Human Cell-Derived Matrix Composite Hydrogels with Diverse Composition for Use in Vasculature-on-chip Models.

Microphysiological and organ-on-chip platforms seek to address critical gaps in human disease models and drug development that underlie poor rates of clinical success for novel interventions. While the fabrication technology and model cells used to synthesize organs-on-chip have advanced considerably, most platforms rely on animal-derived or synthetic extracellular matrix as a cell substrate, limiting mimicry of human physiology and precluding use in modeling diseases in which matrix dynamics play a role in pathogenesis. Here, the development of human cell-derived matrix (hCDM) composite hydrogels for use in 3D microphysiologic models of the vasculature is reported. hCDM composite hydrogels are derived from human donor fibroblasts and maintain a complex milieu of basement membrane, proteoglycans, and nonfibrillar matrix components. The use of hCDM composite hydrogels as 2D and 3D cell culture substrates is demonstrated, and hCDM composite hydrogels are patterned to form engineered human microvessels. Interestingly, hCDM composite hydrogels are enriched in proteins associated with vascular morphogenesis as determined by mass spectrometry, and functional analysis demonstrates proangiogenic signatures in human endothelial cells cultured in these hydrogels. In conclusion, this study suggests that human donor-derived hCDM composite hydrogels could address technical gaps in human organs-on-chip development and serve as substrates to promote vascularization.

Donor-Derived Engineered Microvessels for Cardiovascular Risk Stratification of Patients with Kidney Failure.

Cardiovascular disease is the cause of death in ≈50% of hemodialysis patients. Accumulation of uremic solutes in systemic circulation is thought to be a key driver of the endothelial dysfunction that underlies elevated cardiovascular events. A challenge in understanding the mechanisms relating chronic kidney disease to cardiovascular disease is the lack of in vitro models that allow screening of the effects of the uremic environment on the endothelium. Here, a method is described for microfabrication of human blood vessels from donor cells and perfused with donor serum. The resulting donor-derived microvessels are used to quantify vascular permeability, a hallmark of endothelial dysfunction, in response to serum spiked with pathophysiological levels of indoxyl sulfate, and in response to serum from patients with chronic kidney disease and from uremic pigs. The uremic environment has pronounced effects on microvascular integrity as demonstrated by irregular cell-cell junctions and increased permeability in comparison to cell culture media and healthy serum. Moreover, the engineered microvessels demonstrate an increase in sensitivity compared to traditional 2D assays. Thus, the devices and the methods presented here have the potential to be utilized to risk stratify and to direct personalized treatments for patients with chronic kidney disease.

Identification and Optimization of RNA-Splicing Modulators as Huntingtin Protein-Lowering Agents for the Treatment of Huntington's Disease.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.

Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics.

Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that vEDS patient-derived fibroblasts harboring COL3A1 mutations synthesize ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling. STATEMENT OF SIGNIFICANCE: The role of collagen III ECM mechanics remains unclear, despite reported roles in diseases including fibrosis and cancer. Here, we generate fibrous, collagen-rich ECM from primary donor cells from patients with vascular Ehlers-Danlos syndrome (vEDS), a disease caused by mutations in the gene that encodes collagen III. We observe that ECM grown from vEDS patients is characterized by unique mechanical signatures, including altered viscoelastic properties. By quantifying the structural, biochemical, and mechanical properties of patient-derived ECM, we identify potential drug targets for vEDS, while defining a role for collagen III in ECM mechanics more broadly. Furthermore, the structure/function relationships of collagen III in ECM assembly and mechanics will inform the design of substrates for tissue engineering and regenerative medicine.

Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formation.

Somatic activating mutations of PIK3CA are associated with development of vascular malformations (VMs). Here, we describe a microfluidic model of PIK3CA-driven VMs consisting of human umbilical vein endothelial cells expressing PIK3CA activating mutations embedded in three-dimensional hydrogels. We observed enlarged, irregular vessel phenotypes and the formation of cyst-like structures consistent with clinical signatures and not previously observed in cell culture models. Pathologic morphologies occurred concomitant with up-regulation of Rac1/p21-activated kinase (PAK), mitogen-activated protein kinase cascades (MEK/ERK), and mammalian target of rapamycin (mTORC1/2) signaling networks. We observed differential effects between alpelisib, a PIK3CA inhibitor, and rapamycin, an mTORC1 inhibitor, in mitigating matrix degradation and network topology. While both were effective in preventing vessel enlargement, rapamycin failed to reduce MEK/ERK and mTORC2 activity and resulted in hyperbranching, while inhibiting PAK, MEK1/2, and mTORC1/2 mitigates abnormal growth and vascular dilation. Collectively, these findings demonstrate an in vitro platform for VMs and establish a role of dysregulated Rac1/PAK and mTORC1/2 signaling in PIK3CA-driven VMs.

Developing HDAC4-Selective Protein Degraders To Investigate the Role of HDAC4 in Huntington's Disease Pathology.

Huntington's disease (HD) is a lethal autosomal dominant neurodegenerative disorder resulting from a CAG repeat expansion in the huntingtin (HTT) gene. The product of translation of this gene is a highly aggregation-prone protein containing a polyglutamine tract >35 repeats (mHTT) that has been shown to colocalize with histone deacetylase 4 (HDAC4) in cytoplasmic inclusions in HD mouse models. Genetic reduction of HDAC4 in an HD mouse model resulted in delayed aggregation of mHTT, along with amelioration of neurological phenotypes and extended lifespan. To further investigate the role of HDAC4 in cellular models of HD, we have developed bifunctional degraders of the protein and report the first potent and selective degraders of HDAC4 that show an effect in multiple cell lines, including HD mouse model-derived cortical neurons. These degraders act via the ubiquitin-proteasomal pathway and selectively degrade HDAC4 over other class IIa HDAC isoforms (HDAC5, HDAC7, and HDAC9).

Microfluidic and Organ-on-a-Chip Approaches to Investigate Cellular and Microenvironmental Contributions to Cardiovascular Function and Pathology.

Over the past decade, advances in microfabrication and biomaterials have facilitated the development of microfluidic tissue and organ models to address challenges with conventional animal and cell culture systems. These systems have largely been developed for human disease modeling and preclinical drug development and have been increasingly used to understand cellular and molecular mechanisms, particularly in the cardiovascular system where the characteristic mechanics and architecture are difficult to recapitulate in traditional systems. Here, we review recent microfluidic approaches to model the cardiovascular system and novel insights provided by these systems. Key features of microfluidic approaches include the ability to pattern cells and extracellular matrix (ECM) at cellular length scales and the ability to use patient-derived cells. We focus the review on approaches that have leveraged these features to explore the relationship between genetic mutations and the microenvironment in cardiovascular disease progression. Additionally, we discuss limitations and benefits of the various approaches, and conclude by considering the role further advances in microfabrication technology and biochemistry techniques play in establishing microfluidic cardiovascular disease models as central tools for understanding biological mechanisms and for developing interventional strategies.

Ultrasensitive quantitative measurement of huntingtin phosphorylation at residue S13.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of a CAG triplet repeat (encoding for a polyglutamine tract) within the first exon of the huntingtin gene. Expression of the mutant huntingtin (mHTT) protein can result in the production of N-terminal fragments with a robust propensity to form oligomers and aggregates, which may be causally associated with HD pathology. Several lines of evidence indicate that N17 phosphorylation or pseudophosphorylation at any of the residues T3, S13 or S16, alone or in combination, modulates mHTT aggregation, subcellular localization and toxicity. Consequently, increasing N17 phosphorylation has been proposed as a potential therapeutic approach. However, developing genetic/pharmacological tools to quantify these phosphorylation events is necessary in order to subsequently develop tool modulators, which is difficult given the transient and incompletely penetrant nature of such post-translational modifications. Here we describe the first ultrasensitive sandwich immunoassay that quantifies HTT phosphorylated at residue S13 and demonstrate its utility for specific analyte detection in preclinical models of HD.

Structure-guided Discovery of Dual-recognition Chemibodies.

Small molecules and antibodies each have advantages and limitations as therapeutics. Here, we present for the first time to our knowledge, the structure-guided design of "chemibodies" as small molecule-antibody hybrids that offer dual recognition of a single target by both a small molecule and an antibody, using DPP-IV enzyme as a proof of concept study. Biochemical characterization demonstrates that the chemibodies present superior DPP-IV inhibition compared to either small molecule or antibody component alone. We validated our design by successfully solving a co-crystal structure of a chemibody in complex with DPP-IV, confirming specific binding of the small molecule portion at the interior catalytic site and the Fab portion at the protein surface. The discovery of chemibodies presents considerable potential for novel therapeutics that harness the power of both small molecule and antibody modalities to achieve superior specificity, potency, and pharmacokinetic properties.

What service users with psychotic disorders want in a mental health crisis or relapse: thematic analysis of joint crisis plans.

PURPOSE: Recent legislation and guidance in England emphasises the importance of service user choice in care planning. However, it is not obvious how best to facilitate choices in care planning, and some clinicians are concerned that service users may make 'unwise' decisions. This study aimed to examine mental health service users' preferences and priorities in the event of a future mental health crisis or relapse. METHOD: Thematic analysis of 221 joint crisis plans (JCP) developed by service users and their clinical team as part of the CRIMSON randomised controlled trial. Participants had a diagnosis of a psychotic disorder, at least one psychiatric admission in the past 2 years, contact with a community mental health team, and complex care needs. RESULTS: Two major categories of preferences were identified: first the manner in which crisis care would be delivered; and second, specific treatment interventions. Most service users requested full involvement in decisions about their care, clear and consistent treatment plans, access to familiar clinicians who knew them well, and to be treated with respect and compassion. Some service users requested hospitalisation, but the majority preferred alternatives. The most frequently preferred intervention was care by a home treatment team. Just under half made a treatment refusal, the majority being for specific medications, alternatives were offered. CONCLUSIONS: Joint crisis planning resulted in service users making choices that were clinically reasonable. The technique employed by JCPs appeared to empower service users by engaging them in a productive dialogue with their clinicians.

Nurses' role in care home rehabilitation.

BACKGROUND: Increasing numbers of older people are living in long-term residential care. Due to their often complex physical and medical conditions, there is a need for nursing rehabilitation to improve or maintain their functional status. AIM: The aim of this study was to explore nurses' perceptions of their role in the rehabilitation of older people in long-term care residences. METHODS: Ten nurses working in two long-term care residences participated in this qualitative descriptive design study. Data was collected through semistructured interviews and its thematic content was then analysed. FINDINGS: The nurses considered their roles in caring and promoting enablement and independence to be important, but reported that time constraints and the physical and cognitive limitations in older people made the successful implementation of rehabilitation more difficult. They were positive about multidisciplinary collaboration and reported that multiple skills were needed in rehabilitation nursing, expressing a need for further education and training. They were aware of the need for a sense of homeliness to exist in long-term residences and welcomed working with families to achieve this. CONCLUSION: Rehabilitation nursing of older people in long-term residences focuses on caring and enablement. Participants acknowledged challenges relating to age and infirmities but considered multidisciplinary collaboration, homeliness and families to be important to successful rehabilitation nursing.

Disubstituted 1-aryl-4-aminopiperidine library synthesis using computational drug design and high-throughput batch and flow technologies.

A platform that incorporates computational library design, parallel solution-phase synthesis, continuous flow hydrogenation, and automated high throughput purification and reformatting technologies was applied to the production of a 120-member library of 1-aryl-4-aminopiperidine analogues for drug discovery screening. The application described herein demonstrates the advantages of computational library design coupled with a flexible, modular approach to library synthesis. The enabling technologies described can be readily adopted by the traditional medicinal chemist without extensive training and lengthy process development times.

N-substituted azaindoles as potent inhibitors of Cdc7 kinase.

Cdc7 kinase is responsible for the initiation and regulation of DNA replication and has been proposed as a target for cancer therapy. We have identified a class of Cdc7 inhibitors based on a substituted indole core. Synthesis of focused indole and azaindole analogs yielded potent and selective 5-azaindole Cdc7 inhibitors with improved intrinsic metabolic stability (ie 36). In parallel, quantum mechanical conformational analysis helped to rationalize SAR observations, led to a proposal of the preferred binding conformation in the absence of co-crystallography data, and allowed the design of 7-azaindole 37 as a second lead in this series.

2-Phenylamino-6-cyano-1H-benzimidazole-based isoform selective casein kinase 1 gamma (CK1γ) inhibitors.

Screening of the Amgen compound library led to the identification of 2-phenylamino-6-cyano-1H-benzimidazole 1a as a potent CK1 gamma inhibitor with excellent kinase selectivity and unprecedented CK1 isoform selectivity. Further structure-based optimization of this series resulted in the discovery of 1h which possessed good enzymatic and cellular potency, excellent CK1 isoform and kinase selectivity, and acceptable pharmacokinetic properties.

Rapid development of piperidine carboxamides as potent and selective anaplastic lymphoma kinase inhibitors.

Piperidine carboxamide 1 was identified as a novel inhibitor of anaplastic lymphoma kinase (ALK enzyme assay IC(50) = 0.174 µM) during high throughput screening, with selectivity over the related kinase insulin-like growth factor-1 (IGF1R). The X-ray cocrystal structure of 1 with the ALK kinase domain revealed an unusual DFG-shifted conformation, allowing access to an extended hydrophobic pocket. Structure-activity relationship (SAR) studies were focused on the rapid parallel optimization of both the right- and left-hand side of the molecule, culminating in molecules with improved potency and selectivity over IGF1R.

Discovery of potent and highly selective thienopyridine Janus kinase 2 inhibitors.

Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure-activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.

Evaluation of a commercial packed bed flow hydrogenator for reaction screening, optimization, and synthesis.

The performance of the ThalesNano H-Cube(®), a commercial packed bed flow hydrogenator, was evaluated in the context of small scale reaction screening and optimization. A model reaction, the reduction of styrene to ethylbenzene through a 10% Pd/C catalyst bed, was used to examine performance at various pressure settings, over sequential runs, and with commercial catalyst cartridges. In addition, the consistency of the hydrogen flow was indirectly measured by in-line UV spectroscopy. Finally, system contamination due to catalyst leaching, and the resolution of this issue, is described. The impact of these factors on the run-to-run reproducibility of the H-Cube(®) reactor for screening and reaction optimization is discussed.

Discovery of pyridazinopyridinones as potent and selective p38 mitogen-activated protein kinase inhibitors.

The p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of proinflammatory cytokines, making it an attractive target for the treatment of various inflammatory diseases. A series of pyridazinopyridinone compounds were designed as novel p38 kinase inhibitors. A structure-activity investigation identified several compounds possessing excellent potency in both enzyme and human whole blood assays. Among them, compound 31 exhibited good pharmacokinetic properties and showed excellent selectivity against other related kinases. In addition, 31 demonstrated efficacy in a collagen-induced arthritis disease model in rats.

Structure-activity relationship (SAR) investigations of tetrahydroquinolines as BKCa agonists.

The membrane bound large-conductance, calcium-activated potassium channel (BKCa) is an important regulator of neuronal activity. Here we describe the identification and structure-activity relationship of a novel class of potent tetrahydroquinoline BKCa agonists. An example from this class of BKCa agonists was shown to depress the spontaneous neuronal discharges in an electrophysiological model of migraine.