Early Drug Research Articles

Abstract LB389: Highly potent and selective RET inhibitor with robust anti-tumor activities against wild-type and acquired drug-resistant mutations

Abstract RET gene mutations and fusions are the driving events for the generation and deterioration of many cancer types. The first generation RET inhibitors, selpercatinib (Loxo-292) and pralsetinib (Blu-667), demonstrated clinically acquired resistance (e.g., solvent frontier mutations G810X, double mutations V804X-G810X etc.). Novel treatment that overcomes multiple resistance mutations is needed to improve patient outcomes. Leveraging our in-house AI technology platform, we have discovered the next generation RET inhibitors that overcome multiple resistance mutations including solvent front, gatekeeper and various double mutations. We employ the synergistic advantages of domain expertise and modern technology to accelerate early stage drug discovery. On our deep learning (DL) centric AI platform, extensive scientific computing supports large scale virtual screening, generative design, drug property prediction and multi-objective lead optimization. Only the optimized candidate compounds are passed along for synthesis and testing to save time and cost. In cell proliferation assays, our compound effectively inhibited KIF5B-RET Ba/F3 WT, V804M, G810S, G810R, V804M-G810S with IC50 values of 0.4 nM, 1.3 nM, 1.1 nM, 3.4 nM, 6.3 nM, respectively, demonstrating significant improvement over selpercatinib in the range of several hundred fold in activities. Importantly, the compound is highly selective against VEGFR2, an off-target responsible for cardiovascular adverse effects in the approved RET inhibitors. The superior cellular potencies translated consistently into animal models. In Ba/F3 KIF5B-RET-G810R xenograft model, treatment of 25 mg/kg bid resulted in >90% TGI with a100% survival rate. The compound showed favorable pharmacokinetic properties, lack of drug-drug interaction (DDI), and devoid of hERG liability (IC50 > 10uM). Preclinical safety evaluations in rats exhibited good tolerance margin. We have discovered the second generation compound that potently inhibits a diverse range of RET alterations. The compound exhibits preferable pharmaceutical profiles and is advancing toward IND application. Citation Format: Li Xing, Wei Zhu, Jingbing Wang. Highly potent and selective RET inhibitor with robust anti-tumor activities against wild-type and acquired drug-resistant mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB389.

Bioinformatic Analysis and in Vitro Experimental Verification of the Intervention Effect of Shipi-Xiaoji Recipe on the Progression of non-Alcoholic Fatty Liver Disease to Liver Cancer

Objective This study aimed to screen and identify central genes associated with the progression of non-alcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC), to establish prognostic and clinical prediction models based on the expression of these genes, and to evaluate the intervention of a traditional Chinese medicine (TCM) formula—SPXJF—on the development of NAFLD into HCC through network pharmacology analysis and in vitro and in vivo validation. Methods Datasets for analysis were retrieved from multiple public databases using bioinformatics techniques. Through network pharmacology analysis, SPXJF's active components and their related targets were assessed, while in vitro experiments validated the inhibitory effects of SPXJF on the growth and migration of HepG2 cells. Results The results revealed that the expression levels of the two central genes, regulator of cell cycle gene (RGCC) and atypical chemokine receptor 3 (ACKR3), were closely tied to the survival prognosis of HCC patients. Furthermore, SPXJF significantly inhibited the proliferation and migration of HepG2 cells. Conclusion RGCC and ACKR3, as hub genes, significantly influence HCC patient survival during “NAFLD-HCC” progression. The risk scoring system and prediction model perform well. In vitro experiments showed that SPXJF could inhibit the proliferation and invasion of hepG2 cells. These findings support the discovery of molecular targets for early diagnosis and drug intervention in HCC. They also underscore TCM's potential in anti-HCC strategies and clinical application.

Fabrication of folic acid-modified bovine serum albumin cloaked dual-drug loaded hollow mesoporous silica nanoparticles for pH-responsive and targeted delivery of gastric cancer therapy

Combination therapy is a highly successful way to address the limitations of using a single treatment method and improve therapy's overall efficacy. In this study, we developed a unique hollow mesoporous silica nanoparticle (HMSN) coated with folic acid (FA)-modified bovine serum albumin (FA-BSA). This nanoparticle, referred to as HFB, was designed to target cancer cells and release dual therapeutic drugs, Indocyanine green (ICG) and Paclitaxel (PTX), in response to specific stimuli termed as HFB@IP. The BSA protein acts as a “gatekeeper” to prevent early drug releases and cargo leakage by detaching from BSA in reaction to GSH. The FA facilitates the targeted transport of the drug into cancer cells that express folate receptors (FR), enhancing the effectiveness of chemo-photodynamic treatment (PDT). The drug nanocarrier demonstrated in vitro pH/redox-triggered drug release from HFB@IP due to breaking the imine bonds between aldehyde-functionalized HMSN (CHO-HMSN) and FA-BSA with the disulfide bond inside BSA. In addition, various biological assessments, including cell uptake experiments, demonstrated that HFB@IP effectively targets SGC-7901 cells and induces apoptosis in vitro. Further, it exhibits remarkable efficiency in synergistically killing cancer cells through chemo-photodynamic therapy, as indicated by a combination index (CI) of 0.328. The results showed that combining HMSN with biodegradable stimuli-responsive BSA molecules could offer a promising approach for precise chemo-photodynamic therapy in treating gastric cancer, allowing for the controlled release of drugs as necessary.

Approaches to predict human PK profiles for compounds undergoing transporter mediated clearance in early drug development: From dedrick plot to PBPK modeling

Approaches to predict human PK profiles for compounds undergoing transporter mediated clearance in early drug development: From dedrick plot to PBPK modeling

Applications of the partial-order continual reassessment method in the early development of treatment combinations.

Combination therapy is increasingly being explored as a promising approach for improving cancer treatment outcomes. However, identifying effective dose combinations in early oncology drug development is challenging due to limited sample sizes in early-phase clinical trials. This task becomes even more complex when multiple agents are being escalated simultaneously, potentially leading to a loss of monotonic toxicity order with respect to the dose. Traditional single-agent trial designs are insufficient for this multi-dimensional problem, necessitating the development and implementation of dose-finding methods specifically designed for drug combinations. While, in practice, approaches to this problem have focused on preselecting combinations with a known toxicity order and applying single-agent designs, this limits the number of combinations considered and may miss promising dose combinations. In recent years, several novel designs have been proposed for exploring partially ordered drug combination spaces with the goal of identifying a maximum tolerated dose combination, based on safety, or an optimal dose combination, based on toxicity and efficacy. However, their implementation in clinical practice remains limited. In this article, we describe the application of the partial order continual reassessment method and its extensions for combination therapies in early-phase clinical trials. We present completed trials that use safety endpoints to identify maximum tolerated dose combinations and adaptively use both safety and efficacy endpoints to determine optimal treatment strategies. We discuss the effectiveness of the partial-order continual reassessment method and its extensions in identifying optimal treatment strategies and provide our experience with executing these novel adaptive designs in practice. By utilizing innovative dose-finding methods, researchers and clinicians can more effectively navigate the challenges of combination therapy development, ultimately improving patient outcomes in the treatment of cancer.

A multi-site study on sex differences in cortical thickness in non-demented Parkinson’s disease

Clinical, cognitive, and atrophy characteristics depending on sex have been previously reported in Parkinson’s disease (PD). However, though sex differences in cortical gray matter measures in early drug naïve patients have been described, little is known about differences in cortical thickness (CTh) as the disease advances. Our multi-site sample comprised 211 non-demented PD patients (64.45% males; mean age 65.58 ± 8.44 years old; mean disease duration 6.42 ± 5.11 years) and 86 healthy controls (50% males; mean age 65.49 ± 9.33 years old) with available T1-weighted 3 T MRI data from four international research centers. Sex differences in regional mean CTh estimations were analyzed using generalized linear models. The relation of CTh in regions showing sex differences with age, disease duration, and age of onset was examined through multiple linear regression. PD males showed thinner cortex than PD females in six frontal (bilateral caudal middle frontal, bilateral superior frontal, left precentral and right pars orbitalis), three parietal (bilateral inferior parietal and left supramarginal), and one limbic region (right posterior cingulate). In PD males, lower CTh values in nine out of ten regions were associated with longer disease duration and older age, whereas in PD females, lower CTh was associated with older age but with longer disease duration only in one region. Overall, male patients show a more widespread pattern of reduced CTh compared with female patients. Disease duration seems more relevant to explain reduced CTh in male patients, suggesting worse prognostic over time. Further studies should explore sex-specific cortical atrophy trajectories using large longitudinal multi-site data.

Abstract 1844: Proteomic characterization of decitabine resistance in acute myeloid leukemia reveals signaling pathway crosstalk dampens the effectiveness of combination therapy

Abstract Acute myeloid leukemia (AML) is a deadly form of blood cancer primarily characterized by genetic abnormalities that guide treatment strategies. In recent years, several new therapies have emerged to target these genetic abnormalities such as gilteritinib or quizartinib, which target an internal tandem duplication (ITD) of the FLT3 gene. Additional drugs have been developed to target core cellular processes, such as venetoclax and the decitabine. Despite initial positive responses to these treatments, patients eventually relapse as they develop resistance to the individual drugs. One common approach to drug resistance is to add an additional drug to the treatment protocol. However, patients then develop further resistance to not just a single drug but multiple. As such, understanding the origins of both single and multi-drug resistance is crucial for improving treatment efficacy and clinical outcomes. Here, we present a collection of cell lines that have developed resistance to single, double, and triple drug combinations of gilteritinib, venetoclax, and decitabine. Through comprehensive global and phospho proteomics analyses under multiple conditions together with computational pathway interrogation, we unravel the underlying molecular mechanisms responsible for resistance. Leveraging our previous model of early and late drug resistance, we investigate the emergence of resistance to drug combinations involving gilteritinib alone, gilteritinib with venetoclax, gilteritinib with decitabine, and gilteritinib with venetoclax and decitabine. Through the characterization of molecular signatures for each drug combination, we identify molecular signals that are specific to combination treatments. Specifically, we find that the emergences of decitabine resistance alters the cellular pathways to effectively downregulate targets of venetoclax and gilteritinib, ultimately dampening the response to these drugs, regardless of co-administration. These findings provide a potential explanation for the limited success of combination treatments involving hypomethylating agents, as observed in recent clinical trials. Our results underscore the importance of monitoring proteome-level changes in single and combination drug treatments and shed light on the simple and intuitive reasons behind the lack of synergy often observed in combination therapies. In conclusion, we establish cell line models of resistance to various combinations of venetoclax, gilteritinib, and decitabine. By integrating global and phospo proteomic measurements, we gain a comprehensive understanding of the intricate molecular mechanisms underlying resistance. Citation Format: James C. Pino, Camilo Posso, Setareh Sharzehi, Sara Gosline, Chelsea Hutchinson-Bunch, Elie Traer, Paul D. Piehowski, Karin D. D. Rodland, Jeffrey W. Tyner, Tao Liu, Anupriya Agarwal. Proteomic characterization of decitabine resistance in acute myeloid leukemia reveals signaling pathway crosstalk dampens the effectiveness of combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1844.

Abstract 6778: New microplate platform with cryopreserved 3D tumor spheroids for faster and convenient pre-clinical testing

Abstract Drug discovery and safety testing depend on predictive in vitro models, including multicellular tissues with organotypic functions. While traditional 2D tumor cell cultures have been extensively used in early drug screening due to their user-friendly nature, automation compatibility, and high throughput, they fall short in representing the intricate 3-dimensional structure, physiology, and complex multicellular nature of actual tumors. However, generating such 3D tissues for each test campaign is labor intensive. In addition, not all researchers have the resources to establish these 3D models. To pave the way, we developed a new platform that offers a range of cryopreserved 3D tumor models in micro plate format allowing for high-content screening. It enables users to start testing compounds within a 3-to-5 days simply by thawing an ARCTis™ plate (Always Ready Cryo Tissues) and following clear instructions. Another advantage of these cryopreserved 3D models derives from the fact that they are made in larger batches which means the start point for compound testing is always the same. This improves data quality, maximizes the test window length, and makes the data comparable across work groups. Currently, our growing portfolio consists of 25 tumor cell lines from ATCC representing the most common solid tumors. Their functionality has been determined based on efficacy tests for each cell line with the apoptotic agent Staurosporine. Various cell lines exhibited compact spheroids with a round morphology, less compact aggregates with non-spherical morphology, or loose cell-cell interaction aggregates, which can correlate with their tumor site of origin. Upon treatment with Staurosporine the dose-response profiles indicated growth inhibition, tumor shrinkage, or tumor cell killing effects based on size assessment and cell metabolic analysis (ATP). Moreover, we demonstrated the robust performance of these assays from plates stored for up to 6 months at -80°C.In conclusion, this groundbreaking approach addresses the limitations of traditional 2D cell cultures by providing a high-throughput, in-plate frozen, 3D cell culture platform. By offering a combination of a more physiologically relevant representation of tumors with a robust frozen-storage and time flexible assay platform our method holds great promise for advancing cancer drug discovery by easing the exploration of complex drug testing screens in vitro. Citation Format: Nils Goedecke, Simon Stroebel, Simon Hutter, Tala Issa, Irina Agarkova, Laure-Anne Ligeon, Olivier Frey. New microplate platform with cryopreserved 3D tumor spheroids for faster and convenient pre-clinical testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6778.

Abstract 2816: Comparison of MB-49_luc bladder carcinoma cell clones in the orthotopic superficial bladder tumor model in C57BL/6 female mice

Abstract In preclinical research, cell inoculation via bladder wall injection is a standard for studying invasive bladder cancer into the bladder muscle wall. However, a subset of patients presents with non-muscle invasive (superficial) disease. The standard of care for these patients is excision coupled with BCG treatment via catheterization. There is a need to further establish mouse models of cell inoculation via bladder catheterization for early drug development. While the injection places cells into the bladder wall, catheterization coats the inside of the bladder by first instilling Poly-L-Lysine. After instilling Poly-L-Lysine, instillation of luciferase-tagged cell suspension occurs. MB49-luc cell lines were generated to determine the optimal cell lines for use in analyzing treatments for superficial bladder carcinoma. MB49-luc cells display varying degrees of luciferase activity and growth in vitro. A subset of cell lines were selected for further analysis in vivo, via bioluminescent imaging, survival analysis, flow cytometry and necropsy. The data provides a preclinical reference for the selection of a cell line for analysis of new drug candidates in C57BL/6 female mice inoculated via bladder catheterization. Citation Format: Mollie McArthur, Melissa Tran, Aliccia Koznecki, Helen Ketteringham, Corrine Silvio, Shannan Wenzel, Cheryl Davis, Shorena Nadaraia-Hoke, Chris Holding, Logan Salem, Victoria Caruso, Ben Hoerner, Madison Spencer, Marcus Josey, Alexa Tannu. Comparison of MB-49_luc bladder carcinoma cell clones in the orthotopic superficial bladder tumor model in C57BL/6 female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2816.

Abstract 7178: Neuronal cells derived from iPSCs cell to evaluate neurotoxicity after 48 or 72 hours in high-through put screening format

Abstract Neurotoxicity is a major concern in central nervous system drug discovery and a frequent cause of attrition in clinical trials or approved drug withdrawal. Neuropathy is a side effect defined by the injury of peripheral nerves causing a loss of sensation/motion. Drug development pipelines typically involve testing in cell lines followed by animal investigations with translation to humans. The induced pluripotent stem cells (iPSCs) have widened new model systems to study adverse toxicities. With evolution in regulatory guidance the use of human- iPSC-derived tissue provides high-throughput access and a more relevant testing environment. Neuropathy is one of the most chemotherapy adverse effects. In this study we have screened 8 compounds. Bortezomib (proteasome inhibitor), gemcitabine (approved ovarian cancer treatment), ivermectin (parasitic diseases treatment), flavopiridol (approved CDKs inhibitors), SNS-032 (approved CDKs inhibitor), mefloquine (anti-malaria drug), digitonin (nonionic detergent) and tamoxifen (hormone receptor-positive breast cancer treatment). These molecule toxicities were tested on neuronal-derived iPSCs Dopaneurone, GABAneurone, Glutaneurone, Motorneurone, Microglya and Astrocytes in high throughput screening. For the project, cells were plated in 384 well/plates. DMSO tolerance was performed (up to 10%). Drugs were added, then, after 48h or 72h, total protease and protease activities were measured using CytoTox-GloTM. Compounds were tested at 7 concentrations (from 10−4 to 10−9M).In this study we have validated cell culture and the assay conditions. The signal window range was good after 48h and 72h of treatment. No toxicity was found until 10% DMSO. A specific neurotoxicity signature was measurable for each drug tested. Digitonin, our positive control, induced toxicity in all IPSCs after 48h and 72h of incubation. On the contrary, Tamoxifen, our negative control, did not induce any neurotoxicity. The other molecules generated a signature of severe to mild neurotoxicity. These results are in agreement with their known adverse side-effects. In conclusion, this panel is a good tool to anticipate possible neurotoxicity within the 3Rs respect. It can be used in early drug de-risking or neuroprotection screening, with the aim of preventing/reducing/curing neuropathy in at-risk populations. Citation Format: Fabienne Sabatier, Justine Chaigne, Maryse Martin, Rodolphe Barbeau, Luisa Houssin, Stephane Bonnin, Alastair King, Vanessa Jahnke. Neuronal cells derived from iPSCs cell to evaluate neurotoxicity after 48 or 72 hours in high-through put screening format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7178.

Abstract 2389: Empowering early drug development in neoadjuvant non-small cell lung cancer studies using an innovative proof-of-concept platform trial design

Abstract Several platform trial designs have recently been proposed in oncology. However, developing an efficient one in the neoadjuvant non-small cell lung cancer (NSCLC) is not easy due to the difficulties in patient recruitment and limited relevant data. These obstacles hinder the inclusion of a control arm, thereby losing the ability to make an informed Go/No-Go decision, and to assess prognostic and predictive biomarkers. To overcome these challenges, we have developed an innovative and efficient proof-of-concept platform trial design that aims to empower drug development in early stage of neoadjuvant NSCLC. The proposed platform trial incorporates available experimental agents, allowing for new agents to be added when available. Within the master protocol structure, a small control arm is included, only about 20% of experimental arm size, and is shared by all experimental arms for the analysis purpose. Consequently, the resulting direct comparison with the control arm enables an informed Go/No-Go decision-making, while the total sample size and budget impacts are minimized. The flexibility of Go/No-Go decision-making is further enhanced in a 3-outcome decision-making framework where a consider zone is introduced for physicians to examine the totality of data. Two innovative statistical techniques are incorporated: Bayesian dynamic borrowing for current control, and response-adaptive randomization (RAR). The former helps reduce the sample size, and the latter aims to optimize patient allocation and improve the ability to identify promising agents. Simulation results demonstrate that the direct comparison sharply reduces the false positive rate below 10%, compared to a single-arm design setting. The effective sample size of the control arm is statistically augmented through Bayesian dynamic borrowing. The patient allocation is guided based on the agent’s efficacy data with a well-calibrated RAR. The promising agent receives meaningfully more patients than if a fixed-randomization is used, while the variability in patient allocation is decreased. Compared to a traditional design, our innovative design boosts the probability of identifying promising agent by up to 2-fold. To conclude, this research develops an innovative platform trial design to improve the success of drug development in early stage NSCLC. The master protocol structure, inclusion of a small control arm, and dynamic borrowing, increase the plausibility of conducting a trial in the neoadjuvant population. Advanced statistical features optimize the patient allocation and enhance the ability to identify promising drugs. Lastly, the proposed design can be easily applied to other oncology indications with appropriate modifications. Citation Format: Zhe Chen, Phillip A. Dennis, Ji Lin. Empowering early drug development in neoadjuvant non-small cell lung cancer studies using an innovative proof-of-concept platform trial design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2389.

Pocket Crafter: a 3D generative modeling based workflow for the rapid generation of hit molecules in drug discovery

We present a user-friendly molecular generative pipeline called Pocket Crafter, specifically designed to facilitate hit finding activity in the drug discovery process. This workflow utilized a three-dimensional (3D) generative modeling method Pocket2Mol, for the de novo design of molecules in spatial perspective for the targeted protein structures, followed by filters for chemical-physical properties and drug-likeness, structure–activity relationship analysis, and clustering to generate top virtual hit scaffolds. In our WDR5 case study, we acquired a focused set of 2029 compounds after a targeted searching within Novartis archived library based on the virtual scaffolds. Subsequently, we experimentally profiled these compounds, resulting in a novel chemical scaffold series that demonstrated activity in biochemical and biophysical assays. Pocket Crafter successfully prototyped an effective end-to-end 3D generative chemistry-based workflow for the exploration of new chemical scaffolds, which represents a promising approach in early drug discovery for hit identification.

Beyond Rule of Five and PROTACs in Modern Drug Discovery: Polarity Reducers, Chameleonicity, and the Evolving Physicochemical Landscape.

Developing orally bioavailable drugs demands an understanding of absorption in early drug development. Traditional methods and physicochemical properties optimize absorption for rule of five (Ro5) compounds; beyond rule of five (bRo5) drugs necessitate advanced tools like the experimental measure of exposed polarity (EPSA) and the AbbVie multiparametric score (AB-MPS). Analyzing AB-MPS and EPSA against ∼1000 compounds with human absorption data and ∼10,000 AbbVie tool compounds (∼1000 proteolysis targeting chimeras or PROTACs, ∼7000 Ro5s, and ∼2000 bRo5s) revealed new patterns of physicochemical trends. We introduced a high-throughput "polarity reduction" descriptor: ETR, the EPSA-to-topological polar surface area (TPSA) ratio, highlights unique bRo5 and PROTAC subsets for specialized drug design strategies for effective absorption. Our methods and guidelines refine drug design by providing innovative in vitro approaches, enhancing physicochemical property optimization, and enabling accurate predictions of intestinal absorption in the complex bRo5 domain.

A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides.

Thiopeptides make up a group of structurally complex peptidic natural products holding promise in bioengineering applications. The previously established thiopeptide/mRNA display platform enables de novo discovery of natural product-like thiopeptides with designed bioactivities. However, in contrast to natural thiopeptides, the discovered structures are composed predominantly of proteinogenic amino acids, which results in low metabolic stability in many cases. Here, we redevelop the platform and demonstrate that the utilization of compact reprogrammed genetic codes in mRNA display libraries can lead to the discovery of thiopeptides predominantly composed of nonproteinogenic structural elements. We demonstrate the feasibility of our designs by conducting affinity selections against Traf2- and NCK-interacting kinase (TNIK). The experiment identified a series of thiopeptides with high affinity to the target protein (the best KD = 2.1 nM) and kinase inhibitory activity (the best IC50 = 0.15 μM). The discovered compounds, which bore as many as 15 nonproteinogenic amino acids in an 18-residue macrocycle, demonstrated high metabolic stability in human serum with a half-life of up to 99 h. An X-ray cocrystal structure of TNIK in complex with a discovered thiopeptide revealed how nonproteinogenic building blocks facilitate the target engagement and orchestrate the folding of the thiopeptide into a noncanonical conformation. Altogether, the established platform takes a step toward the discovery of thiopeptides with high metabolic stability for early drug discovery applications.

Characterization workflow for fragments detected in capillary electrophoresis sodium dodecyl sulfate analysis of therapeutic monoclonal antibodies.

Product-related fragments in monoclonal antibodies (mAbs) can have a significant impact on the efficacy and safety of the product. Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is a commonly used method for fragment quantification, but it has challenges in peak identification due to the inability to enrich components and the incompatibility of SDS with mass spectrometry (MS). This article presents a workflow for identifying peaks in CE-SDS analysis. The workflow involves comparing the migration time of peaks with that of standards and utilizing MS analysis to identify fragments. By employing this innovative systematic workflow, we successfully identified the CE-SDS impurity peaks of seven antibody products. Among them, four products exhibited characteristic fragments associated with disulfide bonds (light chain [LC], heavy-light [HL] chain, heavy-heavy [HH] chain, and HH-LC) and a glycosylation-related fragment non-glycosylated heavy chain. Additionally, one product showed a fragment formed by the connection of HC_C130 and HC_C130, which is associated with a thioether bond. Furthermore, two other products displayed amino acid backbone breakage, with one product showing clipping at the HC region of A233-G285 and the other product showing clipping at the HC regions of A97-S158 and N342-T366. This workflow can be applied in early drug research, process development, or during the biologics license application stage to characterize fragments in therapeutic mAbs analyzed by CE-SDS.

Switching photocurrent polarity and mimicking enzyme functions of MIL-53(Fe)@hemin for homogeneous dual-modal ultrasensitive detection of PTP1B

Protein tyrosine phosphatase 1B (PTP1B) as a regulatory enzyme is an important target for the early diagnosis and drug discovery of diabetes, obesity, and cancer. Herein, a homogeneous dual-modal biosensor integrating photoelectrochemical and colorimetric (PEC-CL) strategies was developed in light of the switching photocurrent polarity and mimicking enzyme functions of MIL-53(Fe)@hemin for the sensitive detection of PTP1B activity. Magnetic Fe3O4@Au NPs were synthesized for more sufficient homogeneous biorecognition, and modified with the phosphorylated peptides (p-Peptide), and sequentially connected with MIL-53(Fe)@hemin by the specific coordination of phosphate groups with Fe(III). After PTP1B-mediated specific recognition and dephosphorylation, the separated MIL-53(Fe)@hemin were quickly collected via magnetic separation for further PEC and CL assay. MIL-53(Fe)@hemin not only switched the photocurrent polarity of SnS2 NFs from anode to cathode, but also exhibited excellent peroxidase-like activity for oxidizing 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. As a signal amplifier, MIL-53(Fe) acted as a nanocarrier to load abundant PEC signal switching factor of hemin, and synergistically enhanced its enzyme-mimic property. Consequently, ultrasensitive PEC biosensing (with linear response range of 0.1 pg mL−1 to 3000 ng mL−1 and detection limit of 0.074 pg mL−1) assisted by visual CL strategy (with linear response range of 10 pg mL−1 to 3000 ng mL−1 and detection limit of 0.24 pg mL−1) for PTP1B activity assay were achieved, which not only provided high sensitivity, but also possessed portability, perceptual intuition and reliability.

Advancing amyotrophic lateral sclerosis disease diagnosis: A lab-on-chip electrochemical immunosensor for ultra-sensitive TDP-43 protein detection and monitoring in serum patients’

The global increase in population aging has led to a rise in neurodegenerative diseases (NDs), posing significant challenges to public health. Developing selective and specific biomarkers for early diagnosis and drug development is crucial addressing the growing burden of NDs. In this context, the RNA-binding protein TDP-43 has emerged as a promising biomarker for amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and TDP-43-associated proteinopathies. However, existing detection methods suffer from limitations such as cost, complexity, and operator dependence. Here, we present a novel electrochemical biosensor integrated into a lab-on-chip (LoC) platform to detect TDP-43. The sensor utilizes electrosynthesized polypyrrole derivatives with carboxylic groups for transducer functionalization, enabling targeted immobilization of TDP-43 antibodies. Differential pulsed voltammetry (DPV) is used for the indirect detection and quantification of TDP-43. The chip exhibits rapid response, good reproducibility, a linear detection range, and sensitivity from 0.01 ng/mL to 25 ng/mL of TDP-43 protein concentration with a LOD = 10 pg/mL. Furthermore, successful TDP-43 detection in complex matrices like serum of ALS patients and healthy individuals demonstrates its potential as a point-of-care diagnostic device. This electrochemical biosensor integrated into a chip offers good sensitivity, rapid response, and robust performance, providing a promising avenue for advancing neurodegenerative disease diagnostics and therapeutic development.

An Integrated Hepatocyte Stability Assay for Simultaneous Metabolic Stability Assessment and Metabolite Profiling.

The determination of metabolic stability is critical for drug discovery programs, allowing for the optimization of chemical entities and compound prioritization. As such, it is common to perform high-volume in vitro metabolic stability experiments early in the lead optimization process to understand metabolic liabilities. Additional metabolite identification experiments are subsequently performed for a more comprehensive understanding of the metabolic clearance routes to aid medicinal chemists in the structural design of compounds. Collectively, these experiments require extensive sample preparation and a substantial amount of time and resources. To overcome the challenges, a high-throughput integrated assay for simultaneous hepatocyte metabolic stability assessment and metabolite profiling was developed. This assay platform consists of four parts: 1) an automated liquid-handling system for sample preparation and incubation, 2) a liquid chromatography and high-resolution mass spectrometry-based system to simultaneously monitor the parent compound depletion and metabolite formation, 3) an automated data analysis and report system for hepatic clearance assessment; and 4) streamlined autobatch processing for software-based metabolite profiling. The assay platform was evaluated using eight control compounds with various metabolic rates and biotransformation routes in hepatocytes across three species. Multiple sample preparation and data analysis steps were evaluated and validated for accuracy, repeatability, and metabolite coverage. The combined utility of an automated liquid-handling instrument, a high-resolution mass spectrometer, and multiple streamlined data processing software improves the process of these highly demanding screening assays and allows for simultaneous determination of metabolic stability and metabolite profiles for more efficient lead optimization during early drug discovery. SIGNIFICANCE STATEMENT: Metabolic stability assessment and metabolite profiling are pivotal in drug discovery to fully comprehend metabolic liabilities for chemical entity optimization and lead selection. Process of these assays can be repetitive and resource demanding. Here, we developed an integrated hepatocyte stability assay that combines automation, high-resolution mass spectrometers, and batch-processing software to improve and combine the workflow of these assays. The integrated approach allows simultaneous metabolic stability assessment and metabolite profiling, significantly accelerating screening and lead optimization in a resource-effective manner.

Engineering models of head and neck and oral cancers on-a-chip.

Head and neck cancers (HNCs) rank as the sixth most common cancer globally and result in over 450 000 deaths annually. Despite considerable advancements in diagnostics and treatment, the 5-year survival rate for most types of HNCs remains below 50%. Poor prognoses are often attributed to tumor heterogeneity, drug resistance, and immunosuppression. These characteristics are difficult to replicate using in vitro or in vivo models, culminating in few effective approaches for early detection and therapeutic drug development. Organs-on-a-chip offer a promising avenue for studying HNCs, serving as microphysiological models that closely recapitulate the complexities of biological tissues within highly controllable microfluidic platforms. Such systems have gained interest as advanced experimental tools to investigate human pathophysiology and assess therapeutic efficacy, providing a deeper understanding of cancer pathophysiology. This review outlines current challenges and opportunities in replicating HNCs within microphysiological systems, focusing on mimicking the soft, glandular, and hard tissues of the head and neck. We further delve into the major applications of organ-on-a-chip models for HNCs, including fundamental research, drug discovery, translational approaches, and personalized medicine. This review emphasizes the integration of organs-on-a-chip into the repertoire of biological model systems available to researchers. This integration enables the exploration of unique aspects of HNCs, thereby accelerating discoveries with the potential to improve outcomes for HNC patients.

Genetic predisposition and high exposure to colistin in the early treatment period as independent risk factors for colistin-induced nephrotoxicity.

Colistin is known to cause nephrotoxicity due to its extensive reabsorption and accumulation in renal tubules. Invitro studies have identified the functional role of colistin transporters such as OCTN2, PEPT2, megalin, and P-glycoprotein. However, the role of these transporter gene variants in colistin-induced nephrotoxicity has not been studied. Utilizing targeted next-generation sequencing, we screened for genetic polymorphisms covering the colistin transporters (SLC15A1, SLC15A2, SLC22A5, LRP2, and ABCB1) in 42 critically ill patients who received colistimethate sodium. The genetic variants rs2257212 ((NM_021082.4):c.1048C>G) and rs13397109 ((NM_004525.3):C.7626C > T) were identified as being associated with an increased incidence of acute kidney injury (AKI) on Day 7. Colistin area under the curve (AUC) was predicted using a previously published pharmacokinetic model of colistin. Using logistic regression analysis, the predicted 24-h AUC of colistin was identified as an important contributor for increased odds of AKI on Day 7. Among 42 patients, 4 (9.5%) were identified as having high predisposition to colistin-induced AKI based on the presence of predisposing genetic variants. Determination of the presence of the abovementioned genetic variants and early therapeutic drug monitoring may reduce or prevent colistin-induced nephrotoxicity and facilitate dose optimization of colistimethate sodium.