Small molecule modulation of the 14-3-3 interactome
James H. Frederich
Florida State University
14-3-3 proteins are an abundant family of adapter molecules that regulate the activity of several hundred cellular client proteins by forming transient protein-protein interactions (PPIs). Many of these PPIs are involved in phosphorylation-dependent signaling pathways that govern cellular homeostasis. Consequently, 14-3-3 PPIs are attractive targets for drug discovery; however, the development of a small-molecule that can selectively target one 14-3-3 PPI among several hundred has been a major challenge. Herein, we present our efforts to craft selective 14-3-3 PPI stabilizers from the natural product fusicoccin A.
Exploring (R)-9bMS, a small molecular inhibitor of ACK1-AR epigenetic circuitry in Triple Negative Breast Cancer
Mithila Sawant, Nupam Mahajan
Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612, USA
Department of Oncological Sciences, University of South Florida, Tampa, FL 33612, USA
The highly aggressive Triple Negative Breast Cancers (TNBCs) constitute approximately 20% of total breast cancers. The unavailability of a genuine target in TNBCs, unlike ER/HER2 positive cancers restricts their treatment modalities. Recent studied in our lab have identified ACK1 (also known as TNK2) non-receptor tyrosine kinase as a critical contributing factor towards the aggressiveness and invasive capacity of TNBCs. A positive correlation between the expression of ACK1 and high proliferation rate, invasion and colony forming potential of TNBCs was observed, which could be abolished by genetic ablation of ACK1. In addition, recent studies from our lab have established the existence of positive feedback circuit wherein ACK1 directly phosphorylated histone H4 at Tyr88 residue, upstream of AR gene locus. This novel epigenetic role of ACK1 in regulating AR expression was shown to be crucial for developing drug-resistance in Castration resistant prostate cancer cells (CRPCs). Numerous recent reports have emphasized on the emerging role of Androgen Receptor (AR) as a prospective target in breast cancer. Thus exploring ACK1 and its downstream effector AR as plausible hits to circumvent the problem of lack of expression of targetable receptors may provide clinical benefits in TNBCs. We have designed a small molecular inhibitor of ACK1, (R)-9bMS, which has shown to be potent inhibitor of prostate cancer and TNBCs. (R)-9bMS stalled the cell proliferation of high ACK1 and AR expressing TNBC cell lines. (R)-9bMS was successful in not only diminishing the ACK1 expression, but also exerted an epigenetic shutting-down of AR transcription, reflected in loss of TNBC xenograft tumor growth. Our data strengthens the candidature of (R)-9bMS as potent anti-cancer agent and accentuates that abolishing the ACK1 addiction of TNBCs using ACK1 small molecular inhibitors could serve as a potential therapeutic intervention
Interactions of Ruthenocenyl-Conjugated Antibiotics with CTX-M β-Lactamase
Eric M. Lewandowski
University of South Florida
A series of novel ruthenocenyl-conjugated β-lactams were synthesized in order to investigate the antibacterial properties of the ruthenocenyl moiety against Penicillin Binding Proteins (PBPs). The β-lactams inhibit PBPs by forming a covalent acyl-enzyme complex with the catalytic residue. The active site of PBPs share a high level of homology with that of Class A β-lactamases, with a key difference being the presence of Glu166 in the β-lactamase active site. Glu166 allows β-lactamase to cleave the acyl-enzyme bond, thus allowing for the constant “turn over” of β-lactam antibiotics. We have solved a series of high-resolution X-ray crystal structures of ruthenocenyl-conjugated β-lactams in complex with CTX-M-14 Class A β-lactamase. Using a variety of CTX-M-14 mutants, we were able to capture several versions of hydrolyzed products, as well as intact compounds, in the CTX-M active site. These structures allowed us to make great insight into the CTX-M catalytic mechanism, and therefore, make inferences about the PBP catalytic mechanism. We also discovered that these compounds bind readily away from the active site and at the crystal-packing interface, with the ruthenocenyl moiety making many interactions with the surrounding residues. Some of the ruthenocenyl-conjugated β-lactams found to bind at the crystal-packing interface were compounds that had previously eluded small molecule crystallography efforts, suggesting that the CTX-M crystal system could potentially be used to solve the structure of other hard to crystallize small molecules.
Evolution of hybridization probes to DNA nanorobots for biosensing and gene therapy
Dmitry M. Kolpashchikova,b,c Amanda Cox,a,b Christopher Roldan,a,b Ola Kamar,a Anna F. Fakhardo,c Tatiana A. Lyalina,c Daria D. Nedorezova,c Alexander A. Spelkov,c Nadejda Y. Prokofeva, c Ekaterina A. Bryushkova, c Daria V. Nemirichc
ITMO University, Laboratory of Solution Chemistry of Advanced Materials and Technologies, Lomonosova St. 9, 191002, St. Petersburg, Russian Federation
Chemistry Department, University of Central Florida, Orlando, 32816, Florida, USA.
Burnett School of Biomedical Sciences, University of Central Florida, Orlando, 32816, Florida, USA
Hybridization probes remains one of the most common strategies for biosensing of specific DNA or RNA and have been extensively used in such formats as qualitative PCR, microarrays, florescent in situ hybridization (FISH), to name a few. Moreover, sequence specific recognition of RNA and DNA is on demand by gene silencing approaches including antisense, RNAi and CRISPR/cas9 technologies. However, all commonly used hybridization biosensors suffer from low selectivity at temperatures <40oC, inability to bind to long folded biological RNAs, high cost, low sensitivity. To address these problems, we construct multifunctional DNA nanomachines, in which each particular function is accomplished by a highly specialized component attached to a common DNA-based scaffold. Specialized functional units that are responsible for (i) highly selective sequence recognition; (ii) tight binding to target nucleic acids; (iii) unwinding RNA secondary structure; (iv) signal amplification; (v) fluorescent signaling and others, operate in cooperative manner upon target recognition. We designed DNA nanomachines for highly selective recognition of viral RNA and DNA, cleavage of viral RNA and cancer cell suppression. DNA nanomachines demonstrate improvements in the detection limits, recognition selectivity and efficiency of RNA cleavage in comparison with the traditional hybridization probes and cleaving agents. Possible future evolution of DNA nanomachines to DNA nanorobots for biomedical applications will be discussed.
Funding from NIAID (R15AI10388001A1) and NSF CCF 1423219 is greatly appreciated. D.M.K. was partially supported by the ITMO University Fellowship and Professorship Program.
Structure Studies of Spider Silk Dope
Geoffrey M. Gray and Arjan van der Vaart
Department of Chemistry, University of South Florida
Spider dragline silk possesses a unique combination of strength and elasticity. Its main constituents are two proteins (MaSp1 and MaSp2), that are stored in a high concentration (30-50%) dope before the silk is spun into a fiber. The dope is thought to form micelles that aggregate into liquid crystals, but the microstructure of the proteins in the dope remains unclear. To help elucidate secondary structure characteristics, enhanced sampling simulations were performed on several common dragline silk sequence motifs at various concentrations under dope-like conditions. Enhanced sampling was also performed in octanol to mimic a fiber environment. Additional pulling experiments were used to assess structural changes that occur under spinning-like conditions. Results show that the motifs resemble random coils under aqueous conditions, while showing some turns and helices in octanol. Pulling showed an increase in the 31-helical content, consistent with the idea that these structures form as part of the spinning process.