Biology subjects

Chang, M. C.

Publications and source records attributed to Chang, M. C..

4 recordsLinked to original sources

bioRxiv2023

Investigating impacts of marine sponge derived mycothiazole and its acetylated derivative on mitochondrial function and aging.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), a widely used ETC complex I inhibitor, these two molecules showed cytotoxicity to cancer cells but strikingly demonstrate a lack of toxicity to non-cancer cells, a highly beneficial feature in the development of anti-cancer therapeutics. Furthermore, in vivo experiments with these small molecules utilizing C.elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPRMT) pathway, that extends lifespan of worms when applied in their adult stage. Interestingly, we also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilize the transcription factors ATFS-1 and HSF-1, which are involved in the UPRMT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF-1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

cell biology↗

bioRxiv2023

Synergistic Binding of the Halide and Cationic Prime Substrate of the L-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl- or Br-), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three non-gaseous substrates must bind to activate the enzymes Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2 all coordinate directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H*) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the O_SCPLOWLC_SCPLOW-lysine 4-chlorinase, BesD. After 2OG adds, subsequent coordination of the halide to the cofactor and binding of cationic O_SCPLOWLC_SCPLOW-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon addition of O2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and O_SCPLOWLC_SCPLOW-Lys. The surprising lability of the BesD*[Fe(IV)=O]*Cl*succinate*O_SCPLOWLC_SCPLOW-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in O_SCPLOWLC_SCPLOW-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply that (i) BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between O_SCPLOWLC_SCPLOW-Lys binding and chloride coordination following loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

biochemistry↗

bioRxiv2022

Manufacturing highly potent CD20/CD19-targeted iCasp9 regulatable CAR-T cells using the Quantum pBac-based CAR-T (qCART) system for clinical application

BackgroundCD19-targeted chimeric antigen receptor therapies (CAR19) have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However, >50% of CAR19-treated patients experienced progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore, it remains a challenge to efficiently, cost-effectively, and consistently manufacture clinically relevant number of virally engineered CAR-T cells. MethodsUsing a highly efficient piggyBac transposon-based vector, Quantum pBac, we developed a virus-free cell engineering system, Quantum CART (qCART), for development and production of multiplex CAR-T therapies. ResultsHere, we demonstrated in vitro and in vivo that consistent, robust, and functional CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using the qCART system for clinical application. qCART-manufactured CAR-T cells from cancer patients expanded efficiently, rapidly eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. ConclusionsThe qCART system is an elegant system for the manufacturing of CAR-T products having all the desired CAR-T therapy attributes. We believe that the simplicity of manufacturing multiplex CAR-T cells using the qCART system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future. What is already known on this topicDespite the considerable success which has been achieved with CD19-targeted chimeric antigen receptor therapies (CAR19), >50% of CAR19-treated patients still experienced progressive disease. Therefore, there is a need to further improve CAR19 therapies. Current CAR19 therapies commonly utilize virus-based cell engineering methods. CAR-T production using these methods face multiple hurdles, including difficulties to efficiently, cost-effectively, and consistently manufacture clinically relevant number of CAR-T cells. We have previously used a highly efficient piggyBac transposon-based vector, Quantum pBac, to establish Quantum CART (qCART) which is a virus-free cell engineering system for development and production of multiplex CAR-T therapies. What this study addsIn this report, we further demonstrate in vitro and in vivo that consistent, robust, and functional iCasp9-regulatable, CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using the qCART system for clinical application. These cells possess all the desired attributes for ensuring therapeutic efficacy in CAR-T therapy, including high CAR-TSCM, balanced CD8/CD4 ratio, low exhaustion and senescence marker expressions, and high ex vivo and in vivo expansion capacity. Importantly, we show that qCART-manufactured CAR-T cells from hematological cancer patients expanded efficiently, effectively eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. We believe that the simplicity of manufacturing multiplex CAR-T cells using the qCART system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future. How this study might affect research, practice or policyOur findings demonstrate that qCART is a virus-free CAR-T engineering system for manufacturing CAR-TSCM cells from either healthy donors or hematological cancer patients, that possess all the desired attributes for a successful CAR-T therapy. These cells expanded efficiently, rapidly eradicated tumors, and can be safely controlled via activation of iCasp9. We expect that this simple yet robust system for manufacturing multiplex CAR-T cells will advance the CAR-T field.

cancer biology↗

bioRxiv2021

Engineering site-selective incorporation of fluorine into natural product analogs

While bioactive compounds are commonly derived both by human design as well as from living organisms, man-made and natural products typically display very different structural characteristics. As such, a longstanding goal in the discovery of new molecular function is to develop approaches to incorporate the advantageous elements of both groups of molecules, thereby expanding the molecular space accessible for this purpose. In this work, we report the engineering a fluorine-selective enzyme that can complement mutated acyltransferase (AT) domains of a modular polyketide synthase, which are the main determinants of the identity and location of substituents on polyketides, to produce different fluorinated regioisomers of the erythromycin precursor in vitro. We further show that by engineering cell uptake of fluorinated building blocks, we can control fluorine selectivity in vivo to produce selectively fluorinated polyketides using engineered E. coli. These results demonstrate that it is possible to introduce fluorine, a key synthetic design element for drug development, selectively into the scaffold of a complex natural product and produce these analogs by microbial fermentation.

synthetic biology↗