Browsing by Subject "Small molecule"
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Item Open Access An Asymptotic Model of Electroporation-Mediated Molecular Delivery in Skeletal Muscle Tissue(2014) Cranford, Jonathan PrestonElectroporation is a biological cell's natural reaction to strong electric fields, where transient pores are created in the cell membrane. While electroporation holds promise of being a safe and effective tool for enhancing molecular delivery in numerous medical applications, it remains largely confined to preclinical research and clinical trials due to an incomplete understanding of the exact mechanisms involved. Muscle fibers are an important delivery target, but traditional theoretical studies of electroporation ignore the individual fiber geometry, making it impossible to study the unique transverse and longitudinal effects from the pulse stimulus. In these long, thin muscle fibers, the total reaction of the fiber to the electric field is due to fundamentally different effects from the constituent longitudinal and transverse components of the electric field generated by the pulse stimulus. While effects from the transverse component have been studied to some degree, the effects from the longitudinal component have not been considered.
This study develops a model of electroporation and delivery of small molecules in muscle tissue that includes effects from both the transverse and longitudinal components of the electric field. First, an asymptotic model of electric potential in an individual muscle fiber is derived that separates the full 3D boundary value problem into transverse and a longitudinal problems. The transverse and longitudinal problems each have their own respective source functions: the new "transverse activating function" and the well known longitudinal activating function (AF). This separation enhances analysis of the different effects from these two AFs and drastically reduces computational intensity. Electroporation is added to the asymptotic fiber model, and simplified two-compartment mass transport equations are derived from the full 3D conservation of mass equations to allow simulation of molecular uptake due to diffusion and the electric field. Special emphasis is placed on choosing model geometry, electrical, and pulsing parameters that are in accordance with experiments that study electroporation-mediated delivery of small molecules in the skeletal muscle of small mammals.
Simulations reveal that for fibers close to the electrodes the transverse AF dominates, but for fibers far from the electrodes the longitudinal AF enhances uptake by as much as 2000%. However, on the macroscopic tissue level, the increase in uptake from the longitudinal AF is no more than 10%, given that fibers far from the electrodes contribute so little to the total uptake in the tissue. The mechanism underlying the smaller effect from the longitudinal AF is found to be unique to the process of electroporation itself. Electroporation occurs on the short time scale of polarization via the transverse AF, drastically increases membrane conductance, and effectively precludes further creation of pores from charging of the membrane via the longitudinal AF. The exact value of enhancement in uptake from the longitudinal AF is shown to depend on pulsing, membrane, and tissue parameters. Finally, simulation results reproduce qualitative, and in some cases quantitative, behavior of uptake observed in experiments.
Overall, percent increase in total tissue uptake from the longitudinal AF is on the order of experimental variability, and this study corroborates previous theoretical models that neglect the effects from the longitudinal AF. However, previous models neglect the longitudinal AF without explanation, while the asymptotic fiber model is able to detail the mechanisms involved. Mechanisms revealed by the model offer insight into interpreting experimental results and increasing efficiency of delivery protocols. The model also rigorously derives a new transverse AF based on individual fiber geometry, which affects the spatial distribution of uptake in tissue differently than predicting uptake based on the magnitude of the electric field, as used in many published models. Results of this study are strictly valid for transport of small molecules through small non-growing pores. For gene therapy applications the model must be extended to transport of large DNA molecules through large pores, which may alter the importance of the longitudinal AF. In broader terms, the asymptotic model also provides a new, computationally efficient tool that may be used in studying the effect of transverse and longitudinal components of the field for other types of membrane dynamics in muscle and nerves.
Item Open Access !Development of small molecule therapeutics against anti-infectious and anti-cancer drug resistance via structure-based drug design(2022) Lim, Won Young!Drug discovery typically involves structure-based drug design based on three-dimensional protein structures and hit/lead compound identification and optimization. Herein, this technique was used to overcome several obstacles associated with the developing of antibiotics, anticancer agents, and antifungals and reveal critical insights into the corresponding structure-activity relationships (SARs).Phospho-N-acetyl-muramyl-pentapeptide translocase (MraY) is an important membrane enzyme involved in the early-stage biosynthesis of bacterial peptidoglycans. As the inhibition of MraY leads to bacterial cell lysis, such MraY inhibitors (e.g., muraymycin) hold great promise for antibiotic development. However, the structural complexity of muraymycin makes its synthesis and practical applications challenging. Hence, we synthesized several muraymycin analogs with reduced structural complexity and better synthetic tractability and identified the moieties responsible for their biological activity to facilitate the development of muraymycin-derived antibiotics. Translesion synthesis (TLS) is a major mechanism that enables bypass replication over DNA lesions and promotes the formation of mutagenic DNA. Rev1/Pol ζ–mediated TLS plays an important role in cisplatin-induced mutations, and thus, the Rev1/Pol ζ interface is an attractive target for small-molecule TLS inhibitors. Herein, we aimed to develop TLS inhibitors as potential anticancer agents based on the recently reported inhibitor of the Rev1-Rev7 interaction, JH-RE-06. Despite its high potency, JH-RE-06 is poorly soluble in aqueous media and is therefore a limitation for further development. To overcome this limitation and identify novel anticancer agents, we prepared various JH-RE-06 analogs and studied the related SARs, to determine the critical functional groups for improving the biological activity improvement and aqueous solubility. Currently, fungal infections, which are particularly dangerous to immunocompromised patients, are a frequent cause of a death. However, the similarities between the eukaryotic physiologies of fungal pathogens and their hosts render targeting of the pathogen without causing side effects in the host challenging. Calcineurin (CN) plays a major role in invasive fungal diseases and is therefore a promising target for antifungal drug development. FK506, which is an approved CN inhibitor, exhibits promising activity but an insufficient selectivity because of its strong immunosuppressive effect. Therefore, in developing antifungal agents, we exploited the major structural differences between the CN-FK506-FKBP12 ternary complexes of humans and fungi and developed FK506/520 analogs targeting these complexes. The synthesized analogs retained the parent antifungal efficacy while exhibiting lower immunosuppressive activities and improved therapeutic efficacies both in vivo and in vitro.
Item Embargo Discovery of RNA-Targeted Small Molecules by Quantitative Structure-Activity Relationship (QSAR) Study and Machine Learning(2023) Cai, ZhengguoRNA is a critical macromolecule in many biological processes by encoding both structural and genetic information. It can serve as the physical template for ribosome read-through during protein synthesis and the intermediary interfering gene expression. For example, messenger RNA encodes specific gene sequence, microRNA regulates expression level of the gene, riboswitch controls translation level and RNA splicing, non-coding RNA provides molecular scaffolding for protein recruitment. Undoubtedly, malfunction of cellular RNAs lead to multiple diseases and targeting disease related RNAs has emerged as the new strategy in many drug development campaigns. Indeed, ribosomal RNA has been utilized as the drug target for a long history and fruitful studies on naturally occurred or synthetic ligands were brought to elucidate the mechanism of translation inhibition. It was the past two decades that witnessed growing research on using small-molecule probes to interrogate non-ribosomal RNAs in various disease pathways.RNA molecules bear distinct chemical properties from proteins that make the design of selective and potent chemical probes challenging. The poor chemical diversity of four building units, immensely charged phosphate backbone, shallow and highly hydrophilic binding pocket, dynamic conformations, all combined render a mysterious ligand space to RNA-targeted small molecules that needs further exploration. A deep understanding of privileged chemotypes or physicochemical properties of RNA-targeting ligands will definitely benefit a broad-scope developing novel chemical entities with desired RNA-interfering outcome. In my thesis work, I first applied the computational approach by building the quantitative structure-activity relationship (QSAR) model to predict the binding profiles of a set of biased ligands scaffolding an amiloride core structure against HIV viral RNA elements. The well-performed model predicted the binding parameters of a set of untested molecules and selected the top-ranked one during lead optimization. The study showed the potential of this computational tool in decision-making during synthesis of RNA-targeted ligands. In the following study, we extended the scope of the QSAR study and leveraged the workflow to cater for the context with diverse structures as substrates. We applied explicit algorithms to build the baseline models to allow easy interpretation of binding behaviors of structurally distinct ligands to HIV-1 TAR. The model first time demonstrated molecular factors that contribute to RNA: small molecule recognition, both kinetically and thermodynamically. The general workflow we described will serve as a powerful computational tool to effectively assess underexplored chemical space and guide decision-making for synthesizing RNA-targeted chemical probes. We then bridged our QSAR approach with the generative deep learning model to pursue de novo ligand design to target SARS-CoV-2 frameshifting pseudoknot. The QSAR model that built on the experimentally validated data provided label annotation of the large training sample for deep learning model. A tree graph-based variational auto-encoder was trained to learn the molecular generation process. Annotated label of each training sample was encoded into the continuous latent space where molecules were reduced their dimensionality and projected. Conditions were applied when sampling new entities from the latent space, leading to the new compounds with desired binding properties. The method mentioned here constitutes the first deep learning practice for automatic chemical design against an RNA target and the first-time application of conditional molecular generation via a junction tree-based variational auto-encoder. Overall, the work presented in this thesis explored possibility of data-driven methods such as QSAR studies and deep learning in accelerating ligand discovery for RNA targets. It is anticipated that these workflows will benefit a wide-range studies in understanding and pursuing RNA-centric drug development, yet slight modifications might be needed for tuning into larger data size.
Item Open Access Insights, Assays, and Strategies for Small Molecule-Based Modulation of the MALAT1 RNA Triple Helix(2020) Donlic, AnitaRecent discoveries of myriad of endogenous RNA transcripts that do not code for proteins has led to a paradigm shift in our understanding of human biology. As the subsequent “RNA revolution” continues to elucidate the disease-related functions and structures of mammalian non-coding RNA molecules, interest in developing small molecule probes and drug leads for these targets is on the rise. For example, the long non-coding RNA MALAT1 (Metastasis-Associated-Lung-Adenocarcinoma-Transcript-1) is overexpressed in and is critical for metastasis in a variety of cancers. A recently characterized triple helix structure at the 3'-end of MALAT1 was shown to be pivotal in stabilizing the transcript, and deletions or a single base substitution - proposed to destabilize this structure - led to a significant decrease in MALAT1 accumulation. While this unique structure represents a novel opportunity for therapeutic intervention, there is a gap in knowledge regarding effective strategies for targeting RNA in general and, in particular, triple helices.
Towards elucidating these principles, this work takes a three-pronged approach: i) synthesis and evaluation of scaffold-based small molecule libraries to deduce fundamental ligand-triple helix recognition principles, ii) development of robust and high-throughput screening assays to discover functional probes, and iii) structural characterization of the MALAT1 triple helix conformational landscape for small molecule-based regulation. In the first area, we have discovered first small molecule ligands for the MALAT1 triple helix and gained insights into the role of three-dimensional small molecule shape and intramolecular interactions in binding events. Additionally, we established a correlation between ligand-induced thermal stabilization and prevention of exonucleolytic degradation of this target and elucidated binding properties of ligands with triplex-stabilizing functions. In the second area, we developed a high-throughput screening (HTS) technique based on differential scanning fluorimetry and curated an RNA-targeted library to identify novel small molecules with a destabilizing effect on the triple helix. Such thermal melting profiles were predictive of accelerated degradation profiles of the triple helix, thereby providing a robust discovery tool for identifying functional small molecule modulators of this target. In the third area, we conducted chemical probing experiments to characterize secondary structures that may exist in equilibrium with the triple helix and hence represent attractive targets for small molecule stabilization.
Together, this work has and will continue to build a set of small molecule tools, rapid assays, and novel design strategies to enable chemical biology-based interrogation of the MALAT1 triple helix role in cancer biology. This knowledge and tools can be utilized to interrogate the protective mechanism of the MALAT1 triple helix, to identify and develop therapeutic leads, as well as to apply these strategies for the study of other therapeutically relevant RNA.
Item Open Access Targeting Inducible Heat Shock Protein 70 in Cancer and Dengue Virus Pathogenesis with a Novel Small Molecule Inhibitor(2015) Howe, Matthew K.Inducible Heat shock protein (Hsp70i) is a protein chaperone that is utilized during tumorigenesis and viral infections for efficient propagation. Overexpression of Hsp70i is observed in a wide spectrum of human tumors, and this overexpression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Hsp70i aids in cancer cell propagation through regulation of anti-apoptotic and cell survival pathways. Furthermore, Hsp70i is induced following infection for several viruses and aids viral propagation, in part through regulation of anti-apoptotic pathways as well as promoting the folding of newly synthesized proteins. Due to the parallel role of Hsp70i in both cancer and viral pathogenesis, identification of small-molecule inhibitors selective for Hsp70i could provide tools for the development of novel therapeutics and further elucidate the role of Hsp70i in both cancer and viral infections.
To date, few Hsp70 inhibitors have been identified and characterized, and their efficacy in clinical settings is unknown. Through the fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, an allosteric inhibitor selective for Hsp70i was identified, called HS-72. We show that HS-72 is highly selective for Hsp70i, over the broader purinome and other Hsp70 family members, in particular the closely related constitutively active Hsp70 family member, Hsc70. Additionally, HS-72 acts as an allosteric inhibitor to induce a conformational change and inhibit Hsp70i activity. HS-72 displays hallmarks of Hsp70i inhibition in vitro by promoting Hsp70i substrate protein degradation, protein aggregation, and selective growth inhibition of cancer cells. In wild type mice HS-72 is well tolerated and a limited PK study shows HS-72 is bioavailable. Furthermore, in a MMTV-neu breast cancer mouse model, HS-72 shows efficacy to inhibit tumor growth and promote survival.
Due to the similar utilization of Hsp70i in cancer and viral pathogenesis, this suggests the potential for HS-72 as an antiviral agent. Dengue virus (DENV) is of great public health importance due to estimates of up to 400 million infections per year, coupled with the geographic distribution of the virus, which is now endemic in over 100 countries worldwide. There is also a pressing need for DENV interventions, owing to the lack of approved vaccines or antiviral therapies. DENV is reliant on host factors throughout the viral life cycle and Hsp70i has been implicated as a host factor in DENV pathogenesis. Additionally, the complete role of Hsp70i in DENV pathogenesis remains to be elucidated, highlighting a unique opportunity to use HS-72 as a tool to specifically probe Hsp70i function. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Furthermore, inducing Hsp70i expression prior to infection, correlates with an increase in DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained, through inhibiting the entry stage of the viral life cycle. Following infection, Hsp70i localizes to the cell surface and interacts with the DENV receptor complex to mediate viral entry. While, HS-72 treatment results in a disruption of the interaction of Hsp70i with the DENV receptor complex, yielding a reduction in infected cells.
Collectively this work further supports Hsp70i as an anticancer and anti-dengue virus target, and identifies HS-72, a chemical scaffold that is amenable to resynthesis and iteration, as an ideal starting point for a new generation of therapeutics targeting Hsp70i.