Small Molecule Screening Research Articles

Determination of Protein-Ligand Binding Affinities by Thermal Shift Assay.

Quantification of protein-ligand interactions is crucial for understanding the protein's biological function and for drug discovery. In this study, we employed three distinct approaches for determination of protein-ligand binding affinities by a thermal shift assay using a single ligand concentration. We present the results of the comparison of the performance of the conventional curve fitting (CF) method and two newly introduced methods - assuming zero heat capacity change across small temperature ranges (ZHC) and utilizing the unfolding equilibrium constant (UEC); the latter has the advantage of reducing calculations by obtaining the unfolding equilibrium constant directly from the experimental data. Our results highlight superior performance of the ZHC and UEC methods over the conventional CF method in estimating the binding affinity, irrespective of the ligand concentration. In addition, we evaluated how the new methods can be applied to high-throughput screening for potential binders, when the enthalpy (ΔH L) and molar heat capacity change (ΔC PL) of ligand binding are unknown. Our results suggest that, in this scenario, using the -300 cal K-1 mol-1 assumption for ΔC pL and either -5 kcal mol-1 or the average enthalpy efficiency-based estimation for ΔH L(T) can still provide reasonable estimates of the binding affinity. Incorporating the new methods into the workflow for screening of small drug-like molecules, typically conducted using single-concentration libraries, could greatly simplify and streamline the drug discovery process.

Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn's Disease.

We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.

Cathepsin B causes trogocytosis-mediated CAR T cell dysfunction.

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in cancer treatment. Still, most patients receiving CAR T cells relapse within 5 years of treatment. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism in which cell surface proteins transfer from tumor cells to CAR T cells. CMT results in the emergence of antigen-negative tumor cells, which can evade future CAR detection, and antigen-positive CAR T cells, which has been suggested to cause CAR T cell fratricide and exhaustion. Whether CMT indeed causes CAR T cell dysfunction and the molecular mechanisms conferring CMT remain unknown. Using a selective degrader of trogocytosed antigen in CAR T cells, we show that the presence of trogocytosed antigen on the CAR T cell surface directly causes CAR T cell fratricide and exhaustion. By performing a small molecule screening using a custom high throughput CMT-screening assay, we found that the cysteine protease cathepsin B (CTSB) is essential for CMT and that inhibition of CTSB is sufficient to prevent CAR T cell fratricide and exhaustion. Our data demonstrate that it is feasible to separate CMT from cytotoxic activity and that CAR T cell persistence, a key factor associated with clinical CAR T cell efficacy, is directly linked to CTSB activity in CAR T cells.

In vitro small molecule screening to inform novel candidates for use in fluconazole combination therapy in vivo against Coccidioides.

Developing new drugs, especially for regional orphan diseases, such as Valley Fever, is a slow and costly endeavor. However, there is a wealth of FDA-approved drugs available for repurposing, offering a more economical and expedited approach to improve treatment. Those existing compounds with antifungal properties can become novel therapies with relative ease: a considerable advantage for patients in need of alternative treatment. Despite the scope of remaining tasks, our comprehensive screening of potential candidates has revealed promising combinations for further exploration. This effort outlines a practical pipeline for Valley fever drug screening and identifies viable drug combinations that could impact patients more rapidly than single drug development pathways.

CAG repeat-selective compounds reduce abundance of expanded CAG RNAs in patient cell and murine models of SCAs.

Spinocerebellar ataxias (SCAs) are a genetically heterogenous group of devastating neurodegenerative conditions for which clinical care currently focuses on managing symptoms. Across these diseases there is an unmet need for therapies that address underlying disease mechanisms. We utilised the shared CAG repeat expansion mutation causative for a large subgroup of SCAs, to develop a novel disease-gene independent and mechanism agnostic small molecule screening approach to identify compounds with therapeutic potential across multiple SCAs. Using this approach, we identified the FDA approved microtubule inhibitor Colchicine and a novel CAG-repeat binding compound that reduce expression of disease associated transcripts across SCA1, 3 and 7 patient derived fibroblast lines and the Atxn1 154Q/2Q SCA1 mouse model in a repeat selective manner. Furthermore, our lead candidate rescues dysregulated alternative splicing in Atxn1 154Q/2Q mice. This work provides the first example of small molecules capable of targeting the underlying mechanism of disease across multiple CAG SCAs.

Targeting Super-Enhancer-Driven Transcriptional Dependencies Suppresses Aberrant Hedgehog Pathway Activation and Overcomes Smoothened Inhibitor Resistance.

Aberrant activation of the Hedgehog (Hh) signaling pathway plays important roles in oncogenesis and therapeutic resistance in several types of cancer. The clinical application of FDA-approved Hh-targeted smoothened inhibitors (SMOi) is hindered by the emergence of primary or acquired drug resistance. Epigenetic and transcriptional-targeted therapies represent a promising direction for developing improved anti-Hh therapies. In this study, we integrated epigenetic/transcriptional-targeted small-molecule library screening with CRISPR/Cas9 knockout library screening and identified CDK9 and CDK12, two transcription elongation regulators, as therapeutic targets for antagonizing aberrant Hh activation and overcoming SMOi resistance. Inhibition of CDK9 or CDK12 potently suppressed Hh signaling and tumor growth in various SMOi responsive or resistant Hh-driven tumor models. Systemic epigenomic profiling elucidated the Hh-driven super-enhancer (SE) landscape and identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the insulin-like growth factor (IGF) pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target in Hh-driven tumor models. Collectively, this study identifies SE-driven transcriptional dependencies that represent promising therapeutic vulnerabilities for suppressing the Hh pathway and overcoming SMOi resistance. As CDK9 and IRS inhibitors have already entered human clinical trials for cancer treatment, these findings provide comprehensive preclinical support for developing trials for Hh-driven cancers. Significance: Dissecting transcriptional dependencies driven by super-enhancers uncovers therapeutic targets in Hedgehog-driven cancers and identifies strategies for overcoming resistance to smoothened inhibitors.

Abstract Or121: Druggable Genome CRISPRi/a Screens in iPSC-Derived Cardiomyocytes Identify Therapeutic Targets for Doxorubicin-Induced Cardiotoxicity

Introduction: Doxorubicin is a highly effective chemotherapy drug whose therapeutic utility is significantly hampered by its life-threatening adverse cardiac effects. The need for strategies to mitigate these cardiotoxic effects without diminishing its efficacy against cancer is critical in cardio-oncology. Hypothesis: We hypothesize that through an integrated drug discovery pipeline, utilizing patient iPSC-derived cardiomyocytes (iCMs) and CRISPRi/a bidirectional pooled screens combined with small molecule screening and molecular docking, we can identify therapeutic targets that ameliorate doxorubicin-induced cardiotoxicity (DIC) while preserving its anticancer efficacy. Methods: Our approach included patient iPSC-derived cardiomyocytes (iCMs), CRISPRi/a bidirectional pooled screens targeting druggable genome (~2000 genes) to identify candidate genes implicated in DIC, and small molecule screening alongside molecular docking to find potential inhibitors. Notably, carbonic anhydrase 12 (CA12) was identified as a primary hit for its role in DIC. Results: The CRISPRi/a screens revealed several novel genes, including CA12, as potential contributors to DIC. Genetic inhibition of CA12 in iCMs enhanced cell survival, preserved sarcomere structure, and improved contractile function and calcium handling, effectively protecting against DIC. Furthermore, we discovered that a CA12 antagonist significantly attenuates DIC in both iCMs and mouse models. Mechanistic studies suggested that doxorubicin triggers CA12 activation, leading to a metabolic change towards glycolysis in cardiomyocytes, thereby exacerbating DIC by disrupting cellular metabolism and cardiac functions. Conclusions: Our findings propose a novel therapeutic avenue to safeguard against doxorubicin-induced cardiotoxicity through the targeting of CA12, offering new direction for the development of more refined anticancer therapies that minimize cardiotoxic risk. This study sets the stage of combining CRISPRi/a screen with iPSC technology for future drug discovery endeavors aimed at eliminating off-target cardiotoxicities of chemotherapy agents.

Novel miRNA-inducing drugs enable differentiation of retinoic acid-resistant neuroblastoma cells.

Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA-modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and BDNF-activating bufalin synergistically arrests proliferation, induces differentiation, and maintains the differentiated state of MES SK-N-AS cells for 8 weeks. RNA-seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained MES neuroblastoma GI-ME-N and SH-EP cell lines, as well as glioblastoma LN-229 and U-251 cell lines, for over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.

FOXP4 Is a Direct YAP1 Target That Promotes Gastric Cancer Stemness and Drives Metastasis.

The Hippo-YAP1 pathway is an evolutionally conserved signaling cascade that controls organ size and tissue regeneration. Dysregulation of Hippo-YAP1 signaling promotes initiation and progression of several types of cancer, including gastric cancer. As the Hippo-YAP1 pathway regulates expression of thousands of genes, it is important to establish which target genes contribute to the oncogenic program driven by YAP1 to identify strategies to circumvent it. In this study, we identified a vital role of forkhead box protein 4 (FOXP4) in YAP1-driven gastric carcinogenesis by maintaining stemness and promoting peritoneal metastasis. Loss of FOXP4 impaired gastric cancer spheroid formation and reduced stemness marker expression, whereas FOXP4 upregulation potentiated cancer cell stemness. RNA sequencing analysis revealed SOX12 as a downstream target of FOXP4, and functional studies established that SOX12 supports stemness in YAP1-induced carcinogenesis. A small-molecule screen identified 42-(2-tetrazolyl) rapamycin as a FOXP4 inhibitor, and targeting FOXP4 suppressed gastric cancer tumor growth and enhanced the efficacy of 5-fluorouracil chemotherapy in vivo. Collectively, these findings revealed that FOXP4 upregulation by YAP1 in gastric cancer regulates stemness and tumorigenesis by upregulating SOX12. Targeting the YAP1-FOXP4-SOX12 axis represents a potential therapeutic strategy for gastric cancer. Significance: Hippo-YAP1 signaling maintains stemness in gastric cancer by upregulating FOXP4, identifying FOXP4 as a stemness biomarker and therapeutic target that could help improve patient outcomes.

From hit to vial: Precision discovery and development of an imidazopyrimidine TLR7/8 agonist adjuvant formulation.

Vaccination can help prevent infection and can also be used to treat cancer, allergy, and potentially even drug overdose. Adjuvants enhance vaccine responses, but currently, the path to their advancement and development is incremental. We used a phenotypic small-molecule screen using THP-1 cells to identify nuclear factor-κB (NF-κB)-activating molecules followed by counterscreening lead target libraries with a quantitative tumor necrosis factor immunoassay using primary human peripheral blood mononuclear cells. Screening on primary cells identified an imidazopyrimidine, dubbed PVP-037. Moreover, while PVP-037 did not overtly activate THP-1 cells, it demonstrated broad innate immune activation, including NF-κB and cytokine induction from primary human leukocytes in vitro as well as enhancement of influenza and SARS-CoV-2 antigen-specific humoral responses in mice. Several de novo synthesis structural enhancements iteratively improved PVP-037's in vitro efficacy, potency, species-specific activity, and in vivo adjuvanticity. Overall, we identified imidazopyrimidine Toll-like receptor-7/8 adjuvants that act in synergy with oil-in-water emulsion to enhance immune responses.

Reduction of Z alpha-1 antitrypsin polymers in human iPSC-hepatocytes and mice by LRRK2 inhibitors.

Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by inheritance of the SERPINA1 'Z' genetic variant (PiZ) driving AAT protein misfolding in hepatocytes. There remain no approved medicines for this disease. Here, we report the results of a small molecule screen performed in patient derived iPSC-hepatocytes that identified Leucine-rich repeat kinase-2 (LRRK2) as a potentially new therapeutic target. Of the commercially available LRRK2 inhibitors tested, we identified CZC-25146, a candidate with favorable pharmacokinetic properties, as being capable of reducing polymer load, increasing normal AAT secretion, and reducing inflammatory cytokines in both cells and PiZ mice. Mechanistically, this effect was achieved through induction of autophagy. Our findings support the use of CZC-25146 and LRRK2 inhibitors in hepatic proteinopathy research and their further investigation as novel therapeutic candidates for A1ATD.

A PNPLA3-Deficient iPSC-Derived Hepatocyte Screen Identifies Pathways to Potentially Reduce Steatosis in Metabolic Dysfunction-Associated Fatty Liver Disease.

The incidence of nonalcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated fatty liver disease (MAFLD), is increasing in adults and children. Unfortunately, effective pharmacological treatments remain unavailable. Single nucleotide polymorphisms (SNPs) in the patatin-like phospholipase domain-containing protein (PNPLA3 I148M) have the most significant genetic association with the disease at all stages of its progression. A roadblock to identifying potential treatments for PNPLA3-induced NAFLD is the lack of a human cell platform that recapitulates the PNPLA3 I148M-mediated onset of lipid accumulation. Hepatocyte-like cells were generated from PNPLA3-/- and PNPLA3I148M/M-induced pluripotent stem cells (iPSCs). Lipid levels were measured by staining with BODIPY 493/503 and were found to increase in PNPLA3 variant iPSC-derived hepatocytes. A small-molecule screen identified multiple compounds that target Src/PI3K/Akt signaling and could eradicate lipid accumulation in these cells. We found that drugs currently in clinical trials for cancer treatment that target the same pathways also reduced lipid accumulation in PNPLA3 variant cells.

Predictions of Colloidal Molecular Aggregation Using AI/ML Models.

To facilitate the triage of hits from small molecule screens, we have used various AI/ML techniques and experimentally observed data sets to build models aimed at predicting colloidal aggregation of small organic molecules in aqueous solution. We have found that Naïve Bayesian and deep neural networks outperform logistic regression, recursive partitioning tree, support vector machine, and random forest techniques by having the lowest balanced error rate (BER) for the test set. Derived predictive classification models consistently and successfully discriminated aggregator molecules from nonaggregator hits. An analysis of molecular descriptors in favor of colloidal aggregation confirms previous observations (hydrophobicity, molecular weight, and solubility) in addition to undescribed molecular descriptors such as the fraction of sp3 carbon atoms (Fsp3), and electrotopological state of hydroxyl groups (ES_Sum_sOH). Naïve Bayesian modeling and scaffold tree analysis have revealed chemical features/scaffolds contributing the most to colloidal aggregation and nonaggregation, respectively. These results highlight the importance of scaffolds with high Fsp3 values in promoting nonaggregation. Matched molecular pair analysis (MMPA) has also deciphered context-dependent substitutions, which can be used to design nonaggregator molecules. We found that most matched molecular pairs have a neutral effect on aggregation propensity. We have prospectively applied our predictive models to assist in chemical library triage for optimal plate selection diversity and purchase for high throughput screening (HTS) in drug discovery projects.

Tumor-resident microbiota contributes to colorectal cancer liver metastasis by lactylation and immune modulation

The role of tumor-resident microbiota in modulating tumor immunity remains unclear. Here, we discovered an abundance of intra-tumoral bacteria, such us E.coli, residing and resulting in Colorectal cancer liver metastasis (CRLM). E.coli enhanced lactate production, which mediated M2 macrophage polarization by suppressing nuclear factor-κB -gene binding (NF-κB) signaling through retinoic acid-inducible gene 1 (RIG-I) lactylation. Lactylation of RIG-I suppressed recruitment of NF-κB to the Nlrp3 promoter in macrophages, thereby reducing its transcription. This loss of Nlrp3 affected the immunosuppressive activities of regulatory T cells (Tregs) and the antitumor activities of and CD8+ T cells. Small-molecule compound screening identified a RIG-I lactylation inhibitor that suppressed M2 polarization and sensitized CRLM to 5-fluorouracil (5-FU). Our findings suggest that tumor-resident microbiota may be a potential target for preventing and treating CRLM.

To explore the prognostic characteristics of colon cancer based on tertiary lymphoid structure-related genes and reveal the characteristics of tumor microenvironment and drug prediction

In order to construct a prognostic evaluation model of TLS features in COAD and better realize personalized precision medicine in COAD. Colon adenocarcinoma (COAD) is a common malignant tumor of the digestive system. At present, there is no effective prognostic marker to predict the prognosis of patients. Tertiary lymphoid structure (TLS) affects cancer progression by regulating immune microenvironment. Mining COAD biomarkers based on TLS-related genes helps to improve the prognosis of patients. In order to construct a prognostic evaluation model of TLS features in COAD and better realize personalized precision medicine in COAD. The mRNA expression data and clinical information of COAD and adjacent tissues were downloaded from the Cancer Genome Atlas database. The differentially expressed TLS-related genes of COAD relative to adjacent tissues were obtained by differential analysis. TLS gene co-expression analysis was used to mine genes highly related to TLS, and the intersection of the two was used to obtain candidate genes. Univariate, LASSO, and multivariate Cox regression analysis were performed on candidate genes to screen prognostic markers to construct a risk assessment model. The differences of immune characteristics were evaluated by ESTIMATE, ssGSEA and CIBERSORT in high and low risk groups of prognostic model. The difference of genomic mutation between groups was evaluated by tumor mutation burden score. Screening small molecule drugs through the GDSC library. Finally, a nomogram was drawn to evaluate the clinical value of the prognostic model. Seven TLS-related genes ADAM8, SLC6A1, PAXX, RIMKLB, PTH1R, CD1B, and MMP10 were screened to construct a prognostic model. Survival analysis showed that patients in the high-risk group had significantly lower overall survival rates. Immune microenvironment analysis showed that patients in the high-risk group had higher immune indicators, indicating higher immunity. The genomic mutation patterns of the high-risk and low-risk groups were significantly different, especially the KRAS mutation frequency was significantly higher in the high-risk group. Drug sensitivity analysis showed that the low-risk group was more sensitive to Erlotinib, Savolitinib and VE _ 822, which may be used as a potential drug for COAD treatment. Finally, the nomogram constructed by pathological features combined with RiskScore can accurately evaluate the prognosis of COAD patients. This study constructed and verified a TLS model that can predict COAD. More importantly, it provides a reference standard for guiding the prognosis and immunotherapy of COAD patients.

Identification of a copper-responsive small molecule inhibitor of uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are a major global health problem and are caused predominantly by uropathogenic Escherichia coli (UPEC). UTIs are a leading cause of prescription antimicrobial use. Incessant increase in antimicrobial resistance in UPEC and other uropathogens poses a serious threat to the current treatment practices. Copper is an effector of nutritional immunity that impedes the growth of pathogens during infection. We hypothesized that copper would augment the toxicity of select small molecules against bacterial pathogens. We conducted a small molecule screening campaign with a library of 51,098 molecules to detect hits that inhibit a UPEC ΔtolC mutant in a copper-dependent manner. A molecule, denoted as E. coli inhibitor or ECIN, was identified as a copper-responsive inhibitor of wild-type UPEC strains. Our gene expression and metal content analysis results demonstrate that ECIN works in concert with copper to exacerbate Cu toxicity in UPEC. ECIN has a broad spectrum of activity against pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. Subinhibitory levels of ECIN eliminate UPEC biofilm formation. Transcriptome analysis of UPEC treated with ECIN reveals induction of multiple stress response systems. Furthermore, we demonstrate that L-cysteine rescues the growth of UPEC exposed to ECIN. In summary, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC.IMPORTANCEUrinary tract infection (UTI) is a ubiquitous infectious condition affecting millions of people annually. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. However, UTIs are becoming increasingly difficult to resolve with antimicrobials due to increased antimicrobial resistance in UPEC and other uropathogens. Here, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. In addition to E. coli, this small molecule also inhibits pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus.

7,8-Dihydroxyflavone is a direct inhibitor of human and murine pyridoxal phosphatase

Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

7,8-Dihydroxyflavone is a direct inhibitor of human and murine pyridoxal phosphatase.

Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5'-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

Protein translation rate determines neocortical neuron fate

The mammalian neocortex comprises an enormous diversity regarding cell types, morphology, and connectivity. In this work, we discover a post-transcriptional mechanism of gene expression regulation, protein translation, as a determinant of cortical neuron identity. We find specific upregulation of protein synthesis in the progenitors of later-born neurons and show that translation rates and concomitantly protein half-lives are inherent features of cortical neuron subtypes. In a small molecule screening, we identify Ire1α as a regulator of Satb2 expression and neuronal polarity. In the developing brain, Ire1α regulates global translation rates, coordinates ribosome traffic, and the expression of eIF4A1. Furthermore, we demonstrate that the Satb2 mRNA translation requires eIF4A1 helicase activity towards its 5’-untranslated region. Altogether, we show that cortical neuron diversity is generated by mechanisms operating beyond gene transcription, with Ire1α-safeguarded proteostasis serving as an essential regulator of brain development.

Screening of Functional Small Molecules via Modified Machine Learning Strategy toward Efficient All-Inorganic Perovskite Solar Cells.

Organic small molecules are proven to be capable of passivating the bulk/interfacial defects in inorganic perovskite solar cells. Considering the burdensome situation to screen the functional small molecules, we employ a modified machine learning (ML) strategy to guide screening suitable small molecules toward efficient solar cells through three modified ML algorithms to construct the prediction model: (i) random forest algorithm (RF), (ii) support vector machine algorithm (SVR), and (iii) XGBoost. Among them, the XGBoost algorithm displays a better overall predictive performance, whereby the R2 index reaches 0.939. Accordingly, eight small molecules are selected to modify the interface of perovskite films, and both the theoretical and experimental results certify that the difluorobenzylamine with additional fluorine atoms has a better interface modification effect among the small molecules containing functional groups, e.g., the benzene ring and amino group. The high accuracy of the modified machine learning model enables us to simplify the small-molecule screening process and form an important step for ongoing developments in perovskite solar cells and other optoelectronic devices.