Cryptic enzymatic assembly of peptides armed with ß-lactone warheads.

Nature has evolved biosynthetic pathways to molecules possessing reactive warheads that inspired the development of many therapeutic agents, including penicillin antibiotics. Peptides armed with electrophilic warheads have proven to be particularly effective covalent inhibitors, providing essential antimicrobial, antiviral and anticancer agents. Here we provide a full characterization of the pathways that nature deploys to assemble peptides with ß-lactone warheads, which are potent proteasome inhibitors with promising anticancer activity. Warhead assembly involves a three-step cryptic methylation sequence, which is likely required to reduce unfavorable electrostatic interactions during the sterically demanding ß-lactonization. Amide-bond synthetase and adenosine triphosphate (ATP)-grasp enzymes couple amino acids to the ß-lactone warhead, generating the bioactive peptide products. After reconstituting the entire pathway to ß-lactone peptides in vitro, we go on to deliver a diverse range of analogs through enzymatic cascade reactions. Our approach is more efficient and cleaner than the synthetic methods currently used to produce clinically important warhead-containing peptides.

Throughput-scalable manufacturing of SARS-CoV-2 mRNA lipid nanoparticle vaccines.

Lipid nanoparticles (LNPs) are a potent delivery technology that have made it possible for the recent clinical breakthroughs in mRNA therapeutics and vaccines. A key challenge to the broader implementation of mRNA therapeutics and vaccines is the development of technology to produce precisely defined LNP formulations, with throughput that can scale from discovery to commercial manufacturing and meet the stringent manufacturing standards of the pharmaceutical industry. To address these challenges, we have developed a microfluidic chip that incorporates 1×, 10×, or 256× LNP-generating units that achieve scalable production rates of up to 17 L/h of precisely defined LNPs. Using these chips, we demonstrate that LNP physical properties and potency in vivo are unchanged as throughput is scaled. Our chips are fabricated out of silicon and glass substrates, which have excellent solvent compatibility, compatibility with pharmaceutical manufacturing, and can be fully reset and reused. SARS-CoV-2 mRNA-LNP vaccines formulated by our chips triggered potent antibody responses in a preclinical study. These results demonstrate the feasibility of directly translating microfluidic-generated LNPs to the scale necessary for commercial production.

In vivo bone marrow microenvironment siRNA delivery using lipid-polymer nanoparticles for multiple myeloma therapy.

Multiple myeloma (MM), a hematologic malignancy that preferentially colonizes the bone marrow, remains incurable with a survival rate of 3 to 6 mo for those with advanced disease despite great efforts to develop effective therapies. Thus, there is an urgent clinical need for innovative and more effective MM therapeutics. Insights suggest that endothelial cells within the bone marrow microenvironment play a critical role. Specifically, cyclophilin A (CyPA), a homing factor secreted by bone marrow endothelial cells (BMECs), is critical to MM homing, progression, survival, and chemotherapeutic resistance. Thus, inhibition of CyPA provides a potential strategy to simultaneously inhibit MM progression and sensitize MM to chemotherapeutics, improving therapeutic response. However, inhibiting factors from the bone marrow endothelium remains challenging due to delivery barriers. Here, we utilize both RNA interference (RNAi) and lipid-polymer nanoparticles to engineer a potential MM therapy, which targets CyPA within blood vessels of the bone marrow. We used combinatorial chemistry and high-throughput in vivo screening methods to engineer a nanoparticle platform for small interfering RNA (siRNA) delivery to bone marrow endothelium. We demonstrate that our strategy inhibits CyPA in BMECs, preventing MM cell extravasation in vitro. Finally, we show that siRNA-based silencing of CyPA in a murine xenograft model of MM, either alone or in combination with the Food and Drug Administration (FDA)-approved MM therapeutic bortezomib, reduces tumor burden and extends survival. This nanoparticle platform may provide a broadly enabling technology to deliver nucleic acid therapeutics to other malignancies that home to bone marrow.

Updated variant curation expert panel criteria and pathogenicity classifications for 251 variants for RYR1-related malignant hyperthermia susceptibility.

The ClinGen malignant hyperthermia susceptibility (MHS) variant curation expert panel specified the American College of Medical Genetics and Genomics/Association of Molecular Pathologists (ACMG/AMP) criteria for RYR1-related MHS and a pilot analysis of 84 variants was published. We have now classified an additional 251 variants for RYR1-related MHS according to current ClinGen standards and updated the criteria where necessary. Criterion PS4 was modified such that individuals with multiple RYR1 variants classified as pathogenic (P), likely pathogenic (LP), or variant of uncertain significance (VUS) were not considered as providing evidence for pathogenicity. Criteria PS1 and PM5 were revised to consider LP variants at the same amino-acid residue as providing evidence for pathogenicity at reduced strength. Finally, PM1 was revised such that if PS1 or PM5 are used PM1, if applicable, should be downgraded to supporting. Of the 251 RYR1 variants, 42 were classified as P/LP, 16 as B/LB, and 193 as VUS. The primary driver of 175 VUS classifications was insufficient evidence supporting pathogenicity, rather than evidence against pathogenicity. Functional data supporting PS3/BS3 was identified for only 13 variants. Based on the posterior probabilities of pathogenicity and variant frequencies in gnomAD, we estimated the prevalence of individuals with RYR1-related MHS pathogenic variants to be between 1/300 and 1/1075, considerably higher than current estimates. We have updated ACMG/AMP criteria for RYR1-related MHS and classified 251 variants. We suggest that prioritization of functional studies is needed to resolve the large number of VUS classifications and allow for appropriate risk assessment. RYR1-related MHS pathogenic variants are likely to be more common than currently appreciated.

Rational Design of Bisphosphonate Lipid-like Materials for mRNA Delivery to the Bone Microenvironment.

The development of lipid nanoparticle (LNP) formulations for targeting the bone microenvironment holds significant potential for nucleic acid therapeutic applications including bone regeneration, cancer, and hematopoietic stem cell therapies. However, therapeutic delivery to bone remains a significant challenge due to several biological barriers, such as low blood flow in bone, blood-bone marrow barriers, and low affinity between drugs and bone minerals, which leads to unfavorable therapeutic dosages in the bone microenvironment. Here, we construct a series of bisphosphonate (BP) lipid-like materials possessing a high affinity for bone minerals, as a means to overcome biological barriers to deliver mRNA therapeutics efficiently to the bone microenvironment in vivo. Following in vitro screening of BP lipid-like materials formulated into LNPs, we identified a lead BP-LNP formulation, 490BP-C14, with enhanced mRNA expression and localization in the bone microenvironment of mice in vivo compared to 490-C14 LNPs in the absence of BPs. Moreover, BP-LNPs enhanced mRNA delivery and secretion of therapeutic bone morphogenetic protein-2 from the bone microenvironment upon intravenous administration. These results demonstrate the potential of BP-LNPs for delivery to the bone microenvironment, which could potentially be utilized for a range of mRNA therapeutic applications including regenerative medicine, protein replacement, and gene editing therapies.

Scalable mRNA and siRNA Lipid Nanoparticle Production Using a Parallelized Microfluidic Device.

A major challenge to advance lipid nanoparticles (LNPs) for RNA therapeutics is the development of formulations that can be produced reliably across the various scales of drug development. Microfluidics can generate LNPs with precisely defined properties, but have been limited by challenges in scaling throughput. To address this challenge, we present a scalable, parallelized microfluidic device (PMD) that incorporates an array of 128 mixing channels that operate simultaneously. The PMD achieves a >100× production rate compared to single microfluidic channels, without sacrificing desirable LNP physical properties and potency typical of microfluidic-generated LNPs. In mice, we show superior delivery of LNPs encapsulating either Factor VII siRNA or luciferase-encoding mRNA generated using a PMD compared to conventional mixing, with a 4-fold increase in hepatic gene silencing and 5-fold increase in luciferase expression, respectively. These results suggest that this PMD can generate scalable and reproducible LNP formulations needed for emerging clinical applications, including RNA therapeutics and vaccines.

Development of Halogenase Enzymes for Use in Synthesis.

Nature has evolved halogenase enzymes to regioselectively halogenate a diverse range of biosynthetic precursors, with the halogens introduced often having a profound effect on the biological activity of the resulting natural products. Synthetic endeavors to create non-natural bioactive small molecules for pharmaceutical and agrochemical applications have also arrived at a similar conclusion: halogens can dramatically improve the properties of organic molecules for selective modulation of biological targets in vivo. Consequently, a high proportion of pharmaceuticals and agrochemicals on the market today possess halogens. Halogenated organic compounds are also common intermediates in synthesis and are particularly valuable in metal-catalyzed cross-coupling reactions. Despite the potential utility of organohalogens, traditional nonenzymatic halogenation chemistry utilizes deleterious reagents and often lacks regiocontrol. Reliable, facile, and cleaner methods for the regioselective halogenation of organic compounds are therefore essential in the development of economical and environmentally friendly industrial processes. A potential avenue toward such methods is the use of halogenase enzymes, responsible for the biosynthesis of halogenated natural products, as biocatalysts. This Review will discuss advances in developing halogenases for biocatalysis, potential untapped sources of such biocatalysts and how further optimization of these enzymes is required to achieve the goal of industrial scale biohalogenation.

Engineering Orthogonal Methyltransferases to Create Alternative Bioalkylation Pathways.

S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) catalyse the methylation of a vast array of small metabolites and biomacromolecules. Recently, rare carboxymethylation pathways have been discovered, including carboxymethyltransferase enzymes that utilise a carboxy-SAM (cxSAM) cofactor generated from SAM by a cxSAM synthase (CmoA). We show how MT enzymes can utilise cxSAM to catalyse carboxymethylation of tetrahydroisoquinoline (THIQ) and catechol substrates. Site-directed mutagenesis was used to create orthogonal MTs possessing improved catalytic activity and selectivity for cxSAM, with subsequent coupling to CmoA resulting in more efficient and selective carboxymethylation. An enzymatic approach was also developed to generate a previously undescribed co-factor, carboxy-S-adenosyl-l-ethionine (cxSAE), thereby enabling the stereoselective transfer of a chiral 1-carboxyethyl group to the substrate.

Randomised controlled trial of a repeated consultation support intervention for patients with colorectal cancer.

OBJECTIVE: UK healthcare policy for improving cancer outcomes supports participation of patients in care decisions with clinicians. Consultation Planning, Recording and Summarising (CPRS) has shown evidence of increasing patient decision self-efficacy, reducing uncertainty, and regret of decisions. This is the first trial of CPRS within the colorectal cancer population and delivered over serial medical consultations. METHODS: This randomised controlled trial compared usual care to the addition of CPRS over consecutive oncology consultations with newly diagnosed colorectal cancer (CRC) patients in Edinburgh, Scotland. The study primarily evaluated patients' perception of their decision self-efficacy, preparation for decision-making, decisional conflict, and decisional regret, with secondary measures of anxiety and depression. RESULTS: Compared with usual care, overall, the intervention group reported significantly higher decision self-efficacy (P = 0.001) and preparation for decision-making (P < 0.001) and significantly lower decisional conflict (P = 0.018) and regret (P = 0.039). The repeated intervention patients felt significantly better prepared for each consultation (P < 0.05); reported higher DSE before (P = 0.05) and after (P = 0.031) consultation one, and after consultation three (P = 0.004); and reported lower decisional conflict after consultation two (P = 0.007). Analyses comparing groups over time on decisional variables and anxiety and depression were underpowered because of attrition. CONCLUSIONS: Among colorectal cancer patients, CPRS was associated with decisional benefits before and after each consultation and 3 months after the last consultation. It appears that CPRS patients began their first medical consultation on a better trajectory but did not widen the gap over time. More research is needed on the benefits of CPRS being administered once or consecutively.

The role of the Hes1 crosstalk hub in Notch-Wnt interactions of the intestinal crypt.

The Notch pathway plays a vital role in determining whether cells in the intestinal epithelium adopt a secretory or an absorptive phenotype. Cell fate specification is coordinated via Notch's interaction with the canonical Wnt pathway. Here, we propose a new mathematical model of the Notch and Wnt pathways, in which the Hes1 promoter acts as a hub for pathway crosstalk. Computational simulations of the model can assist in understanding how healthy intestinal tissue is maintained, and predict the likely consequences of biochemical knockouts upon cell fate selection processes. Chemical reaction network theory (CRNT) is a powerful, generalised framework which assesses the capacity of our model for monostability or multistability, by analysing properties of the underlying network structure without recourse to specific parameter values or functional forms for reaction rates. CRNT highlights the role of ß-catenin in stabilising the Notch pathway and damping oscillations, demonstrating that Wnt-mediated actions on the Hes1 promoter can induce dynamic transitions in the Notch system, from multistability to monostability. Time-dependent model simulations of cell pairs reveal the stabilising influence of Wnt upon the Notch pathway, in which ß-catenin- and Dsh-mediated action on the Hes1 promoter are key in shaping the subcellular dynamics. Where Notch-mediated transcription of Hes1 dominates, there is Notch oscillation and maintenance of fate flexibility; Wnt-mediated transcription of Hes1 favours bistability akin to cell fate selection. Cells could therefore regulate the proportion of Wnt- and Notch-mediated control of the Hes1 promoter to coordinate the timing of cell fate selection as they migrate through the intestinal epithelium and are subject to reduced Wnt stimuli. Furthermore, mutant cells characterised by hyperstimulation of the Wnt pathway may, through coupling with Notch, invert cell fate in neighbouring healthy cells, enabling an aberrant cell to maintain its neighbours in mitotically active states.

Pediatric penile porokeratosis: A case report.

We present what we believe to be the second case of pediatric penile porokeratosis and the youngest case reported. A 6-year-old boy presented with a pruritic, verrucous growth at the urethral meatus that recurred after two meatotomies. The diagnosis of porokeratosis was confirmed by biopsy. Porokeratosis should be added to the differential diagnosis of chronic hyperkeratotic penile lesions in children.

Structure and Biocatalytic Scope of Coclaurine N-Methyltransferase.

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse family of plant secondary metabolites, which have been exploited to develop analgesics, antibiotics, antitumor agents, and other therapeutic agents. Biosynthesis of BIAs proceeds via a common pathway from tyrosine to (S)-reticulene at which point the pathway diverges. Coclaurine N-methyltransferase (CNMT) is a key enzyme in the pathway to (S)-reticulene, installing the N-methyl substituent that is essential for the bioactivity of many BIAs. In this paper, we describe the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture. The specificity of CNMT was also explored with a range of natural and synthetic substrates as well as co-factor analogues. Knowledge from this study could be used to generate improved CNMT variants required to produce BIAs or synthetic derivatives.

RadH: A Versatile Halogenase for Integration into Synthetic Pathways.

Flavin-dependent halogenases are useful enzymes for providing halogenated molecules with improved biological activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site-directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high-throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin "non-natural" product in E. coli.

An Enzyme Cascade for Selective Modification of Tyrosine Residues in Structurally Diverse Peptides and Proteins.

Bioorthogonal chemistry enables a specific moiety in a complex biomolecule to be selectively modified in the presence of many reactive functional groups and other cellular entities. Such selectivity has become indispensable in biology, enabling biomolecules to be derivatized, conjugated, labeled, or immobilized for imaging, biochemical assays, or therapeutic applications. Methyltransferase enzymes (MTase) that accept analogues of the cofactor S-adenosyl methionine have been widely deployed for alkyl-diversification and bioorthogonal labeling. However, MTases typically possess tight substrate specificity. Here we introduce a more flexible methodology for selective derivatization of phenolic moieties in complex biomolecules. Our approach relies on the tandem enzymatic reaction of a fungal tyrosinase and the mammalian catechol-O-methyltransferase (COMT), which can effect the sequential hydroxylation of the phenolic group to give an intermediate catechol moiety that is subsequently O-alkylated. When used in this combination, the alkoxylation is highly selective for tyrosine residues in peptides and proteins, yet remarkably tolerant to changes in the peptide sequence. Tyrosinase-COMT are shown to provide highly versatile and regioselective modification of a diverse range of substrates including peptide antitumor agents, hormones, cyclic peptide antibiotics, and model proteins.

A Structure-Guided Switch in the Regioselectivity of a Tryptophan Halogenase.

Flavin-dependent halogenases are potentially useful biocatalysts for the regioselective halogenation of aromatic compounds. Haloaromatic compounds can be utilised in the synthesis and biosynthesis of pharmaceuticals and other valuable products. Here we report the first X-ray crystal structure of a tryptophan 6-halogenase (SttH), which enabled key residues that contribute to the regioselectivity in tryptophan halogenases to be identified. Structure-guided mutagenesis resulted in a triple mutant (L460F/P461E/P462T) that exhibited a complete switch in regioselectivity; with the substrate 3-indolepropionate 75 % 5-chlorination was observed with the mutant in comparison to 90 % 6-chlorination for the wild-type SttH. This is the first clear example of how regiocomplementary halogenases can be created from a single parent enzyme. The biocatalytic repertoire of SttH was also expanded to include a range of indolic and non-indolic substrates.

Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.

Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

Effects of Active-Site Modification and Quaternary Structure on the Regioselectivity of Catechol-O-Methyltransferase.

Catechol-O-methyltransferase (COMT), an important therapeutic target in the treatment of Parkinson's disease, is also being developed for biocatalytic processes, including vanillin production, although lack of regioselectivity has precluded its more widespread application. By using structural and mechanistic information, regiocomplementary COMT variants were engineered that deliver either meta- or para-methylated catechols. X-ray crystallography further revealed how the active-site residues and quaternary structure govern regioselectivity. Finally, analogues of AdoMet are accepted by the regiocomplementary COMT mutants and can be used to prepare alkylated catechols, including ethyl vanillin.

The influence of life stage on supportive care and information needs in cancer patients: does older age matter?

PURPOSE: The purpose of this study is to clarify the effect of older age, on supportive care needs, information satisfaction and service needs in the year following a cancer diagnosis. METHODS: Primary or recurrent prostate, breast, lung or colorectal cancer patients (n = 394) were prospectively surveyed 3 and 9 months post-diagnosis using the Support Care Needs Survey (SCNS-LF59) and Information Satisfaction (ISQ) and Service Needs (SNQ) questionnaires. Two age groups were compared: ≥65 years (senior) versus ≤64 years (junior). RESULTS: Few differences emerged between age groups (SCNS) with the exception of psychological (p < 0.001) and sexuality (p < 0.001) domains where these were greater in the younger patients 3 months post-diagnosis. Sexuality (p < 0.001) and patient care and support (p = 0.023) needs were predicted by age (continuous); younger patients had more needs at 3 months post-diagnosis. For information satisfaction, the older group preferred doctors to make decisions (3 months p < 0.001; 9 months p = 0.008) and preferred positive information (3 months p = 0.006). For the whole group fears about cancer spreading (51 %) and returning (45 %) predominate, alongside patients' concerns about worries of those closest to them (51 %) and uncertainty about their future (42 %) at 3 months. CONCLUSIONS: Older patients differ on information satisfaction showing a preference for doctors to make treatment decisions. For supportive care, there were few age differences; however, the SCNS sexuality and patient care and support domains indicate greater need in younger patients around the 3-month period following diagnosis. With a few exceptions, individual rather than age-specific needs determine supportive and informational care requirements.

A high-throughput assay for arylamine halogenation based on a peroxidase-mediated quinone-amine coupling with applications in the screening of enzymatic halogenations.

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more "green" halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high-throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho-benzoquinone-amine adduct, which is produced by the peroxidase-catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non-halogenated arylamine. This assay is sensitive, rapid and amenable to high-throughput screening platforms. We have also shown this assay to be easily coupled to a flavin-dependent halogenase, which currently lacks any convenient colorimetric assay, in a "one-pot" workflow.

High-throughput barcoding of nanoparticles identifies cationic, degradable lipid-like materials for mRNA delivery to the lungs in female preclinical models.

Lipid nanoparticles for delivering mRNA therapeutics hold immense promise for the treatment of a wide range of lung-associated diseases. However, the lack of effective methodologies capable of identifying the pulmonary delivery profile of chemically distinct lipid libraries poses a significant obstacle to the advancement of mRNA therapeutics. Here we report the implementation of a barcoded high-throughput screening system as a means to identify the lung-targeting efficacy of cationic, degradable lipid-like materials. We combinatorially synthesize 180 cationic, degradable lipids which are initially screened in vitro. We then use barcoding technology to quantify how the selected 96 distinct lipid nanoparticles deliver DNA barcodes in vivo. The top-performing nanoparticle formulation delivering Cas9-based genetic editors exhibits therapeutic potential for antiangiogenic cancer therapy within a lung tumor model in female mice. These data demonstrate that employing high-throughput barcoding technology as a screening tool for identifying nanoparticles with lung tropism holds potential for the development of next-generation extrahepatic delivery platforms.