Small Molecule Targeting Research Articles

Inhibitory effect of small-molecule compound AM679 targeting elongation-factor binding protein 2 on hepatitis B virus in vitro

Objective: To explore the antiviral activity of the small-molecule compound AM679 in hepatitis B virus (HBV) replication and infection cell models. Methods: The positive regulatory effect of AM679 on EFTUD2 expression was validated by qPCR and Western blotting. HepAD38 and HepG2-NTCP cells were treated with AM679 (0.5, 1, and 2 nmol/L). Negative control, positive control, and AM679 combined with the entecavir group were set up. HBV DNA intra-and extracellularly, as well as the expression levels of intracellular HBV total RNAs and 3.5kb-RNA changes, were detected with qPCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured in the cell supernatant by an enzyme-linked immunosorbent assay (ELISA). The t-test method was used for the statistical analysis of the mean difference between groups. Results: EFTUD2 mRNA and protein expression levels were significantly increased in HepAD38 and HepG2-NTCP cells following AM679 treatment, with a statistically significant difference (P < 0.001). Intra-and extracellular indicators such as HBV DNA, HBV RNAs, HBV 3.5kb-RNA, HBsAg, and HBeAg were decreased to varying degrees in both cell models, and the decrease in these indicators was more pronounced with the increase in AM679 concentration and prolonged treatment duration, while the combined use of AM679 and entecavir had a more significant antiviral effect. The HBV DNA inhibition rates in the supernatant of HepAD38 cells with the use of 2 nmol/L AM679 were 21% and 48% on days three and nine, respectively. The AM679 combined with the ETV treatment group had the most significant inhibitory effect (62%), with a P < 0.01. More active HBV replication was observed after silencing EFTUD2, while the antiviral activity of AM679 was significantly weakened. Conclusion: AM679 exerts anti-HBV activity in vitro by targeting the regulation of EFTUD2 expression.

Carbon fiber-sampling combined flame ionization mass spectrometry for direct analysis of drugs in oral fluid

Drug-impaired driving poses a significant risk of collisions and other hazardous accidents, emphasizing the urgent need for simple and rapid roadside detection methods. Oral fluid, as an easily collectible and non-invasive test material, has gained widespread use in detecting drug-impaired driving. In this study, we have devised a method for direct sampling using a carbon fiber bundle combined with flame ionization mass spectrometry. The essence of this method lies in the synergy between the adsorption properties of carbon fiber and the plasma characteristics of the flame. Leveraging the strong adsorption capabilities of the carbon fiber bundle allows for the use of a minimal sample size (<100 μL) during sampling, presenting a distinct advantage in the roadside inspection and sampling process. Throughout the flame ionization process, proteins and salts within the oral fluid matrix adhere well to the carbon fiber bundle, while small molecule targets can be efficiently desorbed and react with charged species in the flame, leading to ionization. The results demonstrate the successful development of carbon fiber-sampling combined flame ionization mass spectrometry, capable of qualitative and quantitative analysis of drugs in oral fluid without the need for sample pre-treatment. Its quantitative capabilities are sufficient for real sample detection, providing an effective analytical method for the roadside detection of drugs in oral fluids.

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy.

Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 μM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

Single-Round Circular Aptamer Discovery Using Bioinspired Magnetosome-Like Magnetic Chain Cross-Linked Graphene Oxide.

Circular aptamers are promising candidates for analytical and therapeutic applications due to their enhanced biological and structural stability. However, the process of circular aptamer selection remains a great challenge, as it requires multiple rounds of binding-separation-amplification that involves issues with nonspecific binding and amplification bias. Here, we develop a highly practical solution for reliable selection of circular aptamers in a single round based on magnetosome-like magnetic chain cross-linked graphene oxide (separation efficiency ≈ 105). High-affinity aptamer candidates can be rapidly selected from a preenriched circular DNA library, while low-affinity candidates are effectively adsorbed and separated by magnetosome-like magnetic chain cross-linked graphene oxide. With lipopolysaccharide as a representative model, the single-round selected lipopolysaccharide circular aptamer has been identified to have a high binding affinity with a Kd value of low to nanomolar range. Using this method, circular aptamers for protein and small-molecule targets were also successfully generated. We envision that this approach will accelerate the discovery of various new circular aptamers and open up a new avenue for analytical and therapeutic studies.

Small-molecule α-lipoic acid targets ELK1 to balance human neutrophil and erythrocyte differentiation

BackgroundErythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation.MethodsIn vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371– HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371– HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371– HPCs were performed to assay the signals changed by ELK1.ResultsHere, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371– hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371– HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment.ConclusionsALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy.

Binding Dynamics of a Stapled Peptide Targeting the Transcription Factor NF-Y.

Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein-protein interaction networks and the frequent lack of well-defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide-based scaffolds have proven useful, in particular with an α-helical active conformation. Peptide-based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α-helical peptides interact with the transcription factor Nuclear Factor-Y (NF-Y). We identified a 13-mer minimal binding core region, for which two crystal structures with an altered C-terminal peptide conformation when bound to NF-Y were obtained. Subsequent molecular dynamics simulations confirmed that the C-terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF-Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.

The Progress of Small Molecule Targeting BCR-ABL in the Treatment of Chronic Myeloid Leukemia.

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease. According to the American Cancer Society's 2021 cancer data report, new cases of CML account for about 15% of all leukemias. CML is generally divided into three stages: chronic phase, accelerated phase, and blast phase. Nearly 90% of patients are diagnosed as a chronic phase. Allogeneic stem cell transplantation and chemotherapeutic drugs, such as interferon IFN-α were used as the earliest treatments for CML. However, they could generate obvious side effects, and scientists had to seek new treatments for CML. A new era of targeted therapy for CML began with the introduction of imatinib, the first-generation BCR-ABL kinase inhibitor. However, the ensuing drug resistance and mutant strains led by T315I limited the further use of imatinib. With the continuous advancement of research, tyrosine kinase inhibitors (TKI) and BCR-ABL protein degraders with novel structures and therapeutic mechanisms have been discovered. From biological macromolecules to classical target protein inhibitors, a growing number of compounds are being developed to treat chronic myelogenous leukemia. In this review, we focus on summarizing the current situation of a series of candidate small-molecule drugs in CML therapy, including TKIs and BCR-ABL protein degrader. The examples provided herein describe the pharmacology activity of small-molecule drugs. These drugs will provide new enlightenment for future treatment directions.

Abstract 4640: Pan-cancer analysis of the influence of ERBB2 alteration on HER2 expression

Abstract Introduction: HER2 (ERBB2) is a target for various anti-cancer therapies, including large (Trastuzumab deruxtecan) or small molecules (Neratinib). Quantitative assessment of HER2 expression at the cellular level may improve therapeutic efficacy prediction. While ERBB2 mutations are known oncogenic drivers, their impact on tumor cell HER2 expression is unclear. In this study, we focused on common ERBB2 mutations, and analyzed HER2 expression via AI-powered image analysis in pan-cancer tumor specimens to identify possible mutation-expression correlations. Methods: We analyzed 183,292 pan-cancer samples from AACR GENIE, and 10,967 from TCGA. A set of 156 cases with both ERBB2 mutation status and HER2 4B5 IHC WSIs was obtained (Neogenomics). An AI-powered HER2 analyzer (Lunit SCOPE HER2) was previously developed using 1,133 HER2 IHC-stained WSIs of breast cancer stained with 4B5. Single-cell HER2 intensity was analyzed and aggregated, reported based on breast ASCO/CAP criteria. ERBB2 RNA levels were analyzed for mutations and amplification. Results: We identified 2,735 cases with ERBB2 mutations (2,603/183,292 [1.4%] in GENIE and 132/10,967 [1.2%] in TCGA). These mutations were categorized for analysis based on their highest frequency and previous characterization: exon 20 insertions (ex20ins, including Y772dup (YVMA) and G776InsVC), S310x, and other pathogenic mutations. In the dataset, ex20ins cases were predominantly non-small cell lung cancer (NSCLC, 381/482 [79.0%]) and S310x cases were enriched in urothelial, NSCLC, and breast cancers (269/683 [39.4%], 64/683 [9.4%], and 47/683 [6.9%], respectively). Of the 156 ERBB2-mutated cases with HER2 IHC images, 59 had ex20ins, 24 had S310x, and 73 had other mutations. AI analysis in each case type indicates a significantly higher proportion of HER2 3+ tumor cells in S310x and ex20ins cases than other mutations (median, 7.5% vs. 6.1% vs. 2.8%, p = 0.003). As such, high HER2 expression was observed in S310x and ex20ins cases compared to other mutation cases (HER2 3+ 10/24 [41.7%], 22/59 [37.3%], and 14/73 [19.2%]). Additionally, in NSCLC, ex20ins and S310x cases exhibited a modest enrichment for high HER2 expression relative to other mutation cases (21/56 [37.5%], 3/9 [33.3%], and 5/20 [27.6%]). RNA analyses from TCGA showed higher levels for ex20ins (median 13.6; interquartile range 13.1-13.7 in log2 scale), compared to S310x (12.4; 11.9-13.6) and other mutations (12.6; 12.1-13.3), but lower than that of amplified ERBB2 (15.5; 13.8-17.0). Conclusion: An AI-powered pan-cancer image analysis of HER2 IHC of tumor cells in conjunction with genomic data reveals a positive correlation between ex20ins and S310x ERBB2 mutation and protein expression. This correlation is also seen at the RNA level, but the lesser levels relative to ERBB2 amplified cases suggests the effect may be mediated at the protein level. Citation Format: Taebum Lee, Sangwon Shin, Heon Song, Seonwook Park, Sergio Pereira, Donggeun Yoo, Juneyoung Ro, Jimin Moon, Soo Ick Cho, Changho Ahn, Chan-Young Ock, Siraj M. Ali. Pan-cancer analysis of the influence of ERBB2 alteration on HER2 expression [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 4640.

Abstract 7145: Identification of Debio 0432 as a potent and selective USP1 inhibitor for cancer therapy

Abstract Introduction: Debio 0432 is a Ubiquitin Specific Protease 1 (USP1) inhibitor at preclinical stage. This study presents the first data on this small molecule and its activity as an anti-cancer therapy in multiple preclinical models. USP1 is a deubiquitinase, member of the deubiquitinating enzyme (DUB) family that plays an important role at the replication fork during DNA replication. Its prominent role in DNA repair, targeting Proliferating Cell Nuclear Antigen (PCNA) that regulates polymerase recruitment in the translesion synthesis (TLS), makes it an emergent target in the DNA damage repair (DDR) field. Methods: In silico docking has been used to identify a likely binding site of Debio 0432. Biochemical and cellular assays have been performed to show its activity on the target and in cell lines of different cancer types. In vivo models in cell line derived xenograft (CDX) and patient derived xenograft (PDX) were then used to confirm the activity of the compound and to study PK/PD relationship. Results: Debio 0432 likely binds USP1 in an allosteric pocket and is very selective across the 58 members of the USP family. Furthermore, it does not affect any kinase. Its biochemical activity on USP1 is in the low nanomolar range and cellular activity ranges between from single digit nM to above 1uM. In vivo, Debio 0432 shows antitumor activity in the BRCA mutant breast cancer model MDA-MB-436. The downstream target Ub-PCNA, selected as pharmacodynamic marker, is modulated in a dose and plasma exposure-dependent manner. Further in vivo experiments in PDX models showed dose-dependent activity in different cancer types, in both BRCA wild type and mutant models. Treatments in all in vivo studies were well tolerated. Conclusion: Overall, we show that Debio 0432 is a selective and potent small molecule targeting USP1, an emerging target in the DDR field. It shows good activity in different preclinical models and is well tolerated at active doses. Debio 0432 is currently undergoing IND-enabling studies and in preparation for entry into clinical development. Citation Format: Noemie Luong, Annett Kunze, Nicolas Quesnot, Erin R. Aho, Loren Berry, Scott Boiko, Alex J. Buckmelter, Sebastien Lofek, Selena Vigano, Christophe Chardonnens. Identification of Debio 0432 as a potent and selective USP1 inhibitor for cancer 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 7145.

Abstract 2091: Pharmacological inhibition of chaperone-mediated autophagy via NCoR1/RARα targeting is effective against non-small cell lung cancer

Abstract Dysregulation of autophagy, the lysosomal degradation of intracellular components, has been universally described in cancer. While activity of macroautophagy changes in a context-dependent manner in cancers, the upregulation of chaperone-mediated autophagy (CMA) is well-described in all solid tumors studied to date. Upregulated CMA directly contributes to both tumor growth and protection against anti-oncogenic interventions in cancers. Selective degradation of anti-oncogenic and pro-apoptotic proteins, such as mutant p53 and PUMA, misfolded proteins, and glycolytic enzymes by CMA allows for increased survival, reduced cell death, increased stress resistance, and enhancement of the Warburg effect in cancer cells, respectively. As such, inhibition of CMA is an attractive target for anti-cancer therapeutics, but the field currently lacks protein targets specific to CMA and selective CMA inhibitors. We have previously demonstrated the regulatory effect of retinoic acid receptor alpha (RARα) and its coregulator, nuclear receptor corepressor 1 (NCoR1), on CMA through transcriptional upregulation of the CMA effector proteins (Bourdenx et al. Cell 2021, Gomez-Sintes et al, Nat. Commun. 2022). Here, we identified a high NCoR1/RARα expression ratio in human non-small cell lung cancer (NSCLC) tumors compared to healthy lung tissue. We further illustrated a dependency on NCoR1 expression and its interaction with RARα to maintain high CMA activity in NSCLC cell lines. Provided the reliance on NCoR1 repression of RARα for high CMA activity in NSCLC, we hypothesized that small molecule targeting of the druggable NCoR1/RARα interaction to disrupt the interaction may result in suppression of CMA effector proteins and CMA inhibition. Through a series of in-silico screens for predicted NCoR1/RARα targeting, and functional screens for CMA inhibition, we identified CIM7, a first-in-class potent and selective CMA inhibitor. We validated CIM7 function through binding to RARα and subsequent disruption of NCoR1 binding, resulting in alterations to CMA-related gene expression. CIM7 inhibits CMA in vivo with no observed toxicity in normal tissues. We additionally demonstrated the therapeutic potential of CIM7 through its ability to reduce tumor growth in vivo in mouse NSCLC xenografts. Our findings support the NCoR1/RARα interaction as a major regulator of CMA in NSCLC and demonstrate the potential of pharmacologic CMA inhibition as an anti-cancer therapeutic strategy. Citation Format: Mericka McCabe, Rabia R. Khawaja, Rajanya Bhattacharyya, Antonio Diaz, Thomas P. Garner, Kristen Lindenau, Rebecca Sereda, Olaya Santiago-Fernández, Ana Maria Cuervo, Evripidis Gavathiotis. Pharmacological inhibition of chaperone-mediated autophagy via NCoR1/RARα targeting is effective against non-small cell lung cancer [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 2091.

Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.

Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

Identification of expression profiles and prognostic value of RFCs in colorectal cancer

Colorectal cancer (CRC) ranks among the most prevalent cancers globally, with its incidence closely tied to DNA damage. The Replication Factor C (RFC) complexes comprises five protein subunits: RFC1, RFC2, RFC3, RFC4, and RFC5. These RFC complexes play crucial roles in DNA replication, repair pathways, activities post DNA damage, and ATP-dependent processes during DNA synthesis. However, the impact of RFC complexes proteins on CRC prognosis remains unclear. To explore this, we employed a computational analysis approach, utilizing platforms such as the DepMap portal, GEPIA, DAVID Bioinformatics for KEGG pathway analysis, Human Protein Atlas (HPA), STRING, and TIMER. Our results indicate that the mRNA levels of RFC1 and RFC5 were the least expressed among CRC cell lines compared to other RFC complex subunits. Notably, low RFC1 and RFC5 expression was correlated with poor prognosis in terms of CRC patients' overall survival (OS). Immunohistochemical results from the Human Protein Atlas demonstrated medium staining for RFC1, RFC2, and RFC5 in CRC tissues. Furthermore, the low expression of RFC1 and RFC5 showed a significant correlation with high expression levels of miR-26a-5p and miR-636, impacting cell proliferation through mismatch repair, DNA replication, and the nucleotide excision repair pathway. Although the precise functions of RFC1 in cancer are still unknown, our findings suggest that the small-molecule single target, CHEMBL430483, and multiple target molecules could be potential treatments for CRC. In conclusion, the elevated expression of miR-26a-5p and miR-636 targeting RFC1 and RFC5 expression holds promise as a potential biomarker for early-stage CRC detection. These insights provide novel directions and strategies for CRC therapies.

Discovering potential WRN inhibitors from natural product database through computational methods

Microsatellite instability (MSI) is a relatively common feature associated with multiple cancers, and Werner syndrome (WRN) ATP-dependent helicase has been recognized as a novel target for treating MSI cancers, such as colorectal cancer. A small-molecule inhibitor targeting WRN would be a promising strategy for treating colorectal cancer with high MSI expression. In this study, we employed a computer-assisted drug discovery strategy to screen over 30,000 natural product molecules. By using a combination of docking, ligand efficiency, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA), and thermodynamic integration (TI) calculations, we identified MOL008980, MOL010740, MOL011832, T4743, TN1166, and TNP-002173 as potential WRN inhibitors. Subsequent molecular dynamics simulation revealed that these screened natural products possessed better binding dynamic characteristics than ATP substrate and were capable of inhibiting the dynamic process of WRN, making them potential strong ATP competitive inhibitors. In conclusion, our computational approach revealed potential WRN inhibitors from a natural product database, providing a theoretical basis for future research.

A generalizable sensing platform based on molecularly imprinted polymer-aptamer double recognition and nanoenzyme assisted photoelectrochemical-colorimetric dual-mode detection

Developing highly sensitive and selective methods that incorporate specific recognition elements is crucial for detecting small molecules because of the limited availability of small molecule antibodies and the challenges in obtaining sensitive signals. In this study, a generalizable photoelectrochemical-colorimetric dual-mode sensing platform was constructed based on the synergistic effects of a molecularly imprinted polymer (MIP)-aptamer sandwich structure and nanoenzymes. The MIP functionalized peroxidase-like Fe3O4 (Fe3O4@MIPs) and alkaline phosphatase mimic Zr-MOF labeled aptamer (Zr-mof@Apt) were used as the recognition elements. By selectively accumulating dibutyl phthalate (DBP), a small molecule target model, on Fe3O4@MIPs, the formation of Zr-MOF@Apt-DBP- Fe3O4@MIPs sandwich structure was triggered. Fe3O4@MIPs oxidized TMB to form blue-colored oxTMB. However, upon selective accumulation of DBP, the catalytic activity of Fe3O4@MIPs was inhibited, resulting in a lighter color that was detectable by the colorimetric method. Additionally, Zr-mof@Apt effectively catalyzed the hydrolysis of L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AAPS), generating ascorbic acid (AA) that could neutralize the photogenerated holes to decrease the photocurrent signals for PEC sensing and reduce oxTMB for colorimetric testing. The dual-mode platform showed strong linearity for different concentrations of DBP from 1.0 pM to 10 μM (PEC) and 0.1 nM to 0.5 μM (colorimetry). The detection limits were 0.263 nM (PEC) and 30.1 nM (colorimetry) (S/N = 3), respectively. The integration of dual-signal measurement mode and sandwich recognition strategy provided a sensitive and accurate platform for the detection of small molecules.

A small-molecule TNIK inhibitor targets fibrosis in preclinical and clinical models.

Idiopathic pulmonary fibrosis (IPF) is an aggressive interstitial lung disease with a high mortality rate. Putative drug targets in IPF have failed to translate into effective therapies at the clinical level. We identify TRAF2- and NCK-interacting kinase (TNIK) as an anti-fibrotic target using a predictive artificial intelligence (AI) approach. Using AI-driven methodology, we generated INS018_055, a small-molecule TNIK inhibitor, which exhibits desirable drug-like properties and anti-fibrotic activity across different organs in vivo through oral, inhaled or topical administration. INS018_055 possesses anti-inflammatory effects in addition to its anti-fibrotic profile, validated in multiple in vivo studies. Its safety and tolerability as well as pharmacokinetics were validated in a randomized, double-blinded, placebo-controlled phase I clinical trial (NCT05154240) involving 78 healthy participants. A separate phase I trial in China, CTR20221542, also demonstrated comparable safety and pharmacokinetic profiles. This work was completed in roughly 18 months from target discovery to preclinical candidate nomination and demonstrates the capabilities of our generative AI-driven drug-discovery pipeline.

Generalized biomolecular modeling and design with RoseTTAFold All-Atom.

Deep-learning methods have revolutionized protein structure prediction and design but are presently limited to protein-only systems. We describe RoseTTAFold All-Atom (RFAA), which combines a residue-based representation of amino acids and DNA bases with an atomic representation of all other groups to model assemblies that contain proteins, nucleic acids, small molecules, metals, and covalent modifications, given their sequences and chemical structures. By fine-tuning on denoising tasks, we developed RFdiffusion All-Atom (RFdiffusionAA), which builds protein structures around small molecules. Starting from random distributions of amino acid residues surrounding target small molecules, we designed and experimentally validated, through crystallography and binding measurements, proteins that bind the cardiac disease therapeutic digoxigenin, the enzymatic cofactor heme, and the light-harvesting molecule bilin.

Constructing Innovative Covalent and Noncovalent Compound Libraries: Insights from 3D Protein-Ligand Interactions.

Noncovalent interactions between small-molecule drugs and protein targets assume a pivotal role in drug design. Moreover, the design of covalent inhibitors, forming covalent bonds with amino acid residues, requires rational reactivity for their covalent warheads, presenting a key challenge as well. Understanding the intricacies of these interactions provides a more comprehensive understanding of molecular binding mechanisms, thereby guiding the rational design of potent inhibitors. In this study, we adopted the fragment-based drug design approach, introducing a novel methodology to extract noncovalent and covalent fragments according to distinct three-dimensional (3D) interaction modes from noncovalent and covalent compound libraries. Additionally, we systematically replaced existing ligands with rational fragment substitutions, based on the spatial orientation of fragments in 3D space. Furthermore, we adopted a molecular generation approach to create innovative covalent inhibitors. This process resulted in the recombination of a noncovalent compound library and several covalent compound libraries, constructed by two commonly encountered covalent amino acids: cysteine and serine. We utilized noncovalent ligands in KLIFS and covalent ligands in CovBinderInPDB as examples to recombine noncovalent and covalent libraries. These recombined compound libraries cover a substantial portion of the chemical space present in the original compound libraries and exhibit superior performance in terms of molecular scaffold diversity compared to the original compound libraries and other 11 commercial libraries. We also recombined BTK-focused libraries, and 23 compounds within our libraries have been validated by former researchers to possess potential biological activity. The establishment of these compound libraries provides valuable resources for virtual screening of covalent and noncovalent drugs targeting similar molecular targets.

Fat mass and obesity-associated protein (FTO) affects midpalatal suture bone remodeling during rapid maxillary expansion.

The fat mass and obesity-associated protein (FTO) is an RNA demethylase that contributes to several physiological processes. Nonetheless, the impact of FTO on bone remodeling in the midpalatal suture while undergoing rapid maxillary expansion (RME) remains unclear. First, to explore the expression of FTO in the midpalatal suture during RME, six rats were randomly divided into two groups: Expansion group and Sham group (springs without being activated). Then, suture mesenchymal stem cells (SuSCs) were isolated as in vitro model. The expression of FTO was knocked down by small interfering RNA to study the effect of FTO on the osteogenic differentiation of SuSCs. Finally, to evaluate the function of FTO in the process of bone remodeling in the midpalatal suture, ten rats were randomly divided into two groups: FB23-2 group (10 μM, a small molecule inhibitor of FTO) and DMSO group (control). Increased arch width and higher expression of OCN and FTO in the midpalatal area were observed in expansion group (P < .05). In the in vitro model, the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b were decreased (P < .05) upon knocking down FTO. Additionally, the protein levels of RUNX2 and OPN were also decreased (P < 0.05). Adding FB23-2, a small-molecule inhibitor targeting FTO, to the medium of SuSCs caused a decrease in the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b (P < 0.05). There was a statistically significant difference in evaluating the expression of OCN and OPN on the palatal suture between the FB23-2 and DMSO groups (P < .05). The molecular mechanisms by which FTO regulates SuSCs osteogenesis remain to be elucidated. The FTO conditional knock out mouse model can be established to further elucidate the role of FTO during RME. FTO contributes to the osteogenic differentiation of SuSCs and plays a promoting role in midpalatal suture bone remodeling during the RME.

Small Molecule-Drug Conjugates Emerge as a New Promising Approach for Cancer Treatment.

Antibody drug conjugates (ADCs) have emerged as a new promising class of anti- cancer agents. However, limitations such as higher costs and unavoidable immunogenicity due to their relatively large structures cannot be ignored. Therefore, the development of lightweight drugs such as small molecule-drug conjugates (SMDCs) based on the ADC design idea has become a new option for targeted therapy. SMDCs are derived from the coupling of small-molecule targeting ligands with cytotoxic drugs. They are composed of three parts: small-molecule targeting ligands, cytotoxic molecules, and linkers. Compared with ADCs, SMDCs can be more rapidly and evenly dispersed into tumor tissues, with low cost and no immunogenicity. In this article, we will give a comprehensive review of different types of SMDCs currently under clinical trials to provide ideas and inspirations for the development of clinically applicable SMDCs.

Targeting the PDK/PDH axis to reverse metabolic abnormalities by structure-based virtual screening with in vitro and in vivo experiments

In humans and animals, the pyruvate dehydrogenase kinase (PDK) family proteins (PDKs 1–4) are excessively activated in metabolic disorders such as obesity, diabetes, and cancer, inhibiting the activity of pyruvate dehydrogenase (PDH) which plays a crucial role in energy and fatty acid metabolism and impairing its function. Intervention and regulation of PDH activity have become important research approaches for the treatment of various metabolic disorders.In this study, a small molecule (g25) targeting PDKs and activating PDH, was identified through multi-level computational screening methods. In vivo and in vitro experiments have shown that g25 activated the activity of PDH and reduced plasma lactate and triglyceride level. Besides, g25 significantly decreased hepatic fat deposition in a diet-induced obesity mouse model. Furthermore, g25 enhanced the tumor-inhibiting activity of cisplatin when used in combination. Molecular dynamics simulations and in vitro kinase assay also revealed the specificity of g25 towards PDK2.Overall, these findings emphasize the importance of targeting the PDK/PDH axis to regulate PDH enzyme activity in the treatment of metabolic disorders, providing directions for future related research. This study provides a possible lead compound for the PDK/PDH axis related diseases and offers insights into the regulatory mechanisms of this pathway in diseases.