C-4 analogues of podophyllotoxin as tubulin inhibitors: synthesis, biological evaluation, and structure-activity relationship.

The diversity of lignan small molecules derived from podophyllotoxin, a non-covalent tubulin inhibitor isolated from the Podophyllum family, has led to the clinical development of FDA-approved anticancer agents etoposide and teniposide. While these two compounds share the same tetracyclic core as podophyllotoxin, two subtle structural changes-4' demethylation on the aromatic ring and stereospecific glycosylation at the C-4 hydroxyl-result in an alternate biological mechanism. Given the immense pharmacological importance of altering the C-4 position, we synthesised and evaluated a systematic library of diversified esters to establish a structure-activity relationship regarding modification at C-4 on the properties of podophyllotoxin. We determined the biological activity of these esters through cell viability assays, computer docking models, tubulin polymerisation assays, and cell cycle analysis. Altogether, we demonstrate that increasing steric hindrance at C-4 leads to a loss in potency against human cancer cells but has a significantly lesser impact on cell-free tubulin inhibition.

TUBA1C orchestrates the immunosuppressive tumor microenvironment and resistance to immune checkpoint blockade in clear cell renal cell carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) poses substantial treatment challenges, especially in advanced stages where the efficacy of immune checkpoint blockade (ICB) therapy varies significantly. Elevated expression of the oncogene TUBA1C has been correlated with poor prognosis in various cancers, however, its role in ccRCC is unclear, especially concerning ICB resistance. Methods: Single-cell analysis was used to examine gene expression variations in malignant cells post-ICB therapy. This included investigating TUBA1C expression across different ICB response groups and its relationship with CD274. A general module of action was identified through pan-cancer and pan-tissue analysis. TUBA1C expression and its association with clinical characteristics and prognosis was further validated. Multiple algorithms were employed to explore immune cell infiltration levels, and the DepMap database was utilized to assess gene dependency and mutation status in kidney cancer cell lines. The in silico knockout of TUBA1C was performed using deep learning model, complemented by immunohistochemical assays, clinical cohort and functional assays validations. Results: TUBA1C expression is elevated in malignant cells following ICB therapy and is correlated with ICB resistance in ccRCC. High TUBA1C expression activates PI3K/AKT pathway and is associated with increased infiltration of regulatory T cells and myeloid-derived suppressor cells, which contributes to an immunosuppressive microenvironment in ccRCC. Patients with high TUBA1C expression exhibit a greater tumor mutation burden and increased genetic variation, which causes a worse prognosis. Additionally, TUBA1C dependency and its effects were evident in kidney cancer cell lines, where mutations conferred resistance to anti-PD-L1 therapy. In silico knockout analyses indicated that treatment targeting TUBA1C shifted malignant cells to a state responsive to ICB therapy. Immunohistochemistry, RT-qPCR and clinical cohort validation further confirmed that TUBA1C expression was upregulated and contributed to poorer outcome in ccRCC. Finaly, wound healing and CCK-8 assays demonstrated the potent oncogenic function of TUBA1C. Conclusions: TUBA1C is a pivotal regulator in ccRCC, affecting both disease progression and the effectiveness of ICB therapy by fostering an immunosuppressive microenvironment mediated by the PI3K/AKT pathway. Additionally, TUBA1C holds promise, both as a prognostic biomarker and a therapeutic target, for enhancing responsiveness to ICB.

The microtubule regulator EFA-6 forms cortical foci dependent on its intrinsically disordered region and interactions with tubulins.

The EFA6 protein family, originally identified as Sec7 guanine nucleotide exchange factors, has also been found to regulate cortical microtubule (MT) dynamics. Here, we find that in the mature C. elegans epidermal epithelium, EFA-6 forms punctate foci in specific regions of the apical cortex, dependent on its intrinsically disordered region (IDR). The EFA-6 IDR can form biomolecular condensates in vitro. In genetic screens for mutants with altered GFP::EFA-6 localization, we identified a gain-of-function (gf) mutation in α-tubulin tba-1 that induces ectopic EFA-6 foci in multiple cell types. Lethality of tba-1(gf) is partially suppressed by loss of function in efa-6. The ability of TBA-1(gf) to trigger ectopic EFA-6 foci requires ß-tubulin TBB-2 and the chaperon EVL-20/Arl2. tba-1(gf)-induced EFA-6 foci display slower turnover, contain the MT-associated protein TAC-1/TACC, and require the EFA-6 MT elimination domain (MTED). Our results reveal functionally important crosstalk between cellular tubulins and cortical MT regulators in vivo.

Highly Cytotoxic Cryptophycin Derivatives with Modification in Unit D for Conjugation.

Cytotoxic payloads for drug conjugates suitable for directed tumor therapy need to be highly potent and require a functional group for conjugation with the homing device (antibody, peptide, or small molecule). Cryptophycins are cyclodepsipeptides that stand out from the realm of natural products due to their extraordinarily high cytotoxicity. However, the installation of a suitable conjugation handle without compromising the toxicity is highly challenging. The unit D, natively 2-hydroxyisocaproic acid (leucic acid), was envisaged as a promising attachment site based on structural information from X-ray analysis. A versatile, scalable and efficient synthetic route towards conjugable cryptophycins with modification in unit D was developed and an array of new cryptophycin analogues was synthesized. Several derivatives, especially those containing lipophilic groups with low steric demand such as alkylated amino groups, exhibit low picomolar cytotoxicity often combined with efficacy against multidrug-resistant tumor cells. The newly established cryptophycin analogues comprise a broad range of relevant functional groups used as conjugation handles, among them amino, hydroxy, carboxy, as well as sulfur-containing derivatives. X-ray crystallographic analysis of a tubulin-bound cryptophycin together with quantitative structure activity relationship manifested rationales for the synthesis of most potent cryptophycin derivatives and further confirmed the suitability of modifications in unit D.

A potent Bioorganic azapodophyllotoxin derivative Suppresses tumor Progression in Triple negative breast Cancer: An Insight into its Inhibitory effect on tubulin polymerization and Disruptive effect on microtubule assembly.

Triple negative breast cancer (TNBC) has long been a challenging disease owing to its high aggressive behaviour, poor prognosis and its limited treatment options. The growing demand of new therapeutics against TNBC enables us to examine the therapeutic efficiency of an emerging class of anticancer compounds, azapodophyllotoxin derivative (HTDQ), a nitrogen analogue of podophyllotoxin, using different biochemical, spectroscopic and computational approaches. The anticancer activities of HTDQ are studied by performing MTT assay in a dose depended manner on Triple negative breast cancer cells using MDA-MB-468 and MDA-MB-231 cell lines with IC50 value 937 nM and 1.13 µM respectively while demonstrating minimal effect on normal epithelial cells. The efficacy of HTDQ was further tested in 3D tumour spheroids formed by the human TNBC cell line MDA-MB468 and also the murine MMTV positive TNBC cell line 4 T1. The shrinkage that observed in the tumor spheroid clearly indicates that HTDQ remarkably decreases the growth of tumor spheroid thereby affirming its cytotoxicity. The 2D cell viability assay shows significant morphological alteration that possibly caused by the cytoskeleton disturbances. Hence the binding interaction of HTDQ with cytoskeleton protein tubulin, its effect on tubulin polymerisation as well as depolymerisation of preformed microtubules along with the conformational alternation in the protein itself have been investigated in detail. Moreover, the apoptotic effects of HTDQ have been examined using a range of apoptotic markers. HTDQ-treated cancer cells showed increased expression of cleaved PARP-1 and pro-caspase-3, suggesting activation of the apoptosis process. HTDQ also upregulated pro-apoptotic Bax expression while inhibiting anti-apoptotic Bcl2 expression, supporting its ability to induce apoptosis in cancer cells. Hence the consolidated biochemical and spectroscopic research described herein may provide enormous information to use azapodophyllotoxin as promising anticancer therapeutics for TNBC cells.

Unveiling the anti-cancer potentiality of phthalimide-based Analogues targeting tubulin polymerization in MCF-7 cancerous Cells: Rational design, chemical Synthesis, and Biological-coupled Computational investigation.

The present study deals with an anti-cancer investigation of an array of phthalimide-1,2,3-triazole molecular conjugates with various sulfonamide fragments against human breast MCF-7 and prostate PC3 cancer cell lines. The targeted 1,2,3-triazole derivatives 4a-l and 6a-c were synthesized from focused phthalimide-based alkyne precursors using a facile click synthesis approach and were thoroughly characterized using several spectroscopic techniques (IR, 1H, 13C NMR, and elemental analysis). The hybrid click adducts 4b, 4 h, and 6c displayed cytotoxic potency (IC50 values of 1.49, 1.07, and 0.56 µM, respectively) against MCF-7 cells. On the contrary, none of the synthesized compounds showed apparent cytotoxic efficacy for PC3 cells (IC50 ranging from 9.87- >100 µM). As a part of the mechanism analysis, compound 6c demonstrated a potent inhibitory effect (78.3 % inhibition) of tubulin polymerization in vitro with an IC50 value of 6.53 µM. In addition, biological assays showed that compound 6c could prompt apoptotic cell death and induce G2/M cell cycle arrest in MCF-7 cells. Accordingly, compound 6c can be further developed as an anti-breast cancer agent through apoptosis-induction.

Partial closure of the γ-tubulin ring complex by CDK5RAP2 activates microtubule nucleation.

Microtubule nucleation is templated by the γ-tubulin ring complex (γ-TuRC), but its structure deviates from the geometry of α-/ß-tubulin in the microtubule, explaining the complex's poor nucleating activity. Several proteins may activate the γ-TuRC, but the mechanisms underlying activation are not known. Here, we determined the structure of the porcine γ-TuRC purified using CDK5RAP2's centrosomin motif 1 (CM1). We identified an unexpected conformation of the γ-TuRC bound to multiple protein modules containing MZT2, GCP2, and CDK5RAP2, resulting in a long-range constriction of the γ-tubulin ring that brings it in closer agreement with the 13-protofilament microtubule. Additional CDK5RAP2 promoted γ-TuRC decoration and stimulated the microtubule-nucleating activities of the porcine γ-TuRC and a reconstituted, CM1-free human complex in single-molecule assays. Our results provide a structural mechanism for the control of microtubule nucleation by CM1 proteins and identify conformational transitions in the γ-TuRC that prime it for microtubule nucleation.

CDK5RAP2 activates microtubule nucleator γTuRC by facilitating template formation and actin release.

To organize microtubules, cells tightly control the activity of the microtubule nucleator γ-tubulin ring complex (γTuRC). The open ring-shaped γTuRC was proposed to nucleate microtubules by a template mechanism. However, its splayed structure does not match microtubule symmetry, leaving it unclear how γTuRC becomes an efficient nucleator. Here, we identify the mechanism of γTuRC activation by CDK5RAP2 centrosomin motif 1 (CM1). Using cryoelectron microscopy (cryo-EM), we find that activation involves binding of multiple CM1 dimers to five distinct sites around the outside of the γTuRC cone, which crucially depends on regulatory modules formed by MZT2 and the N-terminal extensions of GCP2 subunits. CM1 binding promotes lateral interactions between GCP subunits to facilitate microtubule-like conformations and release of luminal actin that is integral to non-activated γTuRC. We propose a model where generation of γTuRC with an expanded conformational range, rather than perfect symmetry, is sufficient to boost nucleation activity.

High-Throughput Image-Based Assay for Identifying In Vitro Hepatocyte Microtubule Disruption.

Disruption of microtubule stability in mammalian cells may lead to genotoxicity and carcinogenesis. The ability to screen for microtubule destabilization or stabilization is therefore a useful and efficient approach to aid in the design of molecules that are safe for human health. In this study, we developed a high-throughput 384-well assay combining immunocytochemistry with high-content imaging to assess microtubule disruption in the metabolically competent human liver cell line: HepaRG. To enhance analysis throughput, we implemented a supervised machine learning approach using a curated training library of 180 compounds. A majority voting ensemble of eight machine learning classifiers was employed for predicting microtubule disruptions. Our prediction model achieved over 99.0% accuracy and a 98.4% F1 score, which reflects the balance between precision and recall for in-sample validation and 93.5% accuracy and a 94.3% F1 score for out-of-sample validation. This automated image-based testing can provide a simple, high-throughput screening method for early stage discovery compounds to reduce the potential risk of genotoxicity for crop protection product development.

Construction and validation of co-expression vector for rice alpha tubulin and microtubule associated protein respectively fused with fluorescent proteins.

Microtubule (MT) consists of α-tubulin and ß-tubulin. The dynamic instability regulated by various microtubule associated proteins (MAPs) is essential for MT functions. To analyze the interaction between tubulin/MT and MAP in vivo, we usually need tubulin and MAP co-expressed. Here, we constructed a dual-transgene vector expressing rice (Oryza sativa) α-tubulin and MAP simultaneously. To construct this vector, plant expression vector pCambia1301 was used as the plasmid backbone and Gibson assembly cloning technology was used. We first fused and cloned the GFP fragment, α-tubulin open reading frame (ORF), and NOS terminator into the vector pCambia1301 to construct the p35S::GFP-α-tubulin vector that expressed GFP-α-tubulin fusion protein. Subsequently, we fused and cloned the CaMV 35S promoter, mCherry fragment, and NOS terminator into the p35S::GFP-α-tubulin vector to generate the universal dual-transgene expression vector (p35S::GFP-α-tubulin-p35S::mCherry vector). With the p35S::GFP-α-tubulin-p35S::mCherry vector, MAP ORF can be cloned into the site of 5' or 3' terminus of mCherry to co-express GFP-α-tubulin and MAP-mCherry/mCherry-MAP. To validate the availability and universality of the dual-transgene expression vector, a series of putative rice MAP genes including GL7, OsKCBP, OsCLASP, and OsMOR1 were cloned into the vector respectively, transformed into Agrobacterium tumefaciens strain, and expressed in Nicotiana benthamiana leaves. The results indicated that all of the MAPs were co-expressed with α-tubulin and localized to MTs, validating the availability and universality of the vector and that GL7, OsKCBP, OsCLASP, and OsMOR1 might be MAPs. The application of the co-expression vector constructed by us would facilitate studies on the interaction between tubulin/MT and MAP in tobacco transient expression systems or transgenic rice.