Target-specific Inhibitors Research Articles

Plasmodium falciparum Mitochondrial Complex III, the Target of Atovaquone, Is Essential for Progression to the Transmissible Sexual Stages.

The Plasmodium falciparum mitochondrial electron transport chain (mETC) is responsible for essential metabolic pathways such as de novo pyrimidine synthesis and ATP synthesis. The mETC complex III (cytochrome bc1 complex) is responsible for transferring electrons from ubiquinol to cytochrome c and generating a proton gradient across the inner mitochondrial membrane, which is necessary for the function of ATP synthase. Recent studies have revealed that the composition of Plasmodium falciparum complex III (PfCIII) is divergent from humans, highlighting its suitability as a target for specific inhibition. Indeed, PfCIII is the target of the clinically used anti-malarial atovaquone and of several inhibitors undergoing pre-clinical trials, yet its role in parasite biology has not been thoroughly studied. We provide evidence that the universally conserved subunit, PfRieske, and the new parasite subunit, PfC3AP2, are part of PfCIII, with the latter providing support for the prediction of its divergent composition. Using inducible depletion, we show that PfRieske, and therefore, PfCIII as a whole, is essential for asexual blood stage parasite survival, in line with previous observations. We further found that depletion of PfRieske results in gametocyte maturation defects. These phenotypes are linked to defects in mitochondrial functions upon PfRieske depletion, including increased sensitivity to mETC inhibitors in asexual stages and decreased cristae abundance alongside abnormal mitochondrial morphology in gametocytes. This is the first study that explores the direct role of the PfCIII in gametogenesis via genetic disruption, paving the way for a better understanding of the role of mETC in the complex life cycle of these important parasites and providing further support for the focus of antimalarial drug development on this pathway.

BRG1 promotes progression of B-cell acute lymphoblastic leukemia by disrupting PPP2R1A transcription

Despite advancements in chemotherapy and the availability of novel therapies, the outcome of adult patients with B-cell acute lymphoblastic leukemia (B-ALL) remains unsatisfactory. Therefore, it is necessary to understand the molecular mechanisms underlying the progression of B-ALL. Brahma-related gene 1 (BRG1) is a poor prognostic factor for multiple cancers. Here, the expression of BRG1 was found to be higher in patients with B-ALL, irrespective of the molecular subtype, than in healthy individuals, and its overexpression was associated with a poor prognosis. Upregulation of BRG1 accelerated cell cycle progression into the S phase, resulting in increased cell proliferation, whereas its downregulation facilitated the apoptosis of B-ALL cells. Mechanistically, BRG1 occupies the transcriptional activation site of PPP2R1A, thereby inhibiting its expression and activating the PI3K/AKT signaling pathway to regulate the proto-oncogenes c-Myc and BCL-2. Consistently, silencing of BRG1 and administration of PFI-3 (a specific inhibitor targeting BRG1) significantly inhibited the progression of leukemia and effectively prolonged survival in cell-derived xenograft mouse models of B-ALL. Altogether, this study demonstrates that BRG1-induced overactivation of the PPP2R1A/PI3K/AKT signaling pathway plays an important role in promoting the progression of B-ALL. Therefore, targeting BRG1 represents a promising strategy for the treatment of B-ALL in adults.

The Leukemic Isocitrate Dehydrogenase (IDH) 1/2 Mutations Impair Myeloid and Erythroid Cell Differentiation of Primary Human Hematopoietic Stem and Progenitor Cells (HSPCs).

How hematopoietic stem and progenitor cell (HSPC) fate decisions are affected by genetic alterations acquired during AML leukemogenesis is poorly understood and mainly explored in animal models. Here, we study isocitrate dehydrogenase (IDH) gene mutations in the human model of HSPC and discuss the available literature on this topic. IDH1/2 mutations occur in ~20% of AML cases, are recognized among the mutations earliest acquired during leukemogenesis, and are targets of specific inhibitors (ivosidenib and enasidenib, respectively). In order to investigate the direct effects of these mutations on HSPCs, we expressed IDH1-R132H or IDH2-R140Q mutants into human CD34+ healthy donor cells via lentiviral transduction and analyzed the colony-forming unit (CFU) ability. CFU ability was dramatically compromised with a complete trilineage block of differentiation. Strikingly, the block was reversed by specific inhibitors, confirming that it was a specific effect induced by the mutants. In line with this observation, the CD34+ leukemic precursors isolated from a patient with IDH2-mutated AML at baseline and during enasidenib treatment showed progressive and marked improvements in their fitness over time, in terms of CFU ability and propensity to differentiate. They attained clonal trilinear reconstitution of hematopoiesis and complete hematological remission.

In silico design of novel multitarget small molecule inhibitors to treat PTEN-mutant endometrial carcinoma.

e15119 Background: Previously we described triple CDK4/6-CDK9 inhibitor, LCI133 for the treatment of endometrial carcinoma (EC). We used in silico modeling to optimize LCI133’s physicochemical properties to increase PI3K p110α potency/ADME to better target epithelial-derived cancers, resulting in nanomolar potent LCI139. We characterized LCI139’s in vitro potency, target specificity, activity in in vitro EC models, safety in normal cell lines. Methods: LCI139 + analogs were designed using computational chemistry based on X-ray crystal structures of each target protein + validated chemistry using nuclear magnetic resonance + high-resolution mass spectrometry. Specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay + selectivity profile. LCI139 (0.001-10μM) was screened against EC cell lines AN3CA, RL95-2, Ishikawa, KLE, HEC-1-A and -B for 48 hours; IC50 values determined by CellTiter-Glo Assay. EC cells were treated with LCI139 or single agent control for 24 hours + stained with Annexin V/7-AAD to assess apoptosis. Representative PTEN wildtype (HEC-1A) + mutant (AN3CA) EC cell lines were chosen for mechanistic studies + treated 6 to 24 hours with multitarget or single agent inhibitor. Cells were stained with Vybrant DyeCycle Violet to determine cell cycle arrest by cytometer. Cellular lysates were prepared for Western blot (WB). Results: LCI139’s has a molecular weight of 502.57Da + is a highly potent PI3Kα-CDK4/6-CDK9 inhibitor by cell-free kinase assays: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). KINOMEscan revealed a favorable S(35) selectivity score of 0.221 screened against 403 non-mutant kinases. PTEN mutant AN3CA (IC50 216.0nM), RL95-2 (IC50 22.8nM), Ishikawa (IC50 2.5nM) had nanomolar LCI139 IC50 values. PTEN-wild type cell lines were insensitive up to 10μM; confirmed by dose-dependent apoptosis + colony-forming assays. WB revealed LCI139 treatment ablated MCL-1 expression, decreased phosphorylation of AKT, Rb, Rpb1 (Ser2), induced significant amounts of cPARP in PTEN mutant AN3CA. In PTEN-wildtype HEC1A WB, dose-dependent expression of pAKT in PTEN siRNA was noted. LCI139 generated heterogenous cell line specific effects on cell cycle progression + arrest. LCI139 displayed favorable toxicity against normal human kidney cells (HEK-293) compared to single agent controls. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor, LCI139, is nanomolar potent against PTEN mutant EC cell lines + displays decreased toxicity compared with CDK9 inhibitor reference compounds, AZD4573 + flavopiridol. Our results merit mechanistic dissection of PTEN interaction with PI3K + CDK9 signaling pathways, cell cycle machinery, CDK9-mediated transcriptional control. These data support extensive in vivo potency, toxicity, PK/PD studies to assess LCI139’s clinical utility in treating EC.

Abstract 1816: In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma

Abstract Background: Previously we described multitarget CDK4/6-CDK9 inhibitor, LCI133 for the treatment of endometrial carcinoma (EC). We have optimized LCI133’s chemical properties to increase PI3K p110α potency to better target epithelial-derived cancers, resulting in nanomolar potent LCI139. We characterized LCI139’s in vitro potency, target specificity, activity in in vitro EC models, safety against normal cell lines. Methods: LCI139’s chemical structure was determined using nuclear magnetic resonance and high-resolution mass spectrometry. Specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay and selectivity profile. LCI139 (0.001-10μM) was screened against EC cell lines AN3CA, RL95-2, Ishikawa, KLE, HEC-1-A and -B for 48 hours; IC50 values determined by CellTiter-Glo Assay. EC cells were treated with LCI139 or single agent control for 24 hours and stained with Annexin V/7-AAD to assess apoptosis by cytometer. Representative PTEN wildtype (HEC-1) and mutant (AN3CA) EC cell lines were chosen for mechanistic studies and treated 6 to 24 hours with multitarget or single agent inhibitor. RNA was isolated to determine gene expression by qRT-PCR, cells were stained with Vybrant DyeCycle Violet to determine cell cycle arrest by cytometer, or cellular lysates were prepared for Western blot (WB). Results: LCI139’s has a molecular weight of 502.57Da and is a highly potent PI3Kα-CDK4/6-CDK9 inhibitor by cell-free kinase assays: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). KINOMEscan revealed a favorable S(35) selectivity score of 0.221 screened against 403 non-mutant kinases. PTEN mutant AN3CA (IC50 243.6nM), RL95-2 (IC50 22.8nM), Ishikawa (IC50 9.8nM) had nanomolar LCI139 IC50 values, while PTEN-wild type cell lines were insensitive up to 10μM; confirmed by dose-dependent apoptosis and colony-forming assays. WB revealed LCI139 treatment ablated MCL-1 expression, decreased phosphorylation of AKT, Rb, Rpb1 (Ser2), induced significant amounts of cPARP. MCL-1 ablation correlated to decreased Mcl1 expression (p<0.0010) assessed 6 hours post treatment. LCI139 generated heterogenous cell line specific effects on cell cycle progression and arrest. LCI139 displayed a favorable toxicity profile against normal human kidney cells (HEK-293) compared to single agent controls. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor, LCI139, is nanomolar potent against PTEN mutant EC cell lines and displays decreased toxicity compared with CDK9 inhibitor reference compounds, AZD4573 and flavopiridol. Our results merit further mechanistic dissection of PTEN interaction with PI3K pathway signaling, cell cycle machinery, CDK9-mediated transcriptional control. These data support extensive in vivo potency, toxicity, PK/PD studies to assess LCI139’s clinical utility in treating EC. Citation Format: Vaidehi Mujumdar, Ritchie Delara, Hailey Dryden, Dhananjaya Pal, Krishnaiah Maddeboina, Page Mangum Arditti, Bharath Yada, Robert Wendel Naumann, Yovanni Casablanca, Erin Crane, Jubilee Brown, Donald Durden, Cody McHale. In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma [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 1816.

Abstract 1976: Targeting metabolism in non-small cell lung cancer (NSCLC) with the novel multitarget small molecule PI3K-CDK4/6-CDK9 inhibitor LCI139

Abstract Introduction: NSCLC is a heterogenous disease estimated to cause the majority of all 2023 United States cancer-related deaths. There is no broadly applicable targeted therapy to treat lung cancer and those FDA-approved drugs target patient-specific biomarker mutations such as EGFR, ALK, ROS1, etc. Targeting multiple oncogenic pathways to induce synthetic lethality is an attractive strategy for anticancer therapeutic development. Here, we describe the effects of novel multitarget small molecule PI3K-CDK4/6-CDK9 inhibitor LCI139 on a panel of human NSCLC cells of mixed histologic and mutational backgrounds. Methods: LCI139 specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay and KINOMEscan. LCI139 (0.001-10μM) was screened against NCI-H1975, NCI-H1703, NCI-H1373, NCI-H1781 and NCI-H226 human cell lines; IC50 values determined by CellTiter-Glo/WST-8 Assay and apoptotic rate by flow cytometry (Annexin V/7-AAD). Metabolomic profile of representative LCI139 sensitive (LCI139S) and resistant (LCI139R) NSCLC cells was evaluated by western blot. Results: LCI139 is a highly potent inhibitor of PI3K-CDK4/6-CDK9: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). NCI-H1703, NCI-H226, NCI-H1373 and NCI-H1975 were highly sensitive to LCI139 (0.68, 3.2, 35.27 and 342.4nM IC50), while NCI-H1781 remained insensitive up to 10μM. Confirming those findings, LCI139 promoted robust apoptotic cell death in NCI-H1703, NCI-H226, NCI-H1373 and NCI-H1975 but not NCI-H1781. LCI139S H1703 cells displayed increased bioenergetic reliance on oxidative phosphorylation (OXPHOS), compared with the LCI139R H1781 cells. In the LCI139S H1703 cells, LCI139 treatment decreased expression of all mitochondrial complexes (I-IV) and glycolytic enzymes (HK2, GAPDH, LDHA). Conversely, LCI139 treatment of LCI139R H1781 cells only inhibited expression of mitochondrial complexes II and III, but increased glucose uptake (Glut1) and retained expression of other glycolytic enzymes. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor LCI139 is nanomolar potent against several NSCLC cells lines of mixed histologic and mutational backgrounds. NSCLC cells that are most reliant on OXPHOS to generate ATP, a classic metabolic adaptation to chemotherapy, appear to be most sensitive to LCI139. Taken together, these data support further drug characterization and mechanistic dissection to assess LCI139’s potential clinical utility in treating advanced stage and recurrent NSCLC. Citation Format: Cody C. McHale, Anna V. Ivanina Foureau, Hailey L. Dryden, Krishnaiah Maddeboina, Dhananjaya Pal, Bharath Yada, Page Mangum Arditti, Fei Guo, Kathryn F. Mileham, David M. Foureau, Donald L. Durden. Targeting metabolism in non-small cell lung cancer (NSCLC) with the novel multitarget small molecule PI3K-CDK4/6-CDK9 inhibitor LCI139 [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 1976.

Discovery of novel polyheterocyclic neuraminidase inhibitors with 1,3,4-oxadiazole thioetheramide as core backbone

Inspired by our earlier findings regarding neuraminidase (NA) inhibitors interacting with 150-cavity or 430-cavity of NA, sixteen novel polyheterocyclic NA inhibitors with 1,3,4-oxadiazole thioetheramide as core backbone were designed and synthesized based on the lead compound ZINC13401480. Of the synthesized compounds, compound N5 targeting 150-cavity exerts the best inhibitory activity against the wild-type H5N1 NA, with IC50 value of 0.14 μM, which is superior to oseltamivir carboxylate (OSC) (IC50 = 0.31 μM). Compound N10 targeting 430-cavity exhibits the best activity against the H5N1–H274Y mutant NA. Although the activity of N10 is comparable to that of OSC for wild-type H5N1 inhibition, it is approximately 60-fold more potent than OSC against the H274Y mutant, suggesting that it is not easy for the virus to develop drug resistance and is attractive for drug development. N10 (EC50 = 0.11 μM) also exhibits excellent antiviral activity against H5N1, which is superior to the positive control OSC (EC50 = 1.47 μM). Molecular docking study shows that the occupation of aromatic fused rings and oxadiazole moiety at the active site and the extension of the substituted phenyl to the 150-cavity or 430-cavity make great contributions to the good potency of this series of polyheterocyclic NA inhibitors. Some advancements in the discovery of effective target-specific NA inhibitors in this study may offer some assistance in the development of more potent anti-influenza drugs.

In silico De Novo Discovery of Novel Target-specific Drug-like Bidentate Inhibitors of PTP 1B

Background: Protein tyrosine phosphatase 1B (PTP 1B) is a recognized legitimate target for type 2 diabetes and obesity, collectively designated as ‘diabesity’, even though first-in-class inhibitor is still awaited. The main cause behind the unachieved target selectivity of investigated inhibitors is the high degree of sharing of structural homology between PTP 1B and other members of the PTP family. Objective: The present work aimed to discover target-specific inhibitors of PTP 1B with bidentate binding features on both the allosteric and active sites. Materials and Methods: We have implicated the amalgamated de novo designing, ADMET screening, and molecular docking simulations to discover novel drug-like allosteric inhibitors of PTP 1B. The LEA3D de novo designing platform was used to design novel thiazolidinediones (TZDs) from scratch in the core of the target on the strict constraints of defined molecular properties of drug-likeness. Molecular modelling and geometry optimization were done using the ChemOffice package. The druglikeness/ ADMET screening was performed using the TSAR package based on Lipinski’s filter. Molegro Virtual Docker (MVD) was used for the prediction of binding cavities in the target, estimation of ligandtarget binding affinities as well as mode of binding interactions. Results and Discussion: Novel TZDs (Molecules 1-8) were de novo designed successfully as drug-like target-specific inhibitors of PTP 1B. The interaction pattern and the energy contribution of ligand (Etotal, Eintra, Epair) and target (Epair) supported that the generated TZDs showed bidentate inhibition. Conclusion: The discovered TZDs can be developed as novel target-specific allosteric inhibitors of PTP 1B after the accomplishment of synthetic and pre-clinical interventions.

Design of target specific peptide inhibitors using generative deep learning and molecular dynamics simulations

We introduce a computational approach for the design of target-specific peptides. Our method integrates a Gated Recurrent Unit-based Variational Autoencoder with Rosetta FlexPepDock for peptide sequence generation and binding affinity assessment. Subsequently, molecular dynamics simulations are employed to narrow down the selection of peptides for experimental assays. We apply this computational strategy to design peptide inhibitors that specifically target β-catenin and NF-κB essential modulator. Among the twelve β-catenin inhibitors, six exhibit improved binding affinity compared to the parent peptide. Notably, the best C-terminal peptide binds β-catenin with an IC50 of 0.010 ± 0.06 μM, which is 15-fold better than the parent peptide. For NF-κB essential modulator, two of the four tested peptides display substantially enhanced binding compared to the parent peptide. Collectively, this study underscores the successful integration of deep learning and structure-based modeling and simulation for target specific peptide design.

Abstract B006: High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC

Abstract KRASG12C inhibitors such as sotorasib and adagrasib have shown efficacy in patients with non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not respond. Therefore, there is a clear need for predictive biomarkers to guide treatment. The proximity ligation assay (PLA) is an immunofluorescent-based approach utilized to analyze endogenous protein-protein interactions. The method allows for the assessment of signaling-associated protein complexes, which can serve as potential targets for specific inhibitors. In particular, KRASG12C inhibitors can disrupt these complexes by interfering with the interaction between RAS and its effector RAF. By providing insights into the presence of these protein complexes, PLA offers a predictive readout for inhibitor sensitivity. We thus conducted a preclinical study to evaluate if a RAS-CRAF PLA could inform on the presence of RAS-RAF protein complexes and predict the response to KRASG12C inhibitors. To assess the specificity of the panRAS-CRAF PLA, we employed RNA interference in a KRASG12C-mutant H358 NSCLC cell line, wherein knockdown of either panRAS or CRAF resulted in a reduction in the panRAS-CRAF PLA signal. Pharmacological inhibition of RAS-RAF interaction with sotorasib also reduced the panRAS-CRAF PLA signal. We next performed panRAS-CRAF PLA in a panel of 11 KRASG12C-mutant NSCLC cell lines, revealing a higher number of panRAS-CRAF PLA spots in H358, LU65, and HCC1171. To further investigate the RAS-RAF interaction, we assessed the abundance of CRAF-bound RAS-GTP by quantifying the binding of RAS-GTP to the RAS-binding domain of CRAF. H358, LU65, and HCC1171 exhibited the highest levels of RAS-GTP among the 11 cell lines. We observed a strong correlation between the number of panRAS-CRAF PLA foci and the levels of CRAF-bound RAS-GTP (r = 0.87, P = 0.0002). These results indicated that the enhanced level of RAS-GTP for CRAF proteins facilitates the formation of RAS-CRAF complexes, thereby augmenting the panRAS-CRAF PLA signals. We then evaluated the in vitro antitumor activity of sotorasib across the G12C-mutated cell lines and found that the 50% inhibitory concentration values of sotorasib for H358, LU65, and HCC1171 were below 30 nmol/L. The panRAS-CRAF PLA was strongly associated with the sensitivity to sotorasib (r = 0.81, P = 0.002). We subsequently assessed the association of panRAS-CRAF PLA with efficacy of adagrasib in a patient-derived xenograft (PDX) cohort. PanRAS-CRAF PLA was assessed on sections of formalin-fixed paraffin-embedded tumors derived from vehicle-treated control groups at the end of efficacy studies in 18 NSCLC PDX models. Our findings revealed a significant association between high panRAS-CRAF PLA signal and increased sensitivity to adagrasib (r = 0.61, P = 0.008). Our results suggest that panRAS-CRAF PLA may serve as a predictive marker to identify patients who could derive optimal benefit from KRASG12C inhibitors, and may direct combination therapy approaches to those patients less likely to benefit from single-agent therapy. Citation Format: Ryoji Kato, Hitendra S. Solanki, Denis Imbody, Anurima Majumder, Yaakov Stern, Liznair Bridenstine, Harika Gundlapalli, Ida Aronchik, Joseph Johnson, Eric B. Haura. High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B006.

Molecular association of Candida albicans and vulvovaginal candidiasis: focusing on a solution.

Candida albicans-mediated vulvovaginal candidiasis (VVC) is a significant challenge in clinical settings, owing to the inefficacy of current antifungals in modulating virulence, development of resistance, and poor penetration into the biofilm matrix. Various predisposition factors are molecular drivers that lead to the dysbiosis of normal microflora of the vagina, upregulation of central metabolic pathways, morphogenesis, hyphal extension, adhesion, invasion, and biofilm formation leading to chronic infection and recurrence. Hence, it is crucial to understand the molecular mechanism behind the virulence pathways driven by those drivers to decode the drug targets. Finding innovative solutions targeting fungal virulence/biofilm may potentiate the antifungals at low concentrations without affecting the recurrence of resistance. With this background, the present review details the critical molecular drivers and associated network of virulence pathways, possible drug targets, target-specific inhibitors, and probable mode of drug delivery to cross the preclinical phase by appropriate in vivo models.

Unveiling Novel Avenues in mTOR-Targeted Therapeutics: Advancements in Glioblastoma Treatment.

The mTOR signaling pathway plays a pivotal and intricate role in the pathogenesis of glioblastoma, driving tumorigenesis and proliferation. Mutations or deletions in the PTEN gene constitutively activate the mTOR pathway by expressing growth factors EGF and PDGF, which activate their respective receptor pathways (e.g., EGFR and PDGFR). The convergence of signaling pathways, such as the PI3K-AKT pathway, intensifies the effect of mTOR activity. The inhibition of mTOR has the potential to disrupt diverse oncogenic processes and improve patient outcomes. However, the complexity of the mTOR signaling, off-target effects, cytotoxicity, suboptimal pharmacokinetics, and drug resistance of the mTOR inhibitors pose ongoing challenges in effectively targeting glioblastoma. Identifying innovative treatment strategies to address these challenges is vital for advancing the field of glioblastoma therapeutics. This review discusses the potential targets of mTOR signaling and the strategies of target-specific mTOR inhibitor development, optimized drug delivery system, and the implementation of personalized treatment approaches to mitigate the complications of mTOR inhibitors. The exploration of precise mTOR-targeted therapies ultimately offers elevated therapeutic outcomes and the development of more effective strategies to combat the deadliest form of adult brain cancer and transform the landscape of glioblastoma therapy.

Combined inhibition of surface CD51 and γ-secretase-mediated CD51 cleavage improves therapeutic efficacy in experimental metastatic hepatocellular carcinoma

Integrin αv (ITGAV, CD51) is regarded as a key component in multiple stages of tumor progression. However, the clinical failure of cilengitide, a specific inhibitor targeting surface CD51, suggests the importance of yet-unknown mechanisms by which CD51 promotes tumor progression. In this study, we used several hepatocellular carcinoma (HCC) cell lines and murine hepatoma cell lines. To investigate the role of CD51 on HCC progression, we used a 3D invasion assay and invivo bioluminescence imaging. We used periostin-knockout transgenic mice to uncover the role of the tumor microenvironment on CD51 cleavage. Moreover, we used several clinically relevant HCC models, including patient-derived organoids and patient-derived xenografts, to evaluate the therapeutic efficacy of cilengitide in combination with the γ-secretase inhibitor LY3039478. We found that CD51 could undergo transmembrane cleavage by γ-secretase to produce a functional intracellular domain (CD51-ICD). The cleaved CD51-ICD facilitated HCC invasion and metastasis by promoting the transcription of oxidative phosphorylation-related genes. Furthermore, we identified cancer-associated fibroblast-derived periostin as the major driver of CD51 cleavage. Lastly, we showed that cilengitide-based therapy led to a dramatic therapeutic effect when supplemented with LY3039478 in both patient-derived organoid and xenograft models. In summary, we revealed previously unrecognized mechanisms by which CD51 is involved in HCC progression and uncovered the underlying cause of cilengitide treatment failure, as well as providing evidence supporting the translational prospects of combined CD51-targeted therapy in the clinic. Integrin αv (CD51) is a widely recognized pro-tumoral molecule that plays a crucial role in various stages of tumor progression, making it a promising therapeutic target. However, despite early promising results, cilengitide, a specific antagonist of CD51, failed in a phase III clinical trial. This prompted further investigation into the underlying mechanisms of CD51's effects. This study reveals that the γ-secretase complex directly cleaves CD51 to produce an intracellular domain (CD51-ICD), which functions as a pro-tumoral transcriptional regulator and can bypass the inhibitory effects of cilengitide by entering the nucleus. Furthermore, the localization of CD51 in the nucleus is significantly associated with the prognosis of patients with HCC. These findings provide a theoretical basis for re-evaluating cilengitide in clinical settings and highlight the importance of identifying a more precise patient subpopulation for future clinical trials targeting CD51.

Association of systemic inflammatory markers with prognosis in erlotinib-treated EGFR-mutant non-small cell lung cancer.

To evaluate the relationship of overall survival (OS) and progression-free survival (PFS) with the derived neutrophil-lymphocyte ratio (dNLR), neutrophil-lymphocyte ratio (NLR), lymphocyte-monocyte ratio (LMR), and platelet-lymphocyte ratio (PLR) in patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). The study included 43 patients with EGFR-mutant metastatic NSCLC. The dNLR, NLR, LMR, and PLR values were calculated using the baseline complete blood counts before and after treatment with erlotinib. The NLR value had the best diagnostic test performance with a sensitivity of 91.3%. dNLR, NLR, LMR, and PLR were found to be significant for the prediction of OS and PFS. While the delta dNLR and NLR values were significant for OS, only the delta NLR value was significant for PFS. The dNLR, NLR, LMR, and PLR values were found to be significant in the prediction of OS and PFS in erlotinib-treated metastatic NSCLC. Further clinical studies are needed to determine the ideal target-specific tyrosine kinase inhibitor in cases of metastatic NSCLC presenting with the EGFR-activating mutation.

Abstract 3636: Liver endothelium secreted LRG1 promotes metastatic colorectal cancer growth through the HER3/RSK/EIF4B AXIS

Abstract Background: 25% of patients diagnosed with colorectal cancer (CRC) have liver metastasis at presentation, and ~80% of all metastatic CRC are developed in the liver. We previously reported that liver endothelial cells (ECs), a key component of the liver microenvironment, secrete LRG1 to promote CRC growth via activating human epidermal growth factor receptor (ERbB3, also known as HER3). However, we found that LRG1-induced HER3 activation is distinct from the canonical neuregulin 1 (NRG1)-induced HER3 pathway. The present study further validated LRG1 as a new HER3 ligand for promoting mCRC growth and elucidated the novel downstream signaling pathway induced by LRG1-HER3. Methods: We first measured the binding affinity between HER3 and LRG1 by Biolayer interferometry (BLI). We then used in vitro and in vivo xenograft approaches to determine the effect of LRG1 monoclonal antibody (15C4) on HER3 activation and CRC growth. To further determine the role of LRG1 in promoting CRC growth in the liver, we used murine CRC cells in a syngeneic orthotropic liver injection model to establish CRC allografts in the liver of LRG1−/− mice with systemic LRG1 knockout and wild-type siblings (LRG1+/+). We also performed unbiased phospho-MS analysis and subsequent validations to determine the downstream signaling pathway activated by LRG1-HER3. Results: We identified that LRG1 binds to HER3 with the affinity at ~100nM. The LRG1 antibody 15C4 completely attenuated LRG1-induced HER3 activation and in vitro and xenograft growth in vivo. Moreover, LRG1−/− mice with CRC allografts in the liver had 2 times longer overall survival than tumor-bearing LRG1+/+ mice. Lastly, unbiased -omics analysis identified eIF4-protein synthesis is significantly activated by LRG1. With target-specific inhibitors, we further determined that LRG1-HER3 activates the PI3K-PDK1-RSK1/3-eIF4 axis independent of AKT. Conclusions: We identified LRG1 as a novel HER3 ligand and demonstrated that the liver microenvironment-derived LRG1 plays a key oncogenic role in mCRC, by activating a novel RSK-eIF4 survival pathway. Our findings highlighted the potential of blocking LRG1-HER3 and involved downstream pathways for treating patients with mCRC. Citation Format: Moeez Ghani Rathore, Michelle Wright, Wei Huang, Derek Taylor, Yamu Li, Jordan Winter, Zhenghe Wang, John Greenwood, Stephen Moss, Rui Wang. Liver endothelium secreted LRG1 promotes metastatic colorectal cancer growth through the HER3/RSK/EIF4B AXIS. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3636.

In silico elucidation of plausible anti-obesity activity by Withaferin-A compound targeting alpha-amylase.

The study aimed to evaluate the Withaferin-A against the drug target α-amylase, revealing its plausible mode of action and molecular-level interactions essential for this specific target inhibitory potential computational approach. In this scenario, we used computational methods, including docking, molecular dynamics simulation, and model-building simulations, to elucidate the atomic-level details responsible for the inhibitory potential of Withaferin-A derived from W. somnifera. The studio visualizer software was used for the visualization of ligands, structures of the receptor, bond length, and rendering of the image. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics of phytochemicals were investigated. Crystal structure of protein receptors and ligands were generated. Semi-flexible docking was done using Autodock software. Docking was performed using the Lamarckian Genetic Algorithm (LGA). Molecular descriptors were evaluated, and the pharmacological properties of the phytochemicals were explored. Molecular dynamic simulations were analyzed at the atomic level. All the simulations were conducted under the same temperature, pressure, and volume circumstances over the simulated time scale. Withaferin-A has shown a strong binding affinity towards α-amylase as demonstrated with -9.79 Kcal/mol with 66.61 estimated nanomolecular IC50 value for plausible anti-obesity activity. Molecular-level relationships and knowledge obtained from this study indicate solid interactions with TYR59, ASP197, and HIS299 residues which are of high importance for future works related to computational screening of target-specific α-amylase inhibitors. The results from the analysis have revealed potential molecular-level interactions useful for further designing/discovering novel α-amylase inhibitors. The framework of the studied phytochemicals enables the rapid development of subsequent modifications that could result in more lead-like compounds with better inhibitory efficacy and selectivity for α-amylase.

Development of machine learning models based on molecular fingerprints for selection of small molecule inhibitors against JAK2 protein.

Janus kinase 2 (JAK2) is emerging as a potential therapeutic target for many inflammatory diseases such as myeloproliferative disorders (MPD), cancer and rheumatoid arthritis (RA). In this study, we have collected experimental data of JAK2 protein containing 6021 unique inhibitors. We then characterized them based on Morgan (ECFP6) fingerprints followed by clustering into training and test set based on their molecular scaffolds. These data were used to build the classification models with various supervised machine learning (ML) algorithms that could prioritize novel inhibitors for future drug development against JAK2 protein. The best model built by Random Forest (RF) and Morgan fingerprints achieved the G-mean value of 0.84 on the external test set. As an application of our classification model, virtual screening was performed against Drugbank molecules in order to identify the potential inhibitors based on the confidence score by RF model. Nine potential molecules were identified, which were further subject to molecular docking studies to evaluate the virtual screening results of the best RF model. This proposed method can prove useful for developing novel target-specific JAK2 inhibitors.

Proinflammatory S100A9 stimulates TLR4/NF-κB signaling pathways causing enhanced phagocytic capacity of microglial cells

Alzheimer's disease (AD) is the main cause of dementia, affecting the increasingly aging population. Growing evidence indicates that neuro-inflammation plays crucial roles, e.g., the association between AD risk genes with innate immune functions. In this study, we demonstrate that moderate concentrations of pro-inflammatory cytokine S100A9 regulate immune response of BV2 microglial cells, i.e., the phagocytic capacity, reflected by elevated number of 1 μm diameter Dsred-stained latex beads in the cytoplasm. In contrast, at high S100A9 concentrations, both the viability and phagocytic capacity of BV2 cells drop substantially. Furthermore, it is uncovered that S100A9 affects phagocytosis of microglia via NF-κB signaling pathways. Application of related target-specific drugs, i.e., IKK and TLR4 inhibitors, effectively suppresses BV2 cells’ immune responses. These results suggest that pro-inflammatory S100A9 activates microglial phagocytosis, and possibly contributes to the clearance of amyloidogenic species at the early stage of AD.

De novo designing of novel drug-like inhibitors of aldose reductase

Aldose reductase (AR, ALR2) is a member of the aldo-keto reductase superfamily. It is the first and rate-limiting enzyme of the polyol pathway. AR plays an essential role in the development of diabetic complications. It is designated as the most legitimate target for managing diabetic complications. One of the key challenges to the successful development of target-specific AR inhibitors is target selectivity. In this research, target-specific drug-like ALR2 inhibito

An insight into therapeutic efficacy of heterocycles as histone‐modifying enzyme inhibitors that targets cancer epigenetic pathways

Histone-modifying enzymes are the key regulators involved in the post-translational modification of histone and non-histone. These enzymes are responsible for the epigenetic control of cellular functions. However, deregulation of the activity of these enzymes results in uncontrolled disorders such as cancer and inflammatory and neurological diseases. The study includes histone acetyltransferases, deacetylases, methyl transferases, demethylases, DNA methyl transferases, and their potent inhibitors which are in a clinical trial and used as medicinal drugs. The present review covers the heterocycles as target-specific inhibitors of histone-modifying enzyme, more specifically histone acetyltransferases. This review also confers more recent reports on heterocycles as potential HAT inhibitors covered from 2016 to 2022 and future perspectives of these heterocycles in epigenetic therapy.