Browsing by Author "Santos, Webster"
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- Developing Sphingosine-1-Phosphate (Spns2) Inhibitors for the Treatment of Multiple SclerosisShrader, Christopher Wayne (Virginia Tech, 2024-02-29)
- Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence CompoundsDye, Keane J.; Vogelaar, Nancy J.; O'Hara, Megan; Sobrado, Pablo; Santos, Webster; Carlier, Paul R.; Yang, Zhaomin (American Society for Microbiology, 2022-12)Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson's disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics.IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.
- Further Exploring the Structure Activity Relationship (SAR) of MMV008138 and MMV1803522Li, Haibo (Virginia Tech, 2023-06-06)The war between human and malaria has never stopped, and the development and application of antimalarial drugs has not eradicated this terrible disease. To fight drug-resistant malaria, many leads have been studied over the years. (1S,3R)-MMV008138 and MMV1803522 are two compounds that have been studied in the Carlier Group. My research focused on the structural variation of each of these compounds, in the hope that greater potency could be realized. Chapter 2 describes my work on (1S,3R)-MMV008138, which inhibits the enzyme PfIspD in the methylerythritol phosphate (MEP) pathway. This compound shows good in vitro potency against the drug resistant Dd2 strain of Plasmodium falciparum. However, this lead showed no activity in mouse models. This lack of activity may be due to poor metabolic stability of the compound. However, a significant increase in in vitro potency could also improve in vivo activity. Towards that end, I focused on further variation of the D-ring and A-rings. With the regard to the D-ring, we made five analogs of MMV008138 that replaced the 2,4-dichlorophenyl ring with dihalogenated thiophen-3-yl and thiophen-2-yl rings. We also explored the effect of installing a cyano group on the A-ring of MMV008138. Unfortunately, none of these new compounds were potent growth inhibitors of Dd2 strain P. falciparum. We conclude that the lead goes into a well-defined pocket within the PfIspD enzyme that only accommodates 2,4-dihalogenated phenyl D-rings. This pocket also cannot accept any substitution larger than F on the A-ring. Interestingly, the crystal structure of 5-cyano-substituted MMV008138 was obtained ((±)-2-50c). This is the first compound out of more than 100 analogs of MMV008138 family to be amenable to crystallization. The solid state conformation of the (±)-2-50c revealed that the C3-carboxyl group was in a pseudoequatorial orientation, and the C1-aryl group was thus in a pseudoaxial orientation. 1H NMR spectroscopic studies in CD3OD-D2O were carried out to determine the solution conformation. As expected from previous studies of ester derivatives of MMV008138, these studies indicated that in solution, 2-5 would adopt both the C3-carboxyl pseudoequatorial and pseudoaxial conformations. In Chapter 3, I describe the synthesis of analogs of the antimalarial drug candidate MMV1803522. This β-carboline-3-carboxamide shows good in vitro growth inhibition potency of Dd2 strain P. falciparum, operating by a still unknown mechanism. To investigate the pharmacophore of this lead, I first sought to determine whether the pyridine N (i.e. N2) of the β-carboline was important for in vitro potency. I prepared series of carbazole analogs of MMV1803522, which replace N2 with a CH. These compounds potently inhibited the growth of Dd2 strain P. falciparum. These results suggest that N2 of MMV1803522 is not involved in any energetically significant interactions with its target protein. To further identify the pharmacophore, we prepared truncated analogs lacking the A- and B- rings (biphenyl analogs), and tricyclic analogs that feature a reversed indole moiety. Unfortunately, the biphenyl analogs and reversed indole analogs show no growth inhibition at 10,000 nM the highest concentration tested. Lastly, I describe analogs of MMV1803522 in which the 3,4-dichlorophenyl ring of MMV1803522 was replaced with halogenated thiophene. This substitution was tolerated, but compounds were roughly half as potent as MMV1803522.
- Fused Heterocycles as Spinster Homolog 2 Inhibitors and Regio- and Stereoselective Copper-Catalyzed Borylation-Protodeboronation of 1,3-Diynes: Access to (Z)-1,3-EnynesBurgio, Ariel Louise (Virginia Tech, 2023-05-15)Sphingosine 1-phosphate (S1P) is a lipid chemoattractant molecule. Once formed, S1P can be transported extracellularly by S1P transporters spinster homolog 2 (Spns2) or major facilitator domain containing 2B (mfsd2b). In the extracellular space, S1P can bind to S1P-specific G-protein coupled receptors (S1PR), which initiate many signaling pathways. A critical role of extracellular S1P is its ability to cause lymphocyte egress, which can have implications for inflammatory and autoimmune diseases. For this reason, there has been a growing interest in exploring potential spns2 inhibitors to further elucidate their therapeutic potential. Initial screenings confirmed that fused heterocycles, including phthalimide and benzoxazoles, demonstrated moderate inhibition of Spns2 using a HeLa cell assay. An extensive structure-activity relationship (SAR) study of these scaffolds was performed to analyze the impact of various amine head groups, regioisomers, and alkyl tails on performance. It was determined that 2-aminobenzoxazoles with secondary amines were potent inhibitors of the transporter. Additionally, the position of the lipophilic tail moiety played a large role in activity. From these modifications, SLB1122168 (2.44p) was found to be our lead compound. It was determined that (2.44p) had an IC50 of 94 ± 6 nM and was shown to be efficacious in decreasing lymphocyte count by 55% in a dose-dependent manner in both rat and mice models. The discovery of (2.44p) can serve as a novel chemical tool to investigate Spns2 biology and use it as a probe to determine the potential of Spns2 as a drug target. Organoboron compounds are useful synthetic intermediates in forming C-X, C-C, and C-H bonds. One way to synthesize these compounds is through copper catalysis. Copper is favorable to other transition metals because it is an Earth-abundant, low-cost metal that can be utilized in regio- and stereoselective reactions. Conjugated 1,3-enynes are important functional groups that iii are found in active natural products, organic synthetic intermediates, and materials. Previous methods used rare transition metals, designer ligands, or harsh acidic conditions to synthesize such compounds. In this dissertation, we developed a stereoselective one-pot copper-catalyzed semi-reduction of 1,3-diynes to produce (Z)-1,3-enynes. This method uses Cu(OAc)2, HBpin and Xantphos to successfully synthesize (Z)-1,3-enynes that were tolerated well over a broad substrate scope, including heterocyclic, alkyl, and aryl substituents. It was determined that this reaction went through a 2-boryl intermediate which was facilitated by a CuH species.
- Improving Potency and Oral Bioavailability of Spinster Homolog 2 (Spns2) Inhibitor: A Structure-Activity Relationship StudyDunnavant, Kyle Jacob (Virginia Tech, 2024-06-13)
- Optimization of Enantiopure tetrahydro-β-carbolines as Potent Antimalarials and Exploration of salicylic acid analogs for combating multidrug-resistant Neisseria gonorrhoeaeAlMolhim, Hanan Suliman (Virginia Tech, 2023-05-15)The emergence of drug resistance towards existing drugs is a constant challenge in the fight against many diseases including Malaria and gonorrhoeae. To evade resistance, new targets must be engaged, and to do that, new structural classes of anti-infective must be prepared and evaluated. During the course of my PhD journey, I had the opportunity to investigate and optimize the antimalarial candidate (±)-2-3b, and salicylic acid (4-1a) as an anti-gonorrhea treatment. Malaria is a life-threatening mosquito-borne disease. In 2021, there were 247 million cases of malaria and the estimated number of malaria deaths stood at 619,000. Because of the rapid development of resistance to all current antimalarials, discovery of antimalarials with unexploited mechanisms of action is critical to reduce malaria mortality. In the Carlier group, our initial approach focused on discovery of inhibitors of the methylerythritol phosphate (MEP) pathway for isoprenoid precursor biosynthesis, since this pathway is essential for Plasmodium falciparum and absent in human. Application of the isopentenyl pyrophosphate (IPP) chemical rescue screen to the compounds of the Malaria Box, a collection of 400 antimalaria candidates with unknown mechanisms of action, identified tetrahydro-β-carboline 2-1 (MMV008138) as an inhibitor of the MEP pathway. Chapter 2 of this work discusses similarity searching of the Novartis portion of the hit set (5K compounds), from the original 20K compound hit set of the Malaria Box, and identifying tetrahydro-β-carboline GNF-Pf-5009, designated as (±)-2-3b. Preparation of pure enantiomers, by resolution, demonstrated the pharmacological superiority of (R)-2-3b over (S)-2-3b, which was found to have good asexual blood stage (ABS) inhibition potency against malarial parasites P. falciparum, and low general cytotoxicity. However, (R)-2-3b was found not orally efficacious in a P. berghei mouse model of malaria. We concluded that the lack of oral efficacy of (R)-2-3b was due to its poor drug-like qualities, in particular its high molecular weight and low solubility. Chapter 3 of this work explores modifications of (R)-2-3b ((R)-3-5Aa) that were expected to improve its properties. We show that the new compounds (R)-3-5Gm and (R)-3-5Gk not only are more potent in vitro than (R)-2-3b ((R)-3-5Aa), but also have molecular weights < 500 g/mol. Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea. Due to the increased rates of infection as well as the prevalence of multidrug-resistant N. gonorrhoeae strains worldwide, the World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) list N. gonorrhoeae at the highest possible threat level to public health. Dual therapy of azithromycin (AZM) and ceftriaxone has been the standard-of-care for treatment of gonococcal infections. However, due to increasing resistance to azithromycin (>33% in some regions) the CDC removed AZM from the treatment regimen for gonorrhea in 2020. Therefore, ceftriaxone remains the only recommended antibiotic for treatment of gonococcal infections. However, increasing resistance to this treatment option has been reported, consequently there is an urgent need to identify novel therapeutics against N. gonorrhoeae. Drug repurposing is a popular strategy that explores new therapeutic opportunities for approved drugs with available information on their pharmacokinetic data, dosages, and toxicity. Salicylic acid is a highly privileged chemical scaffold. Also, the use of salicylic acid to treat sexually transmitted diseases (including gonorrhea) was reported as early as the 19th century. Recently, Dr. Mohamed N. Seleem reported that salicylic acid (4-1a) exhibited modest activity against N. gonorrhoeae strains including the AZM-resistant strain (CDC-181). Chapter 4 of this work illustrates how the anti-gonococcal activity in this scaffold is easily lost by inopportune substitution. However, we found that substituted naphthyl analogs (4-3b,o,p) have superior activity to salicylic acid itself. In addition, the three analogs showed high selectivity, compared to AZM, against N. gonorrhoeae over the vaginal microbiota.
- Stimuli-Responsive Peptide-Based Biomaterials: Design, Synthesis, and ApplicationsZhu, Yumeng (Virginia Tech, 2023-05-15)Peptide-based biomaterials have gained much interest in various applications in drug delivery and tissue engineering in recent years, in large part due to their typically excellent biocompatibility and biodegradability. Composed of different amino acids, peptides can be designed with numerous sequences, providing flexibility and tunability in biomaterials. Peptides are easy to modify with small molecule drugs, inorganic components, and polymer chains to access multiple functions and tune properties relevant to biology and medicine. Stimuli-responsive peptide-based biomaterials can respond to environmental stimuli, such as light and ultrasound, in addition to local environmental factors, such as temperature, enzyme activity, and pH. Under environmental changes, these materials can be triggered to release therapeutic payloads, change conformations, or induce self-assembly in the target sites. In this work, I introduce the design, synthesis, and potential applications of several stimuli-responsive peptide-based biomaterials. The first half of this dissertation is based on enzyme-responsive, peptide-based biomaterials as extracellular matrix (ECM) mimics in tissue engineering. We synthesized linear and dendritic elastin-like peptides (ELPs) as crosslinkers and conjugated them with hyaluronic acid (HA) to form hydrogels. Trypsin was used as the enzyme trigger for cleaving the C-terminal lysine and to study how crosslinker topology affects enzymatic degradation. Hydrogels with dendritic ELPs degraded more slowly than linear ELPs, providing a novel strategy to tune the degradation rate of hydrogels as ECM mimics by the molecular design of crosslinker topology. Building on this peptide-polysaccharide platform for synthetic ECM design, we subsequently prepared hydrogels embedded with bioactive cryptic sites. These novel polymeric hydrogels mimicked native ECM cryptic sites by using depsipeptides that undergo an enzyme-triggered molecular rearrangement, "switching" from a non-functional epitope to a bioactive sequence. Mass spectrometry, 1H and 13C NMR spectroscopy, and fluorescence studies were applied to track structural changes in the peptide. SEM was used to image these polymer-peptide hybrid hydrogels. Finally, in vitro studies were conducted to evaluate cell interactions with the hydrogels. Switch peptide-modified alginate hydrogels showed increased cell adhesion upon induction of enzymatic activity, which provided a "gain of function" of the synthetic ECM. Critically, enzymes associated with the cells themselves could trigger the peptide switch and change in synthetic ECM behavior. With knowledge of stimuli-responsive peptide-based biomaterials applied in tissue engineering, I then studied how this system could be used in drug delivery by designing peptide-hydrogen sulfide (H2S) donor conjugates (PHDCs). H2S is a gasotransmitter that is produced endogenously, which has been explored in recent years with many potential therapeutical applications. We studied H2S release profiles in dual-enzyme-responsive PHDCs, with a further investigation into PHDC–Fe2+ complexes for potential tumor treatments via chemodynamic therapy. The PHDC–Fe2+ complexes were examined in a C6 glioma cell line, exhibiting an improved cell-killing effect compared with controls, by inducing toxic hydroxyl radical generation (•OH) via a Fenton reaction. To this end, we further discovered how side chains influence self-assembling nanostructures, H2S release profiles, and biological activities via three constitutionally isomeric PHDCs. Different morphologies and varied H2S release rates were observed, paving the way for tuning the properties of PHDCs by simple changes in molecular design. Finally, this dissertation discloses conclusions and future directions on stimuli-responsive peptide-based biomaterials using similar platforms with different designs in the drug delivery and tissue engineering fields.
- The Voltage Gated Sodium Channel β1/β1B subunits: Emerging Therapeutic Targets in the HeartWilliams, Zachary James (Virginia Tech, 2024-01-11)Voltage-gated sodium channels are composed of pore-forming α-subunits, and modulatory and multifunctional associated β subunits. While much of the field of cardiac electrophysiology and pathology has focused on treating and preventing cardiac arrhythmias by targeting the α subunit, there is also evidence that targeting the β subunits, particularly SCN1B, the gene that encodes β1 and an alternatively spliced variant β1B, has therapeutic potential. The first attempt at targeting the β1 subunit was with the generation of and treatment with an SCN1B Ig domain mimetic peptide βadp1. Here we describe further investigation into the function and mode-of-action of both βadp1 and novel peptides derived from the original βadp1 sequence. We find that in a heterologous expression system βadp1 initially disrupts β1-mediated trans-homophilic adhesion, but after approximately 30 hours eventually increases adhesion. Novel mimetic dimers increase β1 adhesion up to 48 hours post-treatment. Furthermore, it appears that βadp1 may increase β1 adhesion by upregulating the intramembrane proteolysis of β1, a process which has important downstream implications and effects on translation. Despite these exciting findings, we were unable to translate them into a primary culture of cardiac cells with endogenous expression of β1 because we found that both neonatal rat cardiomyocytes and isolated adult mouse cardiomyocytes do not express β1 at detectable levels, whereas they do appear to express β1B. In summary, we show exciting findings on the function and mode-of-action of SCN1B mimetic peptides and their therapeutic potential in targeting the β1 subunit, but further work is needed to determine the translatability of our findings to in vivo models and eventually to humans.