantify values for the decay constant of a conformational signal as it passes through the molecular structure. This conformational signal is a perturbation of the racemic mixture of  Examples include the helical conformation of DNA under normal conditions8 and structural proteins such as collagen.9 However, many other biopolymers exhibit more complex conformational behaviour in which alternative conformers may be populated. Classic examples10 include allosterically switchable molecules such as haemoglobin11 or phosphorylase enzymes,12 and conformationally switchable proteins involved in signal transduction.14 Yet other biopolymers appear to be almost fully unstructured in a three-dimensional sense, with many energetically similar alternative conformational states being populated.15 Synthetic analogues of these less conformationally uniform biomolecules should form their own group within the general class of foldamers, comprising structures to which more than one well defined ‘specific compact conformation’ is available. Indeed, entropic considerations make it reasonable to expect that even the most conformationally well defined foldamers must spend at least some of their time in minor, less favourable conformations. Studies of the relative tendency towards helicity of alternative monomer units have been made in some foldamer classes by exploring their ability to communicate a screw-sense preference between two helical domains.17 More detailed exploration by classic spectroscopic techniques of the multiple conformers populated by some peptidomimetic structures poses well-established difficulties.20 For this reason, the intrusion of alternative conformers into foldamer structures of uniform primary sequence has rarely been quantified, explored, or exploited.21 Nonetheless, such studies are essential if the broader field of foldamer chemistry is to deliver biomimetic function as well as biomimetic structure.A foldamer is ‘a polymer [or oligomer] with a strong tendency to adopt a specific compact conformation’,etc.23 form a class of foldamers that adopt specific compact conformations that take the form of hydrogen-bonded 310 helices.28 These helices are necessarily equal mixtures of left- and right-handed screw-sense conformers because  the monomers themselves are achiral.29 Nonetheless an equal population of the M and P conformers may be detectably perturbed by incorporation of one or more terminal chiral residues39 or interaction with chiral ligands.44 A helical chain of achiral monomers may be switched between a left- and a right-handed screw-sense preference either by configurational inversion of a single chiral residue at one terminus,45 or by reversible covalent40 or non-covalent47 binding of a chiral ligand to a terminal binding site. Such foldamers exhibit a form of switchability analogous to membrane-bound receptors, in which a conformational response to the binding of a ligand mediates signal transduction through the thickness of the cell membrane to mediate a remote biochemical outcome.13Oligomers of achiral quaternary amino acids, of which 2-aminoisobutyric acid (Aib) is the archetype, but which also include 1-aminocyclohexanecarboxylic acid Ac6c,  along wi48 Two chiral l-α-methylvaline  residues at the terminus of a peptide-like oligomer of achiral quaternary amino acids are sufficient to induce essentially complete control over screw-sense preference which persists far enough to allow reactions to take place with 1,61 asymmetric induction and 88 : 12 diastereoselectivity. Similar reactions over shorter distances  show successively increased levels of stereoselectivity. This chain-length dependence49 indicates that the ability of the helical foldamer to communicate stereochemical information is high, but not perfect  of the reaction is in each case the product of the induced helical excess56 at the N-terminal dimer (hei), the fidelity with which this helical excess is communicated to the C terminus, and the diastereofacial selectivity (ds) with which a pure M or P helix would induce attack on the iminium ion intermediate in the acid-catalysed reaction. As the first and last of these factors are constant in all four reactions, the decay in diastereoselectivity in the reactions of the oligomers 1a–d in 1a–d → 2a–d plotted against the distance n in residues from the inducer of helical preference at residue i to the reaction site. On the assumption that Aib and Ac6c have equivalent conformational preferences,57 we fitted exponential decays to these curves, using the formulan is the diastereoisomeric excess induced in a reaction at residue i + n, hei is the local helical excess induced by the N terminal l-α-MeVal dimer, f is the fraction by which he decreases on passing through each residue in the chain, and ds is the inherent diastereoselectivity of the reaction (i.e. the de that would result from a purely M or purely P helix).The reactions shown in i.e. f = 0.008) under the conditions of the reaction in THF at –50 °C.60 Given that when n = 0, dei = hei × ds, we can use de0 2 of 99% , by measuring the anisochronicity of a pair of signals arising from diastereotopic reporter groups embedded in the helical chain.opic 13C , 1H ) between the CHN signals of the diastereoisomers  and -2a–d decreases with increasing chain length at 23 °C in deuterated THF and at +23 °C or 40 °C in a more polar solvent, deuterated ethanol, and n (the length of the chain lying between the centres) for these solvents. Poor solubility prevented us studying these oligomers in chloroform. We fitted the points to a modification of the formula used earlierδn is the anisochronicity of the CHN signals when they are located at residue i + n and Δδi simply a limiting value to which we assign no chemical significance. Decay constants f (the per-residue decrease in Δδ) can be extracted of 0.1% for THF at 23 °C, 1.9% for ethanol at 23 °C, and 2.5% for ethanol at 40 °C. The near-zero value of the decay constant in THF confirms the observation from 3 .An alternative, simpler measure of signal decay may be obtained by inserting several NMR reporters, each containing a pair of diastereotopic groups giving rise to potentially anisochronous signals, at successive positions along a helical oligomer. Provided that the reporter groups are sufficiently far from the ends of the chain for their anisochronicity to be dictated solely by their local helical environment, the decrease in their anisochronicity along the chain gives a measure of signal decay. The challenge in such an approach is identifying which of several identical reporters is which. This is less difficult if the rate at which anisochronicity decreases is relatively high, because the signals from successive reporters are not superimposed upon one another, and a simplifying assumption can be made that anisochronicity can only decrease along the chain. We applied this principle to oligomer 3 65 which 17 so we expect the values of f for Aib (fAib) and for Gly (fGly) to differ significantly. There are four Aib residues for every Gly in the chain, so the value f obtained from fitting eqn (3) to This time, the additional conformational flexibility complicates the analysis of the decay constants. Gly introduces much more flexibility than Aib into a peptide chain,f obtained exhibit a solvent dependence consistent with that noted for 2, with the more polar methanol allowing greater conformational flexibility than the less polar THF, and ethanol lying somewhere between. Using our previous estimates of values for fAib at +23 °C in THF, in ethanol  indicating that glycine residues introduce a fall in Δδ at +23 °C of 13% in THF, 31% in C2D5OD or 49% in CD3OD, confirming that the Gly residues introduce significantly more conformational flexibility than the Aib residues.The values of  ethanol  and in m1H NMR spectrum. Dispersion in the 13C NMR spectrum is better, and individual residues may be labelled by means of 13C-enriched methyl groups.34 However, difficulties of signal assignment remain, firstly because signals are likely to overlap when different parts of the oligomer exhibit a similar helical excess , and secondly because diastereotopic pairs of signals are harder to identify because they are not related to each other by coupling as they are in the 1H NMR spectra of oligomers containing germinal CH2 groups. We have previously shown, however, that successive members of a series of 13C labels may themselves each be labelled by changing their 13C abundance through successive dilutions with 12C, allowing paired diastereotopic labels to be identified by relative integrals.34Identification of individual residues in an all-Aib oligomer is more challenging, because of the poor dispersion of the Aib methyl groups, which all lie within a <0.35 ppm range in the 7 were synthesized using the strategy illustrated in 4** and 4 were built by standard methods from a starting monomer that was either 100% doubly (4**) or 1.1% singly  13C labelled in its Aib methyl groups. The two samples of trimer 4** and 4 were then mixed in ratios of 50 : 50 or 25 : 75 and these two samples sequentially ligated to the methyl ester of 4** to make a nonamer 5, three of whose residues are labelled with different abundances of 13C, as shown in 5 was extended by ligation with an unlabelled Aib dimer, trimer or tetramer to make the three oligomers 6a–c, which were each coupled at the C terminus to the powerful chiral controlling residue l-AlaNHt-Bu32 to provide 12-mer 7a, 13-mer 7b and 14-mer 7c.We used this approach to explore the communication of conformational information through an Aib oligomer from the C terminus to the N terminus. The multiply labelled compounds i – 3 to i – 11, starting from the C terminal l-AlaNHt-Bu chiral controller at position i  of the label in the chain relative to the C-terminal alaninamide (residue i). Fitting an exponential decay (eqn (3), using positive values of n) to each plot gives signal decay constants f (the per-residue decrease in Δδ) of 6.1% in methanol, 5.4% in 9 : 1 methanol : chloroform, and 0.5% in THF. Earlier work had shown that aggregation effects may become important in solvents such as chloroform,69 but these were shown to be negligible in this case as the anisochronicity was independent of concentration over a 4-fold concentration range (4.6–18 μM). Changes in temperature induced a small variation in the value of f for the oligomers in methanol, which was 5.5% at 0 °C; 6.1% at 23 °C and 6.5% at 40 °C. These values correspond to an enthalpy difference of about 3 kJ mol–1 and an entropy difference of –13 J mol–1 K–1.8a–d  is well separated from those associated with the carbonyl groups in the chain (θmax < 220 nm), and thus we may expect molar ellipticity at 249 nm to be proportional71 to the induced helical excess at the N terminus of the chain. f of 2.5% in THF and 7.6% in MeOH, at +23 °C.In the circular dichroism spectra of 81 Examples include not only oligomers of Aib,29 but also polyacetylenes,84 polyisocyanides,86 polyisocyanates,88 polyaromatic structures of various types91 and even polyacetals.93 The conformations of such helices consist of domains of identical screw-sense which may span the whole length of the oligomer, or alternatively may be interrupted by ‘helical perversions’:94 locations where the screw sense reverses.95 The lengths of the resulting domains of screw-sense uniformity have in some cases been quantified, and in one well studied case, the polyisocyanates, this length can be some 600 monomers at 20 °C.87 In other words, the average number of monomers separating one screw-sense reversal from the next is 600.There are numerous polymeric structures that are helical even though the individual monomers are achiral.P screw sense at one terminus will have P screw sense at the other if it contains zero or an even number of reversals and M screw sense if it contains an odd number of reversals. The likelihood of the two ends of the domain having the same screw sense may be calculated by considering the excess probability of an odd number over an even number of screw sense reversals, assuming an equal probability of screw-sense reversal at each location in the domain.The decay of screw-sense preference on lengthening a helical domain interposed between a screw-sense controller and reporter may be viewed in terms of the chance of helix reversals occurring in that domain. A domain containing a single reversal, or indeed any odd number of reversals, will give rise to opposite screw-sense preferences at the two termini of the domain, while zero or an even number of reversals will lead to identical screw senses at the termini. Thus a helix having N elements, the difference in probability between having an even number of elements in state α and an odd number in state α is65p). Since helical excess (he) = ([P] – [M])/([P] + [M]), interpreting state α as a helix reversal allows the inference that he at position i in a helix built from achiral monomers is related to he at position i + n by this same expressionp may be interpreted as the ‘fidelity’ with which a screw sense preference is transmitted from one monomer to the next .For a domain of f derived from eqn (3)–(5) may be interpreted structurally as a measure of the probability of screw-sense reversal, with f = 2 × (1 – p). In other words with p as the screw-sense fidelity, f/2 may be interpreted as the probability of a helix reversal occurring, and f, the signal decay constant, as the fractional decrease in helical excess as the screw-sense preference passes from one monomer to the next. Values for decay constant f and hence fidelity p obtained from Thus the empirical values for the signal decay constant f for any monomer must be independent of whether the ‘signal’ is passing from the N to the C (as in 2 and 3) or the C to the N (as in 7 and 8) terminus. The values of p obtained for Aib in MeOH from 7 or 8 for a signal travelling from the C terminus to the N terminus  are close to values previously calculated for a signal travelling from the N terminus to the C terminus /(1 – fAib) = 0.59; the corresponding value calculated from the data in This interpretation of the decay of he along a chain in terms of the likelihood of the two termini of a helical domain having the same or opposite screw sense makes it clear that the decay constant  . It is 99 Yet both the observed rate at which the NH signals of the oligomer exchange with D from the deuterated solvent, and the rate at which protonated signals returned on dissolution of deuterated oligomer in protonated solvent, are extremely slow. H/D exchange in CD3OD occurs on a time scale of hours to days at ambient temperature, while helical inversion occurs on the millisecond time scale,64 suggesting that random coil conformations make a negligible contribution to the ensemble of structures adopted by Aib oligomers. Thus we propose that the helical reversals that lead to signal decay are well-defined, local structural features which entail only a small perturbation of the 310 helical structure, and retain a well-defined intramolecularly hydrogen-bonded network. We are currently exploring experimentally the possible detailed structures for these helix reversals. As suggested by Kubasik,63 migration of a well-structured helical reversal along a chain, sequentially breaking and reforming only one or maybe two hydrogen bonds at a time, could provide a mechanism for the remarkably rapid inversion of a 310 helix.The values in 100 of the average length over which hypothetical extended Aib homo-polymers might be expected to adopt a uniform screw sense. The Ising model, first used to describe ferromagnetic and antiferromagnetic arrays of atoms, assumes that the energetic cost of a state reversal (here a change in screw sense) in a linear array is determined solely by nearest neighbours. If the chance of a helix reversal at any one residue is (1 – p), the Boltzmann relation givesJ is the energetic penalty for introducing a helix reversal.The values also allow for the first time an estimate to be made, using the one-dimensional Ising model,n segments as the nth power of tanh(J/kT). This factor is equivalent to our empirical decay factor (1 – f) (eqn (3)), giving as expected f = 2 × (1 – p). The persistence length L  may be obtained from the Ising model by101According to the one-dimensional Ising model, the helical excess will decay exponentially over L are shown in Values for J of a helix reversal. For example, in the case of 2a–d in THF at –50 °C , of the energetic cost 2t –50 °C , J = 10 A screw-sense preference induced by a chiral residue at the terminus of an achiral helical oligomer may be considered as a signal whose strength decays as with decreasing distance from the chiral inducer. The spatial decay rate may be quantified by means of NMR or CD probes of helical excess located at successive positions in an oligomeric structure, or located at the terminus of oligomers of varying lengths. The decay rate depends on solvent and temperature  and on the nature of the achiral oligomers in the chain . Typical values are of the order of 5% for Aib in MeOH, and 1% for Aib in THF, leading to helix persistence lengths in non-polar solvents of around 200 monomers. The decay rate may be interpreted as the result of a helix reversal occurring randomly at any position in the oligomer. Helix reversals appear to be localized, well-ordered structures, and ongoing work seeks to establish their detailed geometry.