Unraveling the folding-assisted unbinding mechanism of TCF with its binding partner ß-catenin.

This study utilizes molecular dynamics simulations aided with multiple walker parallel bias metadynamics to investigate the TCF unbinding mechanism from the ß-catenin interface. The results, consistent with experimental binding affinity calculations, unveil a folding-assisted unbinding mechanism.

Nanostructured lipopeptide-based membranomimetics for stabilizing bacteriorhodopsin.

Nanostructured 7-9-residue cyclic and unstructured lipopeptide-based facial detergents have been engineered to stabilize the model integral membrane protein, bacteriorhodopsin. Formation of a cylindrical-type micelle assembly induced by facial amphipathic lipopeptides resembles a biological membrane more effectively than conventional micelles. The hydrophobic face of this cylindrical-type micelle provides extended stability to the membrane protein and the hydrophilic surface interacts with an aqueous environment. In our present study, we have demonstrated experimentally and computationally that lipopeptide-based facial detergents having an unstructured or ß-turn conformation can stabilize membrane proteins. However, constrained peptide detergents can provide enhanced stability to bacteriorhodopsin. In this study, we have computationally examined the structural stability of bacteriorhodopsin in the presence of helical, beta-strand, and cyclic unstructured peptide detergents, and conventional detergent-like peptides. Our study demonstrates that optimal membranomimetics (detergents) for stabilizing a specific membrane protein can be screened based on the following criteria: (i) hydrodynamic radii of the self-assembled peptide detergents, (ii) stability assay of detergent-encased membrane proteins, (iii) percentage covered area of detergent-encased membrane proteins obtained computationally and (iv) protein-detergent interaction energy.

Integrating Clinical Variability into PBPK Models for Virtual Bioequivalence of Single and Multiple Doses of Tofacitinib Modified-Release Dosage Form.

Tofacitinib is a potent, selective inhibitor of the Janus kinase (JAK) family of kinases with a high degree of selectivity within the human genome's set of protein kinases. Currently approved formulations for tofacitinib citrate are immediate-release (IR) tablets, modified-release (MR) tablets, and IR solution. A once daily MR microsphere formulation was developed for use in pediatric patients. Demonstration of bioequivalence (BE) between the 10 mg once daily (q.d.) MR microsphere formulation and 5 mg twice daily (b.i.d.) IR solution is needed to enable the exposure-response analyses-based bridging to support regulatory approval. To assess BE between MR microsphere and IR solution, an innovative approach was utilized with physiologically-based pharmacokinetic (PBPK) virtual BE trials (VBE) in lieu of a clinical BE trial. A PBPK model was developed to characterize the absorption of different formulations of tofacitinib using Simcyp ADAM module. VBE trials were conducted by simulating PK profiles using the verified PBPK model and integrating the clinically observed intrasubject coefficient of variation (ICV) where BE was assessed with a predetermined sample size and prespecified criteria. The VBE trials demonstrated BE between IR solution 5 mg b.i.d. and MR microsphere 10 mg q.d. after a single dose on day 1 and after multiple doses on day 5. This research presents an innovative approach that incorporates clinically observed ICV in PBPK model-based VBE trials, which could reduce unnecessary drug exposure to healthy volunteers and streamline new formulation development strategies.

A Piecewise Design Approach to Engineering a Miniature ACE2 Mimic to Bind SARS-CoV-2.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues its global spread, the exploration of novel therapeutic and diagnostic strategies is still needed. The virus enters host cells by binding the angiotensin-converting enzyme 2 (ACE2) receptor through the spike protein. Here, we develop an engineered, small, stable, and catalytically inactive version of ACE2, termed miniature ACE2 (mACE2), designed to bind the spike protein with high affinity. Employing a magnetic nanoparticle-based assay, we harnessed the strong binding affinity of mACE2 to develop a sensitive and specific platform for the detection or neutralization of SARS-CoV-2. Our findings highlight the potential of engineered mACE2 as a valuable tool in the fight against SARS-CoV-2. The success of developing such a small reagent based on a piecewise molecular design serves as a proof-of-concept approach for the rapid deployment of such agents to diagnose and fight other viral diseases.

Similar enzymatic functions in distinct bioluminescence systems: evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

From Data to Cure: A Comprehensive Exploration of Multi-omics Data Analysis for Targeted Therapies.

In the dynamic landscape of targeted therapeutics, drug discovery has pivoted towards understanding underlying disease mechanisms, placing a strong emphasis on molecular perturbations and target identification. This paradigm shift, crucial for drug discovery, is underpinned by big data, a transformative force in the current era. Omics data, characterized by its heterogeneity and enormity, has ushered biological and biomedical research into the big data domain. Acknowledging the significance of integrating diverse omics data strata, known as multi-omics studies, researchers delve into the intricate interrelationships among various omics layers. This review navigates the expansive omics landscape, showcasing tailored assays for each molecular layer through genomes to metabolomes. The sheer volume of data generated necessitates sophisticated informatics techniques, with machine-learning (ML) algorithms emerging as robust tools. These datasets not only refine disease classification but also enhance diagnostics and foster the development of targeted therapeutic strategies. Through the integration of high-throughput data, the review focuses on targeting and modeling multiple disease-regulated networks, validating interactions with multiple targets, and enhancing therapeutic potential using network pharmacology approaches. Ultimately, this exploration aims to illuminate the transformative impact of multi-omics in the big data era, shaping the future of biological research.

Model-Informed Assessment of Probability of Phase 3 Success for Ritlecitinib in Patients with Moderate-to-Severe Ulcerative Colitis.

Ritlecitinib, an oral Janus kinase 3/tyrosine kinase expressed in hepatocellular carcinoma family inhibitor, was evaluated in patients with ulcerative colitis (UC) in a phase 2b trial. Model-informed drug development strategies were applied to bridge observations from phase 2b to predictions for a proposed phase 3 study design to assess the probability of achieving the target efficacy outcome. A longitudinal exposure-response model of the time course of the 4 Mayo subscores (rectal bleeding, stool frequency, physician's global assessment, and endoscopic subscore) in patients with UC receiving placebo or ritlecitinib was developed using population modeling approaches and an item response theory framework. The quantitative relationships between the 4 Mayo subscores accommodated the prediction of composite endpoints such as total Mayo score and partial Mayo score (key endpoints from phase 2b), and modified clinical remission and endoscopic remission (proposed phase 3 endpoints). Clinical trial simulations using the final model assessed the probability of candidate ritlecitinib dosing regimens (including those tested in phase 2b and alternative) and phase 3 study designs for achieving target efficacy outcomes benchmarked against an approved treatment for moderate-to-severe UC. The probabilities of achieving target modified clinical remission and endoscopic improvement outcomes at both weeks 8 and 52 for ritlecitinib 100 mg once daily was 74.8%. Model-based assessment mitigated some of the risk associated with proceeding to pivotal phase 3 trials with dosing regimens of which there was limited clinical experience.

Engineering water exchange is a safe and effective method for magnetic resonance imaging in diverse cell types.

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

VISTA checkpoint inhibition by pH-selective antibody SNS-101 with optimized safety and pharmacokinetic profiles enhances PD-1 response.

VISTA, an inhibitory myeloid-T-cell checkpoint, holds promise as a target for cancer immunotherapy. However, its effective targeting has been impeded by issues such as rapid clearance and cytokine release syndrome observed with previous VISTA antibodies. Here we demonstrate that SNS-101, a newly developed pH-selective VISTA antibody, addresses these challenges. Structural and biochemical analyses confirmed the pH-selectivity and unique epitope targeted by SNS-101. These properties confer favorable pharmacokinetic and safety profiles on SNS-101. In syngeneic tumor models utilizing human VISTA knock-in mice, SNS-101 shows in vivo efficacy when combined with a PD-1 inhibitor, modulates cytokine and chemokine signaling, and alters the tumor microenvironment. In summary, SNS-101, currently in Phase I clinical trials, emerges as a promising therapeutic biologic for a wide range of patients whose cancer is refractory to current immunotherapy regimens.

A Dual-Gene Reporter-Amplifier Architecture for Enhancing the Sensitivity of Molecular MRI by Water Exchange.

The development of genetic reporters for magnetic resonance imaging (MRI) is essential for investigating biological functions in vivo. However, current MRI reporters have low sensitivity, making it challenging to create significant contrast against the tissue background, especially when only a small fraction of cells express the reporter. To overcome this limitation, we developed an approach for amplifying the sensitivity of molecular MRI by combining a chemogenetic contrast mechanism with a biophysical approach to increase water diffusion through the co-expression of a dual-gene construct comprising an organic anion transporting polypeptide, Oatp1b3, and a water channel, Aqp1. We first show that the expression of Aqp1 amplifies MRI contrast in cultured cells engineered to express Oatp1b3. We demonstrate that the contrast amplification is caused by Aqp1-driven increase in water exchange, which provides the gadolinium ions internalized by Oatp1b3-expressing cells with access to a larger water pool compared with exchange-limited conditions. We further show that our methodology allows cells to be detected using approximately 10-fold lower concentrations of gadolinium than that in the Aqp1-free scenario. Finally, we show that our approach enables the imaging of mixed-cell cultures containing a low fraction of Oatp1b3-labeled cells that are undetectable on the basis of Oatp1b3 expression alone.

Exploring the potential of water channels for developing MRI reporters and sensors without the need for exogenous contrast agents.

Genetically encoded reporters for magnetic resonance imaging (MRI) offer a valuable technology for making molecular-scale measurements of biological processes within living organisms with high anatomical resolution and whole-organ coverage without relying on ionizing radiation. However, most MRI reporters rely on contrast agents, typically paramagnetic metals and metal complexes, which often need to be supplemented exogenously to create optimal contrast. To eliminate the need for contrast agents, we previously introduced aquaporin-1, a mammalian water channel, as a new reporter gene for the fully autonomous detection of genetically labeled cells using diffusion-weighted MRI. In this study, we aimed to expand the toolbox of diffusion-based genetic reporters by modulating aquaporin membrane trafficking and harnessing the evolutionary diversity of water channels across species. We identified a number of new water channels that functioned as diffusion-weighted reporter genes. In addition, we show that loss-of-function variants of yeast and human aquaporins can be leveraged to design first-in-class diffusion-based sensors for detecting the activity of a model protease within living cells.

Myeloid and dendritic cells enhance therapeutics-induced cytokine release syndrome features in humanized BRGSF-HIS preclinical model.

Objectives: Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings. Methods: BRGSF mice reconstituted with human umbilical cord blood CD34+ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed. Results: OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds. Conclusions: These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

Rational Design of a Circularly Permuted Flavin-Based Fluorescent Protein.

Flavin-based fluorescent proteins are oxygen-independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering.

Tofacitinib pharmacokinetics in children and adolescents with juvenile idiopathic arthritis.

These analyses characterized tofacitinib pharmacokinetics (PKs) in children and adolescents with juvenile idiopathic arthritis (JIA). Data were pooled from phase I (NCT01513902), phase III (NCT02592434), and open-label, long-term extension (NCT01500551) studies of tofacitinib tablet/solution (weight-based doses administered twice daily [b.i.d.]) in patients with JIA aged 2 to less than 18 years. Population PK modeling used a nonlinear mixed-effects approach, with covariates identified using stepwise forward-inclusion backward-deletion procedures. Simulations were performed to derive dosing recommendations for children and adolescents with JIA. Two hundred forty-six pediatric patients were included in the population PK model. A one-compartment model with first-order elimination and absorption with body weight as a covariate for oral clearance and apparent volume of distribution sufficiently described the data. Oral solution was associated with comparable average concentration (Cavg) and slightly higher (113.9%) maximum concentration (Cmax) versus tablet, which was confirmed by a subsequent randomized, open-label, bioavailability study conducted in healthy adult participants (n = 12) by demonstrating adjusted geometric mean ratios (90% confidence interval) between oral solution and tablet of 1.04 (1.00-1.09) and 1.10 (1.00-1.21) for area under the curve extrapolated to infinity and Cmax, respectively (NCT04111614). A dosing regimen of 3.2 mg b.i.d. solution in patients 10 to less than 20 kg, 4 mg b.i.d. solution in patients 20 to less than 40 kg, and 5 mg b.i.d. tablet/solution in patients greater than or equal to 40 kg, irrespective of age, was proposed to achieve constant Cavg across weight groups. In summary, population PK characterization informed a simplified tofacitinib dosing regimen that has been implemented in pediatric patients with JIA.

A dual-gene reporter-amplifier architecture for enhancing the sensitivity of molecular MRI by water exchange.

The development of genetic reporters for magnetic resonance imaging (MRI) is essential for investigating biological functions in intact animals. However, current MRI reporters have low sensitivity, making it challenging to create significant contrast against the tissue background, especially when only a small percentage of cells express the reporter. To overcome this limitation, we developed an approach that amplifies signals by co-expressing an MRI reporter gene, Oatp1b3, with a water channel, aquaporin-1 (Aqp1). We first show that the expression of Aqp1 amplifies the paramagnetic relaxation effect of Oatp1b3 by facilitating transmembrane water exchange. This mechanism provides Oatp1b3-expressing cells with access to a larger water pool compared with typical exchange-limited conditions. We further demonstrated that our methodology allows dual-labeled cells to be detected using approximately 10-fold lower concentrations of contrast agent than that in the Aqp1-free scenario. Finally, we show that our approach enables the imaging of mixed-cell populations containing a low fraction of Oatp1b3-labeled cells that are otherwise undetectable based on Oatp1b3 expression alone.

Targeting RdRp of SARS-CoV-2 with De Novo Molecule Generation.

Viruses are known for their extremely high mutation rates, allowing them to evade both the human immune system and many forms of standard medicine. Despite this, the RNA dependent RNA polymerase (RdRp) of the RNA viruses has been largely conserved, and any significant mutation of this protein is unlikely. The recent COVID-19 pandemic presents a need for therapeutics. We have designed a de novo drug design algorithm that generates strong binding ligands from scratch, based on only the structure of the target protein's receptor. In this paper, we applied our method to target SARS-CoV-2 RdRp and generated several de novo molecules. We then chose some drug molecules based on the structural similarity to some of our strongest binding de novo molecules. Subsequently, we showed, using rigorous all-atom explicit-water free energy calculations in near-microsecond time scales using state-of-the-art well-tempered metadynamics simulations, that some of our de novo generated ligands bind more strongly to RdRp than the recent FDA approved drug remdesivir in its active form, remdesivir triphosphate (RTP). We elucidated the binding mechanism for some of the top binders and compared it with RTP. We believe that this work will be useful both by presenting lead structures for RdRp inhibition and by delivering key insights into the residues of the protein potentially involved in the binding/unbinding of these small molecule drugs, leading to more targeted studies in the future.

Similar enzymatic functions in distinct bioluminescence systems: Evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii , transfer sulfate in vivo to the luciferin substrate, vargulin. We find luciferin sulfotransferases of ostracods are not orthologous to known luciferin sulfotransferases of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Re-use of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Engineering water exchange is a safe and effective method for magnetic resonance imaging in diverse cell types.

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

Molecular Imaging with Aquaporin-Based Reporter Genes: Quantitative Considerations from Monte Carlo Diffusion Simulations.

Aquaporins provide a unique approach for imaging genetic activity in deep tissues by increasing the rate of cellular water diffusion, which generates a magnetic resonance contrast. However, distinguishing aquaporin signals from the tissue background is challenging because water diffusion is influenced by structural factors, such as cell size and packing density. Here, we developed a Monte Carlo model to analyze how cell radius and intracellular volume fraction quantitatively affect aquaporin signals. We demonstrated that a differential imaging approach based on subtracting signals at two diffusion times can improve specificity by unambiguously isolating aquaporin signals from the tissue background. We further used Monte Carlo simulations to analyze the connection between diffusivity and the percentage of cells engineered to express aquaporin and established a mapping that accurately determined the volume fraction of aquaporin-expressing cells in mixed populations. The quantitative framework developed in this study will enable a broad range of applications in biomedical synthetic biology, requiring the use of aquaporins to noninvasively monitor the location and function of genetically engineered devices in live animals.

Solvothermal synthesis of 3D rod-shaped Ti/Al/Cr nano-oxide for photodegradation of wastewater micropollutants under sunlight: a green way to achieve SDG:6.

Waterbodies are day-by-day polluted by the various colored micropollutants, e.g., azo dyes enriched (carcinogenic, non-biodegradable) colored wastewater from textile industries. Water pollution has become a serious global issue as ~ 25% of health diseases are prompted by pollution as reported by WHO. Around 1 billion people will face water scarcity by 2025 and this water crisis is also a prime focus to the UNs' sustainable development goal 6 (SDG6: clean water and sanitation). To prevent the water pollution caused by micropollutants, a mesoporous, 3D rod-like nano-oxide Ti/Al/Cr (abbreviated as TAC) has been synthesized via the solvothermal method. TAC degraded all classes of azo dyes (mono, di, tri, etc.) with > 90% efficiency under renewable energy source solar irradiation within the pH range 2-11. The detailed study was done on the photodegradation of carcinogenic di-azo dye Congo red (CR) which is banned in many countries. TAC showed 90.64 ± 2% degradation efficiency for CR at pH 7. The proposed photodegradation mechanism of CR was confirmed by the high-resolution liquid chromatography-mass spectroscopy (HRLC-MS) analysis obeying the Pirkanniemi path. The photodegradation obeyed the pseudo-1st-order kinetics and was reusable up to successive 5 cycles which can be an efficient tool to meet the UNs' SDG:6.