A survey of cost, access and outcomes for cannabinoid-based medicinal product use by Australians with endometriosis.

People with endometriosis use cannabis to manage their symptoms. This study aimed to identify costs, modes of administration, product composition, and self-reported effectiveness for those accessing medicinal cannabis in Australia. There were 192 survey responses analysed. Most (63.5%) used a 'cannabis clinic' doctor, incurring an initial consultation cost of $100-$200+ (10.2% Medicare bulk-billed) and median cannabinoid medicine costs of $300AUD per month. Cost was a major barrier to access, necessitating reducing dosage (76.1%) and/or consuming illicit cannabis (42.9%), despite a prescription. Most (77%) medical consumers used two or more cannabis products, with delta-9-tetrahydrocannabinol predominant oil and flower products most frequently prescribed.

Harnessing the anti-cancer natural product nimbolide for targeted protein degradation.

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.

First Crystal Structure of a Nonstructural Hepatitis E Viral Protein Identifies a Putative Novel Zinc-Binding Protein.

Hepatitis E virus (HEV) is a 7.2-kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF1 to ORF3. All nonstructural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression, and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a nonclassical, potentially catalytic zinc-binding motif. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome. We present analysis for the first time of this identified nonstructural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology.IMPORTANCE Hepatitis E virus (HEV) is an emerging virus found predominately in developing countries; it causes an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the nonstructural proteins of HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. Here we present the first structural and biophysical characterization of an HEV nonstructural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease.

Comprehensive and High-Throughput Exploration of Chemical Space Using Broadband 19 F†NMR-Based Screening.

Fragment-based lead discovery has become a fundamental approach to identify ligands that efficiently interact with disease-relevant targets. Among the numerous screening techniques, fluorine-detected NMR has gained popularity owing to its high sensitivity, robustness, and ease of use. To effectively explore chemical space, a universal NMR experiment, a rationally designed fragment library, and a sample composition optimized for a maximal number of compounds and minimal measurement time are required. Here, we introduce a comprehensive method that enabled the efficient assembly of a high-quality and diverse library containing nearly 4000 fragments and screening for target-specific binders within days. At the core of the approach is a novel broadband relaxation-edited NMR experiment that covers the entire chemical shift range of drug-like 19 F motifs in a single measurement. Our approach facilitates the identification of diverse binders and the fast ligandability assessment of new targets.

High-Confidence Protein-Ligand Complex Modeling by NMR-Guided Docking Enables Early Hit Optimization.

Structure-based drug design is an integral part of modern day drug discovery and requires detailed structural characterization of protein-ligand interactions, which is most commonly performed by X-ray crystallography. However, the success rate of generating these costructures is often variable, in particular when working with dynamic proteins or weakly binding ligands. As a result, structural information is not routinely obtained in these scenarios, and ligand optimization is challenging or not pursued at all, representing a substantial limitation in chemical scaffolds and diversity. To overcome this impediment, we have developed a robust NMR restraint guided docking protocol to generate high-quality models of protein-ligand complexes. By combining the use of highly methyl-labeled protein with experimentally determined intermolecular distances, a comprehensive set of protein-ligand distances is generated which then drives the docking process and enables the determination of the correct ligand conformation in the bound state. For the first time, the utility and performance of such a method is fully demonstrated by employing the generated models for the successful, prospective optimization of crystallographically intractable fragment hits into more potent binders.

Facilitating unambiguous NMR assignments and enabling higher probe density through selective labeling of all methyl containing amino acids.

The deuteration of proteins and selective labeling of side chain methyl groups has greatly enhanced the molecular weight range of proteins and protein complexes which can be studied using solution NMR spectroscopy. Protocols for the selective labeling of all six methyl group containing amino acids individually are available, however to date, only a maximum of five amino acids have been labeled simultaneously. Here, we describe a new methodology for the simultaneous, selective labeling of all six methyl containing amino acids using the 115 kDa homohexameric enzyme CoaD from E. coli as a model system. The utility of the labeling protocol is demonstrated by efficiently and unambiguously assigning all methyl groups in the enzymatic active site using a single 4D (13)C-resolved HMQC-NOESY-HMQC experiment, in conjunction with a crystal structure. Furthermore, the six fold labeled protein was employed to characterize the interaction between the substrate analogue (R)-pantetheine and CoaD by chemical shift perturbations, demonstrating the benefit of the increased probe density.

APSY-NMR for protein backbone assignment in high-throughput structural biology.

A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h. The results of the automated procedure provided a basis for efficient interactive validation and extension to near-completion of the assignments by reference to the same 3D heteronuclear-resolved [(1)H,(1)H]-NOESY spectra that were subsequently used for the collection of conformational constraints. High-quality structures were obtained for all 30 proteins, using the J-UNIO protocol, which includes extensive automation of NMR structure determination.

Non-Uniform Sampling and J-UNIO Automation for Efficient Protein NMR Structure Determination.

High-resolution structure determination of small proteins in solution is one of the big assets of NMR spectroscopy in structural biology. Improvements in the efficiency of NMR structure determination by advances in NMR experiments and automation of data handling therefore attracts continued interest. Here, non-uniform sampling (NUS) of 3D heteronuclear-resolved [(1)H,(1)H]-NOESY data yielded two- to three-fold savings of instrument time for structure determinations of soluble proteins. With the 152-residue protein NP_372339.1 from Staphylococcus aureus and the 71-residue protein NP_346341.1 from Streptococcus pneumonia we show that high-quality structures can be obtained with NUS NMR data, which are equally well amenable to robust automated analysis as the corresponding uniformly sampled data.

The Place of Cannabinoids in the Treatment of Gynecological Pain.

Cannabis sativa (L), a plant with an extensive history of medicinal usage across numerous cultures, has received increased attention over recent years for its therapeutic potential for gynecological disorders such as endometriosis, chronic pelvic pain, and primary dysmenorrhea, due at least in part to shortcomings with current management options. Despite this growing interest, cannabis inhabits an unusual position in the modern medical pharmacopoeia, being a legal medicine, legal recreational drug, and an illicit drug, depending on jurisdiction. To date, the majority of studies investigating cannabis use have found that most people are using illicit cannabis, with numerous obstacles to medical cannabis adoption having been identified, including outdated drug-driving laws, workplace drug testing policies, the cost of quality-assured medical cannabis products, a lack of cannabis education for healthcare professionals, and significant and persistent stigma. Although currently lacking robust clinical trial data, a growing evidence base of retrospective data, cohort studies, and surveys does support potential use in gynecological pain conditions, with most evidence focusing on endometriosis. Cannabis consumers report substantial reductions in pelvic pain, as well as common comorbid symptoms such as gastrointestinal disturbances, mood disorders such as anxiety and depression, and poor sleep. Substitution effects were reported, with >50% reduction or cessation in opioid and/or non-opioid analgesics being the most common. However, a substantial minority report not disclosing cannabis consumption to their health professional. Therefore, while such deprescribing trends are potentially beneficial, the importance of medical supervision during this process is paramount given the possibility for withdrawal symptoms.

Cannabis, whether purchased illicitly, or obtained through legal means, is commonly used by those with chronic pelvic pain, especially people with endometriosis. People report several benefits from using cannabis, including being able to reduce their normal medications including opioid based painkillers, but often don't tell their health professional about this. This could lead to issues with withdrawal symptoms, so clinicians should be aware of the high prevalence of use of cannabis in this population.

"Should I Inhale?"-Perceptions, Barriers, and Drivers for Medicinal Cannabis Use amongst Australian Women with Primary Dysmenorrhoea: A Qualitative Study.

OBJECTIVE: This study sought to investigate the perceptions, barriers, and drivers associated with medicinal cannabis use among Australian women with primary dysmenorrhea. A qualitative study via virtual focus groups involving 26 women experiencing regular, moderate, or greater menstrual pain explored categories including cost, associated stigma, current drug driving laws, community and workplace ethics, and geographical isolation within the context of patient access under current Australian laws and regulations. RESULTS: A qualitative descriptive analysis identified that dissatisfaction with current management strategies such as over-the-counter analgesic usage was the key driver for wanting to use medicinal cannabis. A number of significant barriers to use were identified including patient access to medical prescribers, medical practitioner bias, current drug driving laws, geographic location, and cost. Community and cultural factors such as the history of cannabis as an illicit drug and the resulting stigma, even when prescribed by a medical doctor, still existed and was of concern to our participants. CONCLUSION: Whilst medicinal cannabis is legal in all states and territories within Australia, several barriers to access exist that require government regulatory attention to assist in increasing patient adoption, including possible subsidisation of cost. The high cost of legal, medicinal cannabis was a key factor in women's choice to use illicit cannabis. Overall, the concerns raised by our participants are consistent with the broader findings of a recent Australian Senate inquiry report into barriers to patient access to medicinal cannabis in Australia, suggesting many of the issues are systematic rather than disease-specific. Given the interest in use of medicinal cannabis amongst women with primary dysmenorrhea, clinical trials in this area are urgently needed.

Endometriosis and Cannabis Consumption During the COVID-19 Pandemic: An International Cross-Sectional Survey.

Introduction: Endometriosis affects 1 in 10 women worldwide, with most experiencing difficulties achieving adequate symptom control. These difficulties have been compounded by the onset of the COVID-19 pandemic due to worldwide shifts in health care resource allocation. As cannabis is a relatively common form of self-management in endometriosis, this study aims to explore the impact of the COVID-19 pandemic on cannabis consumption in those with endometriosis. Methods: An anonymous, cross-sectional online international survey was developed and promoted by endometriosis advocacy/support organizations worldwide. Respondents needed to have a diagnosis of endometriosis and be aged between 18 and 55. Results: A total of 1634 responses were received from 46 different countries. The average age of respondents was 30, with a mean diagnosis age of 25. Eight hundred forty-six respondents (51%) reported consuming cannabis in the past 3 months, with 55% of these reporting use for symptom management only. One in five respondents (20%) reported having consumed cannabis previously, the most common reason for discontinuation (65%) was access difficulties during COVID. Those who had legal access were more likely to consume cannabis than those without (p<0.0001) and were more likely to disclose usage to health care professionals (p<0.0001). The most common reasons for consuming cannabis during COVID was increased stress/anxiety (59%) and lack of access to normal medical care (48%). Pre-pandemic, cannabis was mostly consumed at least once a day (61%) and in inhaled forms (51.6%). Consumption increased for most people (57%) during the pandemic. During the pandemic just under a quarter (23%) of respondents changed their mode of consumption, with a reduction in inhaled forms (39.5%) and an increase in consumption of edibles (40%) or oil (25.2%). Conclusions: Cannabis consumption, especially for symptom relief, was relatively common among those with endometriosis, with some people starting their consumption of cannabis due to health care restrictions that occurred due to the COVID-19 pandemic. Difficulties accessing cannabis and unpleasant/unwanted side effects were the most common reasons for lack of current cannabis consumption in those who had previously consumed it. Cannabis consumption may form an important part of endometriosis management especially when access to routine medical care is restricted.

Selective Methyl Labeling of Proteins: Enabling Structural and Mechanistic Studies As Well As Drug Discovery Applications by Solution-State NMR.

Escherichia coli expression protocols for selective labeling of methyl groups in proteins have been essential in expanding the size range of targets that can be studied by biomolecular NMR. Based on the initial work achieving selective labeling of isoleucine, leucine, and valine residues, additional methods were developed over the past years which enabled the individual and/or simultaneous combinatorial labeling of all methyl containing amino acids. Together with the introduction of new methyl-optimized NMR experiments, this now allows the detailed characterization of protein-ligand interactions as well as mechanistic and dynamic processes of protein-protein complexes up to 1MDa in size. In this chapter, we provide a general introduction to selective labeling of proteins using E. coli-based expression systems, describe the considerations taken into account prior to the selective labeling of a protein, and include the protocols used to produce such proteins. An overview of applications using selectively labeled proteins with an emphasis on examples relevant to the drug discovery process is then presented.

Dynamic Local Polymorphisms in the Gbx1 Homeodomain Induced by DNA Binding.

The Gastrulation Brain Homeobox 1 (Gbx1) gene encodes the Gbx1 homeodomain that targets TAATTA motifs in double-stranded DNA (dsDNA). Residues Glu17 and Arg52 in Gbx1 form a salt bridge, which is preserved in crystal structures and molecular dynamics simulations of homologous homeodomain-DNA complexes. In contrast, our nuclear magnetic resonance (NMR) studies show that DNA binding to Gbx1 induces dynamic local polymorphisms, which include breaking of the Glu17-Arg52 salt bridge. To study this interaction, we produced a variant with Glu17Arg and Arg52Glu mutations, which exhibited the same fold as the wild-type protein, but a 2-fold reduction in affinity for dsDNA. Analysis of the NMR structures of the Gbx1 homeodomain in the free form, the Gbx1[E17R,R52E] variant, and a Gbx1 homeodomain-DNA complex showed that stabilizing interactions of the Arg52 side chain with the DNA backbone are facilitated by transient breakage of the Glu17-Arg52 salt bridge in the DNA-bound Gbx1.

Dlx5 Homeodomain:DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome.

The Dlx5 homeodomain is a transcription factor related to the Drosophila distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including split hand and foot malformation 1 in humans. Our characterization of a Dlx5 homeodomain:(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A Q186H mutation linked to split hand and foot malformation 1 likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and they likely destabilize the formation of functional complexes.

NMR in structural genomics to increase structural coverage of the protein universe: Delivered by Prof. Kurt Wüthrich on 7 July 2013 at the 38th FEBS Congress in St. Petersburg, Russia.

For more than a decade, the Joint Center for Structural Genomics (JCSG; www.jcsg.org) worked toward increased three-dimensional structure coverage of the protein universe. This coordinated quest was one of the main goals of the four high-throughput (HT) structure determination centers of the Protein Structure Initiative (PSI; www.nigms.nih.gov/Research/specificareas/PSI). To achieve the goals of the PSI, the JCSG made use of the complementarity of structure determination by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy to increase and diversify the range of targets entering the HT structure determination pipeline. The overall strategy, for both techniques, was to determine atomic resolution structures for representatives of large protein families, as defined by the Pfam database, which had no structural coverage and could make significant contributions to biological and biomedical research. Furthermore, the experimental structures could be leveraged by homology modeling to further expand the structural coverage of the protein universe and increase biological insights. Here, we describe what could be achieved by this structural genomics approach, using as an illustration the contributions from 20 NMR structure determinations out of a total of 98 JCSG NMR structures, which were selected because they are the first three-dimensional structure representations of the respective Pfam protein families. The information from this small sample is representative for the overall results from crystal and NMR structure determination in the JCSG. There are five new folds, which were classified as domains of unknown functions (DUF), three of the proteins could be functionally annotated based on three-dimensional structure similarity with previously characterized proteins, and 12 proteins showed only limited similarity with previous deposits in the Protein Data Bank (PDB) and were classified as DUFs.