Platform For Drug Development Research Articles

Systemic computational investigation to identify potential inhibitors against cancer by targeting P21-activated kinase 4 and D(CGATCG)

Cancer accounts for more than 10 million deaths in the year 2020. Development of drugs that specifically target cancer signaling pathways and proteins attain significant importance in the recent past. The p21-activated kinase 4 enzyme, which plays diverse functions in cancer and is reported in elevated expression makes this enzyme an attractive anti-cancer drug target. Similarly, cancer cells’ DNA could also serve as a good platform for anti-cancer drug development. Herein, a robust in silico framework is designed to virtually screen multiple drug libraries from diverse sources to identify potential binders of the mentioned cancer targets. The virtual screening process identified three compounds (BAS_01059603, ASN_10027856, and ASN_06916672) as best docked molecules with a binding energy score of ≤ −10 kcal/mol for p21-activated kinase 4 and ≤ −6 kcal/mol for D(CGATCG). In the docking analysis, the filtered compounds revealed stable binding to the same site to which controls bind in X-ray structures. The binding interactions of the compounds with receptors are dominated by van der Waals interactions. The average root mean square deviation (rmsd) value for p21-activated kinase 4 systems is noticed at ∼2 Å, while for D(CGATCG), the average rmsd is 2.7 Å. The MMGB/PBSA interpreted ASN_12674021 to show strong intermolecular binding energy compared to the other two systems and control in both receptors. Moreover, the entropy energy contribution is less than the mean binding energy. In short, the compounds are showing promising binding to the biomolecules and therefore must be evaluated for anti-cancer activity in experimental studies. Communicated by Ramaswamy H. Sarma

Humanized cerebral organoids-based ischemic stroke model for discovering of potential anti-stroke agents.

Establishing a stoke experimental model, which is better in line with the physiology and function of human brain, is the bottleneck for the development of effective anti-stroke drugs. A three-dimensional cerebral organoids (COs) from human pluripotent stem cells can mimic cell composition, cortical structure, brain neural connectivity and epigenetic genomics of in-vivo human brain, which provides a promising application in establishing humanized ischemic stroke model. COs have been used for modeling low oxygen condition-induced hypoxic injury, but there is no report on the changes of COs in response to in vitro oxygen-glucose deprivation (OGD)-induced damage of ischemic stroke as well as its application in testing anti-stroke drugs. In this study we compared the cell composition of COs at different culture time and explored the cell types, cell ratios and volume size of COs at 85 days (85 d-CO). The 85 d-CO with diameter more than 2 mm was chosen for establishing humanized ischemic stroke model of OGD. By determining the time-injury relationship of the model, we observed aggravated ischemic injury of COs with OGD exposure time, obtaining first-hand evidence for the damage degree of COs under different OGD condition. The sensitivity of the model to ischemic injury and related treatment was validated by the proven pan-Caspase inhibitor Z-VAD-FMK (20 μM) and Bcl-2 inhibitor navitoclax (0.5 μM). Neuroprotective agents edaravone, butylphthalide, P7C3-A20 and ZL006 (10 μM for each) exerted similar beneficial effects in this model. Taken together, this study establishes a humanized ischemic stroke model based on COs, and provides evidence as a new research platform for anti-stroke drug development.

Need for a Standardized Translational Drug Development Platform: Lessons Learned from the Repurposing of Drugs for COVID-19.

In the absence of drugs to treat or prevent COVID-19, drug repurposing can be a valuable strategy. Despite a substantial number of clinical trials, drug repurposing did not deliver on its promise. While success was observed with some repurposed drugs (e.g., remdesivir, dexamethasone, tocilizumab, baricitinib), others failed to show clinical efficacy. One reason is the lack of clear translational processes based on adequate preclinical profiling before clinical evaluation. Combined with limitations of existing in vitro and in vivo models, there is a need for a systematic approach to urgent antiviral drug development in the context of a global pandemic. We implemented a methodology to test repurposed and experimental drugs to generate robust preclinical evidence for further clinical development. This translational drug development platform comprises in vitro, ex vivo, and in vivo models of SARS-CoV-2, along with pharmacokinetic modeling and simulation approaches to evaluate exposure levels in plasma and target organs. Here, we provide examples of identified repurposed antiviral drugs tested within our multidisciplinary collaboration to highlight lessons learned in urgent antiviral drug development during the COVID-19 pandemic. Our data confirm the importance of assessing in vitro and in vivo potency in multiple assays to boost the translatability of pre-clinical data. The value of pharmacokinetic modeling and simulations for compound prioritization is also discussed. We advocate the need for a standardized translational drug development platform for mild-to-moderate COVID-19 to generate preclinical evidence in support of clinical trials. We propose clear prerequisites for progression of drug candidates for repurposing into clinical trials. Further research is needed to gain a deeper understanding of the scope and limitations of the presented translational drug development platform.

CRISPR/Cas9-mediated knock-out of AGXT1 in HepG2 cells as a new in vitro model of Primary Hyperoxaluria Type 1

AGXT1 encodes alanine:glyoxylate aminotransferase 1 (AGT1), a liver peroxisomal pyridoxal 5′-phosphate dependent-enzyme whose deficit causes Primary Hyperoxaluria Type 1 (PH1). PH1 is a rare disease characterized by overproduction of oxalate, first leading to kidney stones formation, and possibly evolving to life-threatening systemic oxalosis. A minority of PH1 patients is responsive to pyridoxine, while the option for non-responders is liver-kidney transplantation. Therefore, huge efforts are currently focused on the identification of new therapies, including the promising approaches based on RNA silencing recently approved. Many PH1-associated mutations are missense and lead to a variety of kinetic and/or folding defects on AGT1. In this context, the availability of a reliable in vitro disease model would be essential to better understand the phenotype of known or newly-identified pathogenic variants as well as to test novel drug candidates.Here, we took advantage of the CRISPR/Cas9 technology to specifically knock-out AGXT1 in HepG2 cells, a hepatoma-derived cell model exhibiting a conserved glyoxylate metabolism. AGXT1-KO HepG2 displayed null AGT1 expression and significantly reduced transaminase activity leading to an enhanced secretion of oxalate upon glycolate challenge. Known pathogenic AGT1 variants expressed in AGXT1-KO HepG2 cells showed alteration in both protein levels and specific transaminase activity, as well as a partial mitochondrial mistargeting when associated with a common polymorphism. Notably, pyridoxine treatment was able to partially rescue activity and localization of clinically-responsive variants. Overall, our data validate AGXT1-KO HepG2 cells as a novel cellular model to investigate PH1 pathophysiology, and as a platform for drug discovery and development.

The Amyloid Cascade Hypothesis 2.0: On the Possibility of Once-in-a-Lifetime-Only Treatment for Prevention of Alzheimer's Disease and for Its Potential Cure at Symptomatic Stages.

We posit that Alzheimer’s disease (AD) is driven by amyloid-β (Aβ) generated in the amyloid-β protein precursor (AβPP) independent pathway activated by AβPP-derived Aβ accumulated intraneuronally in a life-long process. This interpretation constitutes the Amyloid Cascade Hypothesis 2.0 (ACH2.0). It defines a tandem intraneuronal-Aβ (iAβ)-anchored cascade occurrence: intraneuronally-accumulated, AβPP-derived iAβ triggers relatively benign cascade that activates the AβPP-independent iAβ-generating pathway, which, in turn, initiates the second, devastating cascade that includes tau pathology and leads to neuronal loss. The entire output of the AβPP-independent iAβ-generating pathway is retained intraneuronally and perpetuates the pathway’s operation. This process constitutes a self-propagating, autonomous engine that drives AD and ultimately kills its host cells. Once activated, the AD Engine is self-reliant and independent from Aβ production in the AβPP proteolytic pathway; operation of the former renders the latter irrelevant to the progression of AD by relegating its iAβ contribution to insignificant, and brands its manipulation for therapeutic purposes, such as BACE (beta-site AβPP-cleaving enzyme) inhibition, as futile. In the proposed AD paradigm, the only valid direct therapeutic strategy is targeting the engine’s components, and the most effective feasible approach appears to be the activation of BACE1 and/or of its homolog BACE2, with the aim of exploiting their Aβ-cleaving activities. Such treatment would collapse the iAβ population and ‘reset’ its levels below those required for the operation of the AD Engine. Any sufficiently selective iAβ-depleting treatment would be equally effective. Remarkably, this approach opens the possibility of a short-duration, once-in-a-lifetime-only or very infrequent, preventive or curative therapy for AD; this therapy would be also effective for prevention and treatment of the ‘common’ pervasive aging-associated cognitive decline. The ACH2.0 clarifies all ACH-unresolved inconsistencies, explains the widespread ‘resilience to AD’ phenomenon, predicts occurrences of a category of AD-afflicted individuals without excessive Aβ plaque load and of a novel type of familial insusceptibility to AD; it also predicts the lifespan-dependent inevitability of AD in humans if untreated preventively. The article details strategy and methodology to generate an adequate AD model and validate the hypothesis; the proposed AD model may also serve as a research and drug development platform.

(Invited) Multi-Modality Input/Output Interfaces with Tissue and Cells Using Nanocarbons

My team’s efforts are focused on three major thrusts: (i) synthesis and in depth mechanistic investigation of the unique emergent optical, thermal, electrical and electrochemical properties of novel hybrid-nanomaterials and nanomaterials topologies composed on one-dimensional and two-dimensional building blocks, (ii) application and characterization of hybrid-nanomaterials interfaces with cells and tissue, and (iii) development and engineering of nanomaterials-based platforms to interrogate and affect the electrical properties of tissue and cells such as cardiomyocytes, and neurons, with a specific focus to understand electrical signal transduction in complex 3D cellular assemblies. Major questions we strive to answer are: Can we make materials and platforms that are tailored to allow seamless and stable integration with cells and tissue enabling sensing and actuation? Can hybrid-nanomaterials allow new insights about biological processes, e.g., tissue development and disease progression?In this talk I will describe our recent efforts in tackling these challenges. Our highly flexible bottom-up nanomaterials synthesis capabilities allow us to form unique hybrid-nanomaterials that can be used in various input/output bioelectrical interfaces, i.e., bioelectrical platforms for chemical and physical sensing and actuation. We developed a breakthrough bioelectrical interface, a 3D self-rolled biosensor arrays (3D-SR-BAs) of either active field effect transistors or passive microelectrodes to measure both cardiac and neural spheroids electrophysiology in 3D. This approach enables electrophysiological investigation and monitoring of the complex signal transduction in 3D cellular assemblies toward an organ-on-an-electronic-chip (organ-on-e-chip) platform for tissue maturation investigations and development of drugs for disease treatment. Utilizing graphene, a two-dimensional (2D) atomically thin carbon allotrope, we demonstrated a new technique to simultaneously record the intracellular electrical activity of multiple excitable cells with ultra-microelectrodes that can be as small as the size as an axon ca. 2µm in size. The outstanding electrochemical properties of our hybrid-nanomaterials allowed us to develop electrical sensors and actuators, e.g., sensors to explore the brain chemistry and sensors/actuators that are deployed in a large volumetric muscle loss animal model. Finally, using the unique optical properties of nanocarbons, e.g., graphene-based hybrid-nanomaterials and 2D nanocarbides (MXene), we have formed remote, non-genetic bioelectrical interfaces with excitable cells and modulated cellular and network activity with high precision and low needed energy. In summary, the exceptional synthetic control and flexible assembly of nanomaterials provide powerful tools for fundamental studies and applications in life science and potentially seamlessly merge nanomaterials-based platforms with cells, fusing nonliving and living systems together.

Cerebral organoids as an in vitro model to study autism spectrum disorders.

Autism spectrum disorders (ASDs) are a set of disorders characterised by social and communication deficits caused by numerous genetic lesions affecting brain development. Progress in ASD research has been hampered by the lack of appropriate models, as both 2D cell culture as well as animal models cannot fully recapitulate the developing human brain or the pathogenesis of ASD. Recently, cerebral organoids have been developed to provide a more accurate, 3D in vitro model of human brain development. Cerebral organoids have been shown to recapitulate the foetal brain gene expression profile, transcriptome, epigenome, as well as disease dynamics of both idiopathic and syndromic ASDs. They are thus an excellent tool to investigate development of foetal stage ASDs, as well as interventions that can reverse or rescue the altered phenotypes observed. In this review, we discuss the development of cerebral organoids, their recent applications in the study of both syndromic and idiopathic ASDs, their use as an ASD drug development platform, as well as limitations of their use in ASD research.

Therapeutic Delivery: Industry Update Covering January 2022

This industry update covers the period from January 1 through January 31, 2022, and is based on information sourced from company press releases, scientific literature, patents andnews websites. January 2022 saw Janssen and Midatech expand their collaboration on bioresorbable polymer microsphere technology for drug delivery. Takeda announced its plans to acquire UK-based Adaptate Biotherapeutics and Gandeeva raised further investment funds to support its drug discovery and development platform focused on the evaluation of protein-drug interactions. Biogen announced that it will sell its stake in a biosimilars joint ventureand ABL Bio and Sanofi announced a collaboration around a novel treatment for Parkinson's disease. New regulatory announcements this month included USFDA approvals of a new insomnia treatment for Idorsia and a treatment for atopic dermatitis developed by Pfizer. Insulet gained FDA clearance for a closed-loop insulin pump andAscendis Pharmafollowed up its United States approval last year for a once-weekly treatment for growth hormone deficiency with European approval. Pfizer and Ionis announced the discontinuation of the clinical development of a novel cardiovascular drug. In terms of collaborations, Novartis and Alnylam announced they will work together to explore targeted therapies to restore liver function; Scorpion Therapeutics partnered with AstraZeneca to develop novel cancer treatments and Nutriband Inc. and Kindeva Drug Deliverywill work together to develop a transdermal fentanyl patch. Collaborations were also announced betweenCentury Therapeutics and Bristol Myers Squibb andLillyand Entos Pharmaceuticalsin the areas of stem cell therapies for cancer treatment and neurology, respectively. A team from the Massachusetts Institute of Technologyreported progress in developing oral mRNA treatments and West Pharmaceutical Services published a blog describing the development of a proof-of-principle system for a closed-loop feedback system targeting opioid overdose. A report on the BBC website highlighted the benefits of more sustainable inhalers.

Functional genomics and the future of iPSCs in disease modeling.

SummaryInduced pluripotent stem cells (iPSCs) are valuable in disease modeling because of their potential to expand and differentiate into virtually any cell type and recapitulate key aspects of human biology. Functional genomics are genome-wide studies that aim to discover genotype-phenotype relationships, thereby revealing the impact of human genetic diversity on normal and pathophysiology. In this review, we make the case that human iPSCs (hiPSCs) are a powerful tool for functional genomics, since they provide an in vitro platform for the study of population genetics. We describe cutting-edge tools and strategies now available to researchers, including multi-omics technologies, advances in hiPSC culture techniques, and innovations in drug development. Functional genomics approaches based on hiPSCs hold great promise for advancing drug discovery, disease etiology, and the impact of genetic variation on human biology.

The Role of Nanotechnology in the Fight against COVID-19

COVID-19 has lately emerged as one of the most difficult pandemics of the twentieth century, with lethal consequences and a high rate of replication. It emphasizes the critical importance of developing effective vaccines to prevent virus infection, early and rapid diagnosis using high sensitivity and selectivity diagnostic kits, and effective antiviral and protective therapeutics to reduce and eliminate viral load and tissue damage-related side effects. As a result, non-toxic antiviral nanoparticles (NPs) are being developed for therapeutic use in the prevention and treatment of COVID-19. Nanoparticles have shown considerable promise in the development of nano vaccines to combat viral diseases. In this paper, we look at the potential of nanoparticles as a medicine or as a platform for drug and vaccine repurposing and development. Meanwhile, sophisticated virus detection methodologies based on NPs will be detailed, with the goal of inspiring scientists to develop cost-effective Nano platforms for prevention, diagnosis, and therapy.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

<em>Ex Vivo</em> and <em>In Vivo</em> Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

In Silico Development of Combinatorial Therapeutic Approaches Targeting Key Signaling Pathways in Metabolic Syndrome

Dysregulations of key signaling pathways in metabolic syndrome are multifactorial, eventually leading to cardiovascular events. Hyperglycemia in conjunction with dyslipidemia induces insulin resistance and provokes release of proinflammatory cytokines resulting in chronic inflammation, accelerated lipid peroxidation with further development of atherosclerotic alterations and diabetes. We have proposed a novel combinatorial approach using FDA approved compounds targeting IL-17a and DPP4 to ameliorate a significant portion of the clustered clinical risks in patients with metabolic syndrome. In our current research we have modeled the outcomes of metabolic syndrome treatment using two distinct drug classes. Targets were chosen based on the clustered clinical risks in metabolic syndrome: dyslipidemia, insulin resistance, impaired glucose control, and chronic inflammation. Drug development platform, BIOiSIM™, was used to narrow down two different drug classes with distinct modes of action and modalities. Pharmacokinetic and pharmacodynamic profiles of the most promising drugs were modeling showing predicted outcomes of combinatorial therapeutic interventions. Preliminary studies demonstrated that the most promising drugs belong to DPP-4 inhibitors and IL-17A inhibitors. Evogliptin was chosen to be a candidate for regulating glucose control with long term collateral benefit of weight loss and improved lipid profiles. Secukinumab, an IL-17A sequestering agent used in treating psoriasis, was selected as a repurposed candidate to address the sequential inflammatory disorders that follow the first metabolic insult. Our analysis suggests this novel combinatorial therapeutic approach inducing DPP4 and Il-17a suppression has a high likelihood of ameliorating a significant portion of the clustered clinical risk in metabolic syndrome.

A repositioning screen using an FGFR2 splicing reporter reveals compounds that regulate epithelial-mesenchymal transitions and inhibit growth of prostate cancer xenografts

Research in the area of hallmarks of cancer has opened the possibility of designing new therapies based on modulating these cancer properties. We present here a screen designed to find chemicals that modulate epithelial-mesenchymal transitions (EMTs) in prostate cancer. For screening, we used a repurposing library and, as a readout, an FGFR2-based splicing reporter, which has been shown previously to be a sensor for EMTs. Various properties of cancer cells were assessed, signaling pathways investigated, and in vivo experiments in nude mice xenografts performed. The screen yielded three hit compounds (a T-type Ca channel inhibitor, an L-type Ca channel inhibitor, and an opioid antagonist) that switch FGFR2 splicing and induce an epithelial phenotype in prostate cancer cells. The compounds affected differently various properties of cancer cells, but all of them decreased cell migration, which is in line with modulating EMTs. We further present mechanistic insights into one of the compounds, nemadipine-A. The administration of nemadipine-A intraperitoneally in a nude mouse xenograft model of prostate cancer slowed tumor growth. To conclude, we show that knowledge of the molecular mechanisms that connect alternative splicing and various cancer properties may be used as a platform for drug development.

Antisense Oligonucleotide: A Potential Therapeutic Intervention for Chronic Kidney Disease

Chronic kidney disease (CKD) is a global public health issue that places an increasing burden on the healthcare systems of both the developed and developing countries. CKD is a progressive and irreversible condition, affecting approximately 10% of the population worldwide. Patients that have progressed to end-stage renal disease (ESRD) require expensive renal replacement therapy, i.e., dialysis or kidney transplantation. Current CKD therapy largely relies on the use of angiotensin-converting enzyme inhibitors (ACEis) and angiotensin receptor blockers (ARBs). However, these treatments by no means halt the progression of CKD to ESRD. Therefore, the development of new therapies is urgently needed. Antisense oligonucleotide (ASO) has recently attracted considerable interest as a drug development platform. Thus far, eight ASO-based drugs have been granted approval by the US Food and Drug Administration for the treatment of various diseases. Herein, we review the ASOs developed for the identification of CKD-relevant genes and/or the simultaneous development of the ASOs as potential therapeutics towards treating CKD.

Recent Advances in Modeling Mitochondrial Cardiomyopathy Using Human Induced Pluripotent Stem Cells.

Around one third of patients with mitochondrial disorders develop a kind of cardiomyopathy. In these cases, severity is quite variable ranging from asymptomatic status to severe manifestations including heart failure, arrhythmias, and sudden cardiac death. ATP is primarily generated in the mitochondrial respiratory chain via oxidative phosphorylation by utilizing fatty acids and carbohydrates. Genes in both the nuclear and the mitochondrial DNA encode components of this metabolic route and, although mutations in these genes are extremely rare, the risk to develop cardiac symptoms is significantly higher in this patient cohort. Additionally, infants with cardiovascular compromise in mitochondrial deficiency display a worse late survival compared to patients without cardiac symptoms. At this point, the mechanisms behind cardiac disease progression related to mitochondrial gene mutations are poorly understood and current therapies are unable to substantially restore the cardiac performance and to reduce the disease burden. Therefore, new strategies are needed to uncover the pathophysiological mechanisms and to identify new therapeutic options for mitochondrial cardiomyopathies. Here, human induced pluripotent stem cell (iPSC) technology has emerged to provide a suitable patient-specific model system by recapitulating major characteristics of the disease in vitro, as well as to offer a powerful platform for pre-clinical drug development and for the testing of novel therapeutic options. In the present review, we summarize recent advances in iPSC-based disease modeling of mitochondrial cardiomyopathies and explore the patho-mechanistic insights as well as new therapeutic approaches that were uncovered with this experimental platform. Further, we discuss the challenges and limitations of this technology and provide an overview of the latest techniques to promote metabolic and functional maturation of iPSC-derived cardiomyocytes that might be necessary for modeling of mitochondrial disorders.

Generation of hiPSC line UMi030-A from an individual with the hearing loss-related GJB2 mutation c.109G>A.

Genetic variants in the GJB2 gene which encodes for the Connexin 26 protein account for∼60% of cases of genetic hearing loss. A novel hiPSC line was generated from an individual with the hearing loss-related variant c.109G>A in GJB2 leading to the p.V37I alteration in the Connexin26 protein. These cells will help to delineate the role of GJB2 in hearing loss pathogenesis and serve as a platform for drug discovery and development.

Overview, Generation, and Significance of Variable New Antigen Receptors (VNARs) as a Platform for Drug and Diagnostic Development.

The approval of the first VHH-based drug caplacizumab (anti-von Willebrand factor) has validated a two-decade long commitment in time and research effort to realize the clinical potential of single-domain antibodies. The variable domain (VNAR) of the immunoglobulin new antigen receptor (IgNAR) found in sharks provides an alternative small binding domain to conventional monoclonal antibodies and their fragments and heavy-chain antibody-derived VHHs. Evolutionarily distinct from mammalian antibody variable domains, VNARs have enhanced thermostability and unusual convex paratopes. This predisposition to bind cryptic and recessed epitopes has facilitated both the targeting of new antigens and new (neutralizing) epitopes on existing antigens. Together these unique properties position the VNAR platform as an alternative non-antibody binding domain for therapeutic drug, diagnostic and reagent development. In this introductory chapter, we highlight recent VNAR advancements that further underline the exciting potential of this discovery platform.

Analysis of Cell Proliferation by Three-Dimensional Culture.

Three-dimensional (3D) cell culture technology is a powerful tool in cancer research and drug development. It retains several critical components of the in vivo environment, including cell-cell and cell-extracellular matrix (ECM) interactions in a 3D fashion, gradients of oxygen, nutrients and metabolic waste, and it is thus more physiologically relevant than traditional two-dimensional (2D) cell culture. Here, we describe a simple and versatile method using commercially available chamber slides and Matrigel, a surrogate ECM hydrogel, to set up a 3D culture model for breast cancer cells. In this 3D culture model, cells form aggregates or spheroids on top of a thin layer of Matrigel, which can be fixed directly onto the chamber slides for cell imaging and immunofluorescence staining. Alternatively, RNA and protein can be extracted from the cells for further investigation. This 3D cell culture model provides a useful platform for cancer research and drug development, in which the effects of novel compounds or genetic modifications can be tested.

Microengineered Multi-Organoid System from hiPSCs to Recapitulate Human Liver-Islet Axis in Normal and Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) is a systematic multi‐organ metabolic disease, which is characterized by the dynamic interplay among different organs. The increasing incidence of T2DM reflects an urgent need for the development of in vitro human‐relevant models for disease study and drug therapy. Here, a new microfluidic multi‐organoid system is developed that recapitulates the human liver‐pancreatic islet axis in normal and disease states. The system contains two compartmentalized regions connected by a microchannel network, enabling 3D co‐culture of human induced pluripotent stem cells (hiPSC)‐derived liver and islet organoids for up to 30 days under circulatory perfusion conditions. The co‐cultured liver and islet organoids exhibit favorable growth and improved tissue‐specific functions. Transcriptional analyses reveal the activation of metabolically relevant signaling pathways in the co‐cultured organoids. Notably, the co‐culture system facilitates sensitive glucose‐stimulated insulin secretion from islet organoids and increased glucose utilization in liver organoids by glucose tolerance tests. Both liver and islet organoids display mitochondrial dysfunction and decreased glucose transport capacity under high glucose conditions, which can be alleviated by metformin treatment. This novel multi‐organoid system can recapitulate human‐relevant liver‐islet axis under both physiological and pathological conditions, providing a unique platform for future T2DM research and drug development.