Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases.

Terpene synthases (TS) catalyze complex reactions to produce a diverse array of terpene skeletons from linear isoprenyl diphosphates. Patchoulol synthase (PTS) from Pogostemon cablin converts farnesyl diphosphate into patchoulol. Using simulation-guided engineering, we obtained PTS variants that eliminate water capture. Further, we demonstrate that modifying the structurally conserved Hα-1 loop also reduces hydroxylation in PTS, as well as in germacradiene-11-ol synthase (Gd11olS), leading to cyclic neutral intermediates as products, including α-bulnesene (PTS) and isolepidozene (Gd11olS). Hα-1 loop modification could be a general strategy for engineering sesquiterpene synthases to produce complex cyclic hydrocarbons without the need for structure determination or modeling.

The emerging potential of interactive virtual reality in drug discovery.

INTRODUCTION: The potential of virtual reality (VR) to contribute to drug design and development has been recognized for many years. A recent advance is to use VR not only to visualize and interact with molecules, but also to interact with molecular dynamics simulations 'on the fly' (interactive molecular dynamics in VR, IMD-VR), which is useful for flexible docking and examining binding processes and conformational changes. AREAS COVERED: The authors use the term 'interactive VR' to refer to software where interactivity is an inherent part of the user VR experience e.g. in making structural modifications or interacting with a physically rigorous molecular dynamics (MD) simulation, as opposed to using VR controllers to rotate and translate the molecule for enhanced visualization. Here, they describe these methods and their application to problems relevant to drug discovery, highlighting the possibilities that they offer in this arena. EXPERT OPINION: The ease of viewing and manipulating molecular structures and dynamics, using accessible VR hardware, and the ability to modify structures on the fly (e.g. adding or deleting atoms) - and for groups of researchers to work together in the same virtual environment - makes modern interactive VR a valuable tool to add to the armory of drug design and development methods.

Discovery of SARS-CoV-2 Mpro peptide inhibitors from modelling substrate and ligand binding.

The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective 'peptibitors' inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.

Interactive Molecular Dynamics in Virtual Reality Is an Effective Tool for Flexible Substrate and Inhibitor Docking to the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the SARS-CoV-2 virus is one focus of drug development efforts for COVID-19. Here, we show that interactive molecular dynamics in virtual reality (iMD-VR) is a useful and effective tool for creating Mpro complexes. We make these tools and models freely available. iMD-VR provides an immersive environment in which users can interact with MD simulations and so build protein complexes in a physically rigorous and flexible way. Recently, we have demonstrated that iMD-VR is an effective method for interactive, flexible docking of small molecule drugs into their protein targets (Deeks et al. PLoS One 2020, 15, e0228461). Here, we apply this approach to both an Mpro inhibitor and an oligopeptide substrate, using experimentally determined crystal structures. For the oligopeptide, we test against a crystallographic structure of the original SARS Mpro. Docking with iMD-VR gives models in agreement with experimentally observed (crystal) structures. The docked structures are also tested in MD simulations and found to be stable. Different protocols for iMD-VR docking are explored, e.g., with and without restraints on protein backbone, and we provide recommendations for its use. We find that it is important for the user to focus on forming binding interactions, such as hydrogen bonds, and not to rely on using simple metrics (such as RMSD), in order to create realistic, stable complexes. We also test the use of apo (uncomplexed) crystal structures for docking and find that they can give good results. This is because of the flexibility and dynamic response allowed by the physically rigorous, atomically detailed simulation approach of iMD-VR. We make our models (and interactive simulations) freely available. The software framework that we use, Narupa, is open source, and uses commodity VR hardware, so these tools are readily accessible to the wider research community working on Mpro (and other COVID-19 targets). These should be widely useful in drug development, in education applications, e.g., on viral enzyme structure and function, and in scientific communication more generally.

Interactive molecular dynamics in virtual reality for accurate flexible protein-ligand docking.

Simulating drug binding and unbinding is a challenge, as the rugged energy landscapes that separate bound and unbound states require extensive sampling that consumes significant computational resources. Here, we describe the use of interactive molecular dynamics in virtual reality (iMD-VR) as an accurate low-cost strategy for flexible protein-ligand docking. We outline an experimental protocol which enables expert iMD-VR users to guide ligands into and out of the binding pockets of trypsin, neuraminidase, and HIV-1 protease, and recreate their respective crystallographic protein-ligand binding poses within 5-10 minutes. Following a brief training phase, our studies shown that iMD-VR novices were able to generate unbinding and rebinding pathways on similar timescales as iMD-VR experts, with the majority able to recover binding poses within 2.15 Å RMSD of the crystallographic binding pose. These results indicate that iMD-VR affords sufficient control for users to carry out the detailed atomic manipulations required to dock flexible ligands into dynamic enzyme active sites and recover crystallographic poses, offering an interesting new approach for simulating drug docking and generating binding hypotheses.

Interactive molecular dynamics in virtual reality from quantum chemistry to drug binding: An open-source multi-person framework.

As molecular scientists have made progress in their ability to engineer nanoscale molecular structure, we face new challenges in our ability to engineer molecular dynamics (MD) and flexibility. Dynamics at the molecular scale differs from the familiar mechanics of everyday objects because it involves a complicated, highly correlated, and three-dimensional many-body dynamical choreography which is often nonintuitive even for highly trained researchers. We recently described how interactive molecular dynamics in virtual reality (iMD-VR) can help to meet this challenge, enabling researchers to manipulate real-time MD simulations of flexible structures in 3D. In this article, we outline various efforts to extend immersive technologies to the molecular sciences, and we introduce "Narupa," a flexible, open-source, multiperson iMD-VR software framework which enables groups of researchers to simultaneously cohabit real-time simulation environments to interactively visualize and manipulate the dynamics of molecular structures with atomic-level precision. We outline several application domains where iMD-VR is facilitating research, communication, and creative approaches within the molecular sciences, including training machines to learn potential energy functions, biomolecular conformational sampling, protein-ligand binding, reaction discovery using "on-the-fly" quantum chemistry, and transport dynamics in materials. We touch on iMD-VR's various cognitive and perceptual affordances and outline how these provide research insight for molecular systems. By synergistically combining human spatial reasoning and design insight with computational automation, technologies such as iMD-VR have the potential to improve our ability to understand, engineer, and communicate microscopic dynamical behavior, offering the potential to usher in a new paradigm for engineering molecules and nano-architectures.

Applying sociodramatic methods in teaching transition to palliative care.

We introduce the technique of sociodrama, describe its key components, and illustrate how this simulation method was applied in a workshop format to address the challenge of discussing transition to palliative care. We describe how warm-up exercises prepared 15 learners who provide direct clinical care to patients with cancer for a dramatic portrayal of this dilemma. We then show how small-group brainstorming led to the creation of a challenging scenario wherein highly optimistic family members of a 20-year-old young man with terminal acute lymphocytic leukemia responded to information about the lack of further anticancer treatment with anger and blame toward the staff. We illustrate how the facilitators, using sociodramatic techniques of doubling and role reversal, helped learners to understand and articulate the hidden feelings of fear and loss behind the family's emotional reactions. By modeling effective communication skills, the facilitators demonstrated how key communication skills, such as empathic responses to anger and blame and using "wish" statements, could transform the conversation from one of conflict to one of problem solving with the family. We also describe how we set up practice dyads to give the learners an opportunity to try out new skills with each other. An evaluation of the workshop and similar workshops we conducted is presented.

Using sociodrama and psychodrama to teach communication in end-of-life care.

End-of-life discussions can be stressful and can elicit strong emotions in the provider as well as the patient and family. In palliative care, understanding and effectively addressing emotions is a key skill that can enhance professional competency and patient/family satisfaction with care. We illustrate how in coursework for a Master's degree in palliative medicine we used dramatic "action methods" derived from sociodrama and psychodrama in the portrayal of two challenging cases to train providers in the emotional aspects of caring for patients with advanced cancer. We describe the specific techniques of constructing and enacting case scenarios using warm-ups, role-creation, doubling and role-reversal. In particular, we illustrate how these techniques and others were used to reveal and address the "hidden" emotions, attitudes, and values that were central to the communication dilemma. Finally, we present an evaluation completed by the 26 participants who attended the course.

Preventing and treating discitis: cephazolin penetration in ovine lumbar intervertebral disc.

Infection can occur after any spinal procedure that violates the disc and although it is not common, the potential consequences are serious. Treatment of discitis is not always successful and the key to management is prevention. Intradiscal prophylaxis with antibiotic is routinely used in spinal surgery, but there is a limited understanding of how well antibiotics can enter the avascular disc after intravenous injection. An in vivo ovine study to optimise prophylactic and parenteral treatment of discitis is described to assess the effectiveness of cephazolin in preventing and treating infection. The concentration of cephazolin was measured in disc tissue from normal and degenerate sheep discs to determine if cephazolin can enter the disc and if disc degeneration affects antibiotic uptake. Fourteen sheep were deliberately inoculated with bacteria to induce discitis. Eight sheep ("prophylaxis" group) were given either a 0, 1, 2 or 3 g dose of prophylactic cephazolin before inoculation while the remaining sheep ("treatment" group) were treated with cephazolin commencing 7 days after inoculation for 21 days at a dose of 50 mg/kg/day. Histopathology and radiography were used to assess the effect of the different treatments. Cephazolin was given 30 min prior to sacrifice and the intradiscal concentration was measured by biochemistry. In the "prophylaxis" group all doses of antibiotic provided some protection against infection, although it was not dose dependent. In the "treatment" group discitis was confirmed radiologically and histologically in all animals from 2 weeks onwards. Biochemical assay confirmed that antibiotic is distributed throughout the disc but was present in higher concentration in the anulus fibrosus than the nucleus pulposus. This study demonstrated that whilst the incidence of iatrogenic discitis can be reduced by antibiotic prophylaxis, it could not be abolished in all incidences with a broad-spectrum antibiotic such as cephazolin. Furthermore, antibiotics were ineffective at preventing endplate destruction once an intradiscal inoculum was established.

Penetration of cephazolin in human lumbar intervertebral disc.

STUDY DESIGN: Levels of the antibiotic cephazolin were measured in disc and blood from a consecutive series of patients undergoing lumbar spinal fusion. OBJECTIVES: To determine if levels of cephazolin in the human disc reach the stated minimum inhibitory concentration against Staphylococcus aureus (1 mg/L) following intravenous administration, and to determine if there is a therapeutic relationship between serum concentration and disc concentration. SUMMARY OF BACKGROUND DATA: Disc space infection is a potential complication of surgical procedures that violates the intervertebral disc. Bacteria, often from normal skin flora, can be introduced into the disc space causing inflammation with destruction of the adjacent vertebral endplate. Although there is substantial evidence from animal studies that prophylactic antibiotics reduce the incidence of discitis, similar evidence in human beings is not strong. METHODS: A total of 30 patients, including 15 females and 15 males, with a mean age 42 years (range 21-63), received 1-g cephazolin (intravenous) during 1 or 2-level lumbar spinal fusion surgery. Venous blood was collected before administration of cephazolin and again at disc removal. Disc cephazolin concentrations were measured at the same time serum levels were measured for each patient. RESULTS: The interval between cephazolin administration and tissue sampling ranged from 7 to 137 minutes. Cephazolin concentration in the serum (range 31.1-148 mg/L) was higher than in the disc (range 0-9.5 mg/L). The concentration of cephazolin peaked in the serum at 7 minutes, and in the disc between 37 and 53 minutes. More than 70% of the disc samples had detectable levels of cephazolin at the time the disc was removed, although only half of these samples had cephazolin levels higher than 1 mg/L. All serum cephazolin concentrations were higher than 1 mg/L. Serum cephazolin concentration did not relate to disc concentration at a given time. CONCLUSIONS: The time when antibiotic concentration is highest in the disc varies among patients. More than 70% of disc samples had detectable levels of cephazolin, although only half of the discs contained cephazolin higher than the stated minimum inhibitory concentration for S. aureus(>1 mg/L). Factors such as molecular charge, degree of disc degeneration, and disc size may influence antibiotic penetration into the disc.

Prophylactic cephazolin to prevent discitis in an ovine model.

STUDY DESIGN: An ovine model of discitis was used to determine the efficacy of the antibiotic cephazolin for prophylactic use in spinal surgery. OBJECTIVES: To determine in juvenile and adult sheep spines if 2-g cephazolin given at intervals over a 4-hour period would prevent iatrogenic discitis, and determine the concentration of cephazolin in nucleus and anulus tissue after administration of a bolus dose. SUMMARY OF BACKGROUND DATA: It is standard practice at our institutions to give patients undergoing spinal surgery a single prophylactic (1-2 g) dose of cephazolin with a second dose for prolonged (>4 hours) procedures. Although this regimen provides therapeutic serum levels, the levels of antibiotic in the intervertebral disc are significantly lower. Because cephazolin is a negatively charged molecule, it is thought to diffuse poorly into the disc, raising questions about its efficacy as a prophylactic antibiotic for spinal procedures. METHODS: There were 18 animals, including 9 lambs and 9 sheep, that received a single 2-g dose of cephazolin intravenously at 30-minute intervals over a 4-hour period. Two control animals (1 sheep and 1 lamb) did not receive antibiotic. All animals had diskograms at 2 previously incised lumbar levels and 2 nonincised levels using contrast that was deliberately contaminated with bacteria. Lateral spine radiographs were taken at postoperative intervals. After 12 weeks, all animals received another 2-g dose of cephazolin intravenously at intervals before the spines were removed for pathologic and biochemical analysis. RESULTS: Discitis was detected in all control animals. Of those animals given cephazolin, discitis developed in 1 sheep and 4 lambs. Discitis did not develop in any of the sheep that received cephazolin 30 minutes before inoculation. Cephazolin was detected throughout the disc but was more concentrated in the anulus fibrosus. Disc levels of cephazolin peaked at 15 minutes, at which time serum levels were up to 50 times higher. Cephazolin levels in nonoperated and incised discs were not significantly different. CONCLUSIONS: A single prophylactic 2-g dose of cephazolin administered anytime over a 4-hour period prevented discitis in almost all animals but was not as effective in lambs. Although lambs have a higher vascular supply to deliver antibiotics to the disc, it is likely that some other physiologic factor may be responsible for the increased infection rate. This study supports that timing of antibiotic prophylaxis is critical to prevent iatrogenic disc infection.

Effects of intervertebral disc infection on the developing ovine spine.

STUDY DESIGN: A prospective in vivo animal study. OBJECTIVES: To determine whether infection in the juvenile spine influences spinal development. SUMMARY OF BACKGROUND DATA: Discitis is thought to occur in children when blood-borne infection penetrates the highly vascular immature disc. The condition generally resolves without apparent complication, but little is known about the long-term effects on the growing spine. METHODS: Twenty-nine 6-week-old lambs underwent discography at multiple lumbar levels using radiographic contrast deliberately inoculated with Staphylococcus epidermidis. No antibiotics were given. Plain radiographs were taken at intervals up to 52 weeks after inoculation for morphometric analysis of the vertebral bodies and discs, and the lumbar spines were prepared for histology. RESULTS: Sixteen of 44 inoculated discs showed radiological evidence of discitis between 2 and 6 weeks after inoculation. Disc height and disc area were significantly reduced from 2 weeks, and did not recover during the study period. Vertebral body dimensions and overall lumbar spine length were not significantly affected. CONCLUSION: Infection of juvenile ovine discs impedes disc development but has no significant effect on vertebral body growth.

Does intradiscal electrothermal therapy denervate and repair experimentally induced posterolateral annular tears in an animal model?

STUDY DESIGN: The effects of intradiscal electrothermal therapy (IDET) on intervertebral discs in sheep were studied experimentally. OBJECTIVES: Posterolateral annular lesions were experimentally induced and allowed to mature for 12 weeks in the intervertebral discs of sheep. IDET was performed in an attempt to denervate and repair the annular lesion. The histologic and immunohistochemical effects of IDET were studied. SUMMARY OF BACKGROUND DATA: IDET continues to be used as a minimally invasive treatment for chronic discogenic low back pain, with success rates reported in up to 70% of cases. The mechanism of action by which IDET exerts its effect is poorly understood. Proposed mechanisms include the contraction of collagen and the coagulation of annular nociceptors. An ovine model was used firstly to induce a posterolateral annular lesion, secondly to assess the innervation of such a lesion, and thirdly to assess the effect of IDET on this innervation. MATERIALS AND METHODS: Posterolateral annular incisions were made in 40 lumbar discs of 20 sheep. Twelve weeks were allowed for each annular lesion to mature. IDET was then performed in the disc with the posterolateral annular tear and in another control level. IDET was performed using a modified intradiscal catheter (Spine-CATH; Oratec Interventions, Menlo Park, CA). Temperatures were recorded in the nucleus (TN) and the posterior annulus (TPA). The spines were harvested at predetermined intervals up to 18 months. Histologic sections of the discs were graded for disc morphology to assess degeneration and immunohistochemical staining to assess potential denervation. RESULTS: Vascular granulation tissue consistent with a healing response was observed in the posterior annular tear of all incised discs from 12 weeks. Protein gene product 9.5 (PGP 9.5) positive nerve fibers were clearly identified in the adjacent periannular tissue and the outer few lamellae of the posterior annulus. During the IDET procedure, the mean maximum TPA was 63.6C and the mean maximum TN was 67.C.At sacrifice, the number of nerve fibers identified in the posterior annular tear was the same for those specimens that had undergone IDET and those that had not. From 6 weeks after IDET, there was evidence of thermal necrosisin the inner annulus and adjacent nucleus but sparing the periphery of the disc. CONCLUSIONS: Vascular granulation tissue and posterior annular neo-innervation was observed in the experimentally induced posterolateral annular lesion. IDET delivered at 90C in the sheep consistently heated the posterior annulus and the nucleus to a temperature normally associated with coagulation of nociceptors and collagen contraction. IDET did not denervate the posterior annular lesion. Thermal necrosis was observed within the inner annulus and adjacent nucleus from 6 weeks after IDET. The reported benefits from IDET appear to be related to factors other than denervation and repair.

The biologic response to particles from a lumbar disc prosthesis.

STUDY DESIGN: Particles of a proprietary polyolefin rubber compound used in a lumbar disc prosthesis were generated in vitro and tested for biocompatibility in two animal models. OBJECTIVE To characterize any tissue response to polyolefin rubber particles. SUMMARY OF BACKGROUND DATA: Intervertebral disc prostheses are emerging as alternatives to fusion techniques for the treatment of symptomatic disc degeneration. The biocompatibility of all novel components used in the construction of these devices must be verified before they can be considered for general use. METHODS: Laboratory-generated polyolefin rubber particles were either injected into dorsal subcutaneous air pouches of 30 rats or placed directly onto the lumbosacral dura and nerve roots of 9 sheep. Histologic sections of tissues from, and remote from, the site of implantation were examined for evidence of inflammation and wound-healing responses. RESULTS: Polyolefin rubber particle debris induced a tissue response that was consistent with a normal foreign body reaction to large particles. The response was not significantly greater than that seen with similar size particles of ultrahigh molecular weight polyethylene. There was no evidence of particle migration from the site of implantation, and there was no evidence of local or systemic toxic effects. CONCLUSION: Polyolefin rubber particles induce only localized tissue response that is consistent with a normal foreign body reaction to large nontoxic particles.