Recent Advances in Combretastatin A-4 Inspired Inhibitors of Tubulin Polymerization-An Update

Cancer is one of the leading causes of fatality and mortality worldwide. Investigations on developing therapeutic strategies for cancer are supported throughout the world. The massive achievements in molecular sciences involving biochemistry, molecular chemistry, medicine, and pharmacy, and high throughput techniques such as genomics and proteomics have helped to create new potential drug targets for cancer treatment. Microtubules are very attractive targets for cancer therapy because of the crucial roles they play in cell division. In recent years, lots of efforts have been put into the identification of new microtubule-targeting agents (MTAs) in anticancer therapy. Combretastatin A-4 (CA-4) is a natural compound that binds to microtubules’ colchicine binding site and inhibits microtubule polymerization. Due to CA-4’s structural simplicity, many analogs have been synthesized. This article summarizes the new molecule development efforts to reach CA-4 analogs by modifications on its pharmacophore groups, published since 2015.

The TOG protein Stu2 is regulated by acetylation

Stu2 in S. cerevisiae is a member of the XMAP215/Dis1/Alp14/Msps/CKAP5/ch-TOG family of MAPs and has multiple functions in controlling microtubules, including microtubule polymerization, microtubule depolymerization, linking chromosomes to the kinetochore, and assembly of γ-TuSCs at the SPB. Whereas phosphorylation has been shown to be critical for Stu2 localization at the kinetochore, other regulatory mechanisms that control Stu2 function are still poorly understood. Here, we show that a novel form of Stu2 regulation occurs through the acetylation of three lysine residues at K252, K469, and K870, which are located in three distinct domains of Stu2. Alteration of acetylation through acetyl-mimetic and acetyl-blocking mutations did not impact the essential function of Stu2. Instead, these mutations lead to both positive and negative changes in chromosome stability, as well as changes in resistance to the microtubule depolymerization drug, benomyl. In agreement with our in silico modeling, several acetylation-mimetic mutants displayed increased interactions with γ-tubulin. Taken together, these data suggest that Stu2 acetylation can govern multiple Stu2 functions in both a positive and negative manner, including chromosome stability and interactions at the SPB.

Characterization of a recently synthesized microtubule-targeting compound that disrupts mitotic spindle poles in human cells

We reveal the effects of a new microtubule-destabilizing compound in human cells. C75 has a core thienoisoquinoline scaffold with several functional groups amenable to modification. Previously we found that sub micromolar concentrations of C75 caused cytotoxicity. We also found that C75 inhibited microtubule polymerization and competed with colchicine for tubulin-binding in vitro. However, here we found that the two compounds synergized suggesting differences in their mechanism of action. Indeed, live imaging revealed that C75 causes different spindle phenotypes compared to colchicine. Spindles remained bipolar and collapsed after colchicine treatment, while C75 caused bipolar spindles to become multipolar. Importantly, microtubules rapidly disappeared after C75-treatment, but then grew back unevenly and from multiple poles. The C75 spindle phenotype is reminiscent of phenotypes caused by depletion of ch-TOG, a microtubule polymerase, suggesting that C75 blocks microtubule polymerization in metaphase cells. C75 also caused an increase in the number of spindle poles in paclitaxel-treated cells, and combining low amounts of C75 and paclitaxel caused greater regression of multicellular tumour spheroids compared to each compound on their own. These findings warrant further exploration of C75’s anti-cancer potential.

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end

Microtubule polymerization dynamics result from the biochemical interactions of αβ-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αβ-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently "poisoned" growth, yielding a wider range of growth rate compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP tubulin at the growing microtubule end may be an important early event determining catastrophe.

Microtubule Polymerization Inhibition Enhances Human Hematopoietic Stem Cell Homing and Engraftment

Hematopoietic stem cells (HSCs) serve as the origin of the hematopoietic system, with the ability to differentiate into all blood cell lineages and self-renewal to sustain the hematopoiesis throughout life. Hematopoietic stem cell transplantation (HSCT) currently represents the most effective therapeutic strategies to treat hematological and non-hematological diseases. However, limited numbers of HSCs or poor homing capabilities into the bone marrow are still major hurdles for successful HSCT. Moreover, graft failure and delayed reconstitution due to inefficient engraftment, remains an important complication because of the high morbidity and mortality. Although ex vivo expansion of HSCs has been well studied for decades, which displays huge potentials for clinical application, exploration of novel targets to improve HSC homing and engraftment will provide new insights to enhance HSCT efficacy. To explore the chemical compounds enhancing the capabilities of homing and engraftment, we used CXCR4 (CD184) as the readout bio-marker, which is considered as the most essential chemokine receptor of HSPCs interacting with CXCL12 (SDF1) secreted in BM niche to support HSPCs homing, migration, proliferation and survival. We first performed chemical screening of 139 small molecules that can increase CD184 expression on cord blood (CB) CD34 + hematopoietic stem and progenitor cells (HSPCs). We concluded that treatment of CB CD34 + HSPCs for 16 hours with Lexibulin (Lex) or Vinblastine Sulfate (VS), both of which were microtubule polymerization (MP) inhibitors, could significantly promote the CD184 expression. Next, we optimized the MP inhibitors treatment conditions including dosage, treatment duration and culture time prior to treatment. The results proved that treatment with Lex or VS for 16 hours at 1μM was the optimal conditions to significantly enhance the CD184 expression of CD34 + HPSCs and LT-HSCs (CD34 +CD90 +CD45RA -), while maintaining robust cell survival, when compared with the DMSO control group. Moreover, we found that only when HSPCs were under culture within two days prior to small molecules treatment, CD184 expression was significantly increased by MP inhibitors while maintaining high viability, compared with DMSO control group. In order to assess the in vivo repopulating potential of the CB CD34 + HSPCs post treatment with MP inhibitors, we transplanted CB-HSPCs 16 hours post-treatment with Lex and VS respectively into irradiated nonobese diabetic (NOD)/Prkdc scid/IL-2Rγ null (NPG) mice. All transplanted mice of MP inhibitors-treated groups presented efficient engraftment, in multiple immune organs at 4-16 weeks post-transplantation, suggesting greater engraftment potential than the mock group, as measured by human CD45 of total CD45. Furthermore, hematopoietic reconstitution analysis indicated that the MP inhibitors -treated cells maintained different lineage distribution in peripheral blood (PB), bone marrow (BM) and spleen. Moreover, the equivalent phenotypes of pre- and post-treatment reveal the better reconstitution by MP inhibitors was independent of HSC-enrichment, Thus, short-term MP inhibitors treatment of CB CD34 + HSPCs enhances their homing and long-term engraftment. In conclusion, we demonstrated that short-term microtubule polymerization inhibition on human CB CD34 + HSPCs could not only enhance CD184 cell surface expression but also the capabilities of in vivo human HSCs homing and reconstitution via screening chemical compounds to increase CD184 expression and the following function evaluation study. Vinblastine Sulfate and Lexbulin were applied or registered as anti-cancer drugs for clinical use. Our study also indicates that MP inhibitors pretreatment of cells possesses significant translational implications, designating MP inhibitors as promising drug candidates to facilitate clinical HSCT. Figure 1 Figure 1. Disclosures Fang: EdiGene, Inc.: Current Employment.

The ASC Speck and NLRP3 Inflammasome Function Are Spatially and Temporally Distinct

Although considered the ternary inflammasome structure, whether the singular, perinuclear NLRP3:ASC speck is synonymous with the NLRP3 inflammasome is unclear. Herein, we report that the NLRP3:ASC speck is not required for nigericin-induced inflammasome activation but facilitates and maximizes IL-1β processing. Furthermore, the NLRP3 agonists H2O2 and MSU elicited IL-1β maturation without inducing specks. Notably, caspase-1 activity is spatially distinct from the speck, occurring at multiple cytoplasmic sites. Additionally, caspase-1 activity negatively regulates speck frequency and speck size, while speck numbers and IL-1β processing are negatively correlated, cyclical and can be uncoupled by NLRP3 mutations or inhibiting microtubule polymerization. Finally, when specks are present, caspase-1 is likely activated after leaving the speck structure. Thus, the speck is not the NLRP3 inflammasome itself, but is instead a dynamic structure which may amplify the NLRP3 response to weak stimuli by facilitating the formation and release of small NLRP3:ASC complexes which in turn activate caspase-1.

PIAS1 Regulates Hepatitis C Virus-Induced Lipid Droplet Accumulation by Controlling Septin 9 and Microtubule Filament Assembly

Chronic hepatitis C virus (HCV) infection often leads to fibrosis and chronic hepatitis, then cirrhosis and ultimately hepatocellular carcinoma (HCC). The processes of the HVC life cycle involve intimate interactions between viral and host cell proteins and lipid metabolism. However, the molecules and mechanisms involved in this tripartite interaction remain poorly understood. Herein, we show that the infection of HCC-derived Huh7.5 cells with HCV promotes upregulation of the protein inhibitor of activated STAT1 (PIAS1). Reciprocally, PIAS1 regulated the expression of HCV core protein and HCV-induced LD accumulation and impaired HCV replication. Furthermore, PIAS1 controlled HCV-promoted septin 9 filament formation and microtubule polymerization. Subsequently, we found that PIAS1 interacted with septin 9 and controlled its assembly on filaments, which thus affected septin 9-induced lipid droplet accumulation. Taken together, these data reveal that PIAS1 regulates the accumulation of lipid droplets and offer a meaningful insight into how HCV interacts with host proteins.

Inhibitor of DNA binding 2 (Id2) mediates microtubule polymerization in the brain by regulating αK40 acetylation of α-tubulin

Acetylation of α-tubulin lysine 40 (αK40) contributes to microtubule (MT) stability and is essential for neuronal development and function, whereas excessive αK40 deacetylation is observed in neurodegenerative disorders including Alzheimer’s disease (AD). Here we identified inhibitor of DNA binding 2 (Id2) as a novel MT-binding partner that interacts with α-tubulin and enhances αK40 acetylation, leading to MT polymerization in the neurons. Commensurate with our finding that the low levels of Id2 expression along with a reduced αK40 acetylation in the postmortem human AD patient and 5X-FAD, AD model mice brain, Id2 upregulation in the hippocampus of 5X-FAD, which exhibit high levels of Sirt2 expression, increased αK40 acetylation and reconstitutes axon growth. Hence our study suggests that Id2 is critical for maintaining MT stability during neural development and the potential of Id2 to counteract pathogenic Sirt2 activity in AD.

Functional fluorescently-tagged human profilin

Profilin is an essential regulator of actin and microtubule dynamics and therefore a critical control point for the normal division, motility, and morphology of cells. Most studies of profilin have focused on biochemical investigations using purified protein because high cellular concentrations (121 µM) present challenges for conventional imaging modalities. In addition, past studies that employed direct labeling or conventional fusion protein strategies compromised different facets of profilin function. We engineered a fluorescently-labeled profilin that retains native activities with respect to phosphoinositide lipids, actin monomers, formin-mediated actin assembly, and microtubule polymerization. This fluorescent profilin directly binds to dimers of tubulin (kD = 1.7 µM) and the microtubule lattice (kD = 10 µM) to stimulate microtubule assembly. In cells, our tagged profilin fully rescues profilin-1(-/-) cells from knockout-induced perturbations to cell shape, actin filament architecture, and microtubule arrays. Thus, this labeled profilin-1 is a reliable tool to investigate the dynamic interactions of profilin with actin or microtubules in live cell and in vitro applications.