Herman O. Sintim, DPhil - US grants

With this CAREER Award, the Organic and Macromolecular Chemistry program and the Cellular Systems Cluster program are supporting the research of Professor Herman O. Sintim of the University of Maryland, College Park. Professor Sintim will develop new chemical tools to study 3'-5'-cyclic diguanylic acid (c-di-GMP) signaling in bacteria. C-di-GMP, a common chemical messenger present uniquely in bacteria, plays a central role in bacterial biofilm formation and regulation of virulence-related factors in diverse bacteria. Several questions remain unanswered in c-di-GMP signaling in bacteria. For example, the environmental cues that modulate c-di-GMP signaling in bacteria and the identities of the adaptor proteins that respond to fluctuations in intracellular concentration of c-di-GMP remain largely uncharacterized. The first objective is to develop a fluorescent sensor for c-di-GMP. Such a sensor will be useful for establishing a link between different environmental cues and the intracellular concentrations of c-di-GMP. Secondly, a new solid-phase methodology will be used to prepare photo-affinity c-di-GMP analogs for the identification of c-di-GMP adaptor proteins. The identification of c-di-GMP binding proteins should help unravel the complex signaling network that c-di-GMP apparently regulates.

Professor Sintim will also establish four Percy Julian undergraduate research fellowships at the University of Maryland. This research fellowship will be open to underrepresented minorities in the sciences. Percy Julian fellows will be supported to conduct research in the laboratory of an established investigator at the University of Maryland. This fellowship provides the opportunity to nurture minority students at an early stage of their careers and encourage such students to consider a career in the sciences.

Professors Michael P. Doyle, Jeffrey T. Davis, Daniel E. Falvey, Lyle D. Issacs and Herman O. Sintim of the University of Maryland have submitted a proposal in response to the CRIF: MU solicitation to acquire a high resolution double sector mass spectrometer. The research projects it will support are related to a variety of studies including: (i) synthesis and application of unnatural amino acids; (ii) characterization of small molecule intermediates used in supramolecular chemistry, molecular recognition, and the assembly of receptors for transmembrane ion-transport; (iii) development of highly selective and efficient catalytic processes for the synthesis of biologically relevant compounds; (iv) the study of photo induced electron transfer reactions and the generation of novel high spin organic species; (v) development of small molecule probes for electron and electrophile migration along strands of DNA; and, (vi) total synthesis of antibiotics of the family of Platensimycin.

Mass spectrometry (MS) is used to identify the chemical composition of a sample and determine its purity. A high resolution mass spectrometer has the capability of performing accurate elemental composition analysis of compounds. This makes it a powerful tool for identification of known and unknown or new compounds. This acquisition will benefit undergraduate and graduate students in their research and in a new experimental course to be developed. Students and faculty at Howard University Virginia State University and Catholic University will also use it to analyze samples.

With this award, the Chemistry of Life Processes program is supporting the research of Professors T. Kwaku Dayie and Herman O. Sintim of the University of Maryland at College Park. Professors Dayie and Sintim will examine the structural and dynamic basis of riboswitch RNA regulation, i.e. turning on and off gene regulatory circuits in bacteria. While X-ray crystallography has become a powerful tool to unravel the nature of the riboswitch RNA bound to its ligand, it usually fails to characterize the unbound state. This proposal aims to develop tools such as nuclear magnetic resonance (NMR) spectroscopy and small angle x-ray scattering (SAXS) to provide complementary approaches to better understand the nature of riboswitch transitions from the unbound state to a competently folded and functional state. A critical component of these efforts will be to develop techniques to more efficiently generate isotopically enriched RNAs for NMR studies.

The Broader Impacts of this project include deepening our understanding of RNA signaling at the heart of gene regulation. Perhaps most important is the education and training of students who work on this project, Undergraduate and graduate student researchers on the team will be exposed to a multi-disciplinary training environment, including the use of NMR and computational modeling, synthetic organic chemistry and enzymology, with the goal of understanding how RNA biomolecules control important cellular processes of catalysis and gene regulation at the molecular level.

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Sintim, Herman O.

This award is supporting the research of Professors Herman O. Sintim, William Bentley and Gary Rubloff of the University of Maryland at College Park. The team will develop multimodal and multifunctional microfluidic systems for studying cell signaling by integrating stimuli-responsive biofabrication with device-imposed, complex gradient generation of bacterial signaling molecules. This device will then be used to systematically investigate the response of bacterial cells to quorum sensing molecules and environmental cues. Additionally, the device will be used to identify new molecules that inhibit bacterial chemotaxis.

Due to the central role that bacterial cell signaling plays in bacterial physiology, there is a high interest in understanding or unraveling the various factors that control bacterial response to signaling molecules. Transformative technologies that can aid the screening of molecules that inhibit bacterial communication would have potential applications in medicine, agriculture and industry. The broader impact of this project includes development of a new technological platform to study how bacteria interact with each other and the environment. This project is highly multidisciplinary, involving chemical biologists, bioengineers and biosystems engineers. Therefore, the students who will be involved with the project will be trained to solve scientific problems using diverse approaches.

This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.

With this award, the Chemistry of Life Processes program is supporting the research of Professor Herman O. Sintim of the University of Maryland at College Park. Professor Sintim will develop new tools to study the molecular mechanisms that govern biofilm formation and development in bacteria. Dinucleotides act as second messengers in bacteria to regulate bacterial biofilm and virulence factors production but many receptors that are involved in dinucleotide signaling are yet to be identified or fully characterized. This proposal aims to develop probes for the identification and characterization of new nucleotide receptors in bacteria. Efforts will also be directed towards the development of synthetic molecules that strongly associate with second messenger nucleotides to disrupt the biological activities of the signaling nucleotides. These molecules could be used to modulate the transition between the biofilm and planktonic states.

Microbial biofilms are sometimes responsible for the clogging of pipes at homes or industrial settings. Additionally, biofilms that form in humans or livestock contribute to the persistence of microbial infections. Currently there are only a handful of non-toxic compounds that can effectively disperse microbial biofilms. Therefore studies that shed more insights into biofilm formation by microbes could lead to the identification of new strategies to eradicate biofilms. The broader Impacts of this project include increasing our understanding of how bacteria form biofilms, which could be useful for the development of strategies to curb bacterial biofilm formation. With regards to STEM education, high school students and teachers, undergraduate and graduate student researchers who work on this project will develop diverse skill sets, including chemistry, molecular biology and microbiology. This multi-disciplinary experience will prepare such students for diverse careers.

This award is co-funded by the Systems and Synthetic Biology Cluster in the Molecular and Cellular Biosciences (MCB) Division of the Biological Sciences (BIO) Directorate.

DESCRIPTION (provided by applicant): Identification of cellular sensors for key parameters such as lateral pressure in the lipid bilayer and degree of cytoplasm hydration will not only advance our basic understanding of cell physiology and mechanics, but can also be used in the development of bio-inspired sensor devices for probing the environment and for screening potential pharmaceuticals. Mechanosensitive Channel of Small Conductance (MscS) is a ubiquitous osmolyte release channel found in all phyla of organisms with cell walls. Esherichia coli MscS, the best understood representative, is directly activated by membrane tension and inhibited by increased crowding pressure of polymers in the cytoplasm. Crystal structures predict that the transmembrane domain of MscS senses tension, whereas the hollow cytoplasmic domain (cage) perceives crowding pressure and adjusts tension sensitivity and duration of opening according to the degree of cytoplasmic hydration. Additionally, due to the asymmetric position of the gate relative to the membrane midplane, MscS is more sensitive to lateral pressure/tension in the inner leaflet. Activating tensions are strongly influenced by amphipathic substances, and therefore the channel can be used as an endogenous sensor of drug partitioning into the native bacterial membrane. In this project we combine experimental and computational efforts of three groups aimed to explore different sensing modalities of MscS and approach the practical design of a lateral pressure sensor. More specifically, we propose to (1) simulate intercalation of several biologically active compounds into the lipid bilayer and compute changes in lateral pressure profiles using Molecular Dynamics. We will then use these results to simulate MscS expansion to identify intermolecular interactions in the channel that can influence sensitivity to asymmetric tension. (2) Based on these results, we will re-engineer MscS for higher sensitivity and stability. The channel will be calibrated in the presence of substances causing known pressure shifts determined using independent surface chemistry techniques, and then used for practical screening and characterization of several antibiotics and their synthetic analogs. In order to understand the mechanism of MscS inactivation by cytoplasmic crowding, we will (3) computationally explore the conformational dynamics of the hollow cage domain, excluded volumes and compressibilities in different conformations, and the coupling with the pore-lining helices. (4) Conformations identified by computations as functionally important will be tested experimentally through mutagenesis and detailed patch-clamp analysis in the presence of crowding agents. The project will establish the very first sensor-based platform for monitoring incorporation and permeation of amphipathic substances through native bacterial membranes. It will also reveal the allosteric interplay between the membrane-embedded and cytoplasmic domains of MscS, and the biophysical principle by which cells measure the extent of cytoplasmic hydration, thus opening the opportunity for the design of bio-inspired osmosensors.

Lyme disease has emerged as a major public health threat in the US. Currently, no human vaccine is available. Although targeting tick populations with chemical acaricides is an effective way of reducing the risk of infection, the use of acaricides has adverse health effects and raises environmental concerns. A host-targeted approach by deploying doxycycline hyclate-laden baits to the field showed dramatic reduction of infection rates in both rodent reservoirs and Ixodes scapularis ticks by Borrelia burgdorferi, the agent of Lyme disease. However, antibiotics such as doxycycline are not suitable for such purpose as they are used for treating patients. Nevertheless, such proof of concept study demonstrates that a host-targeted approach using alternative compounds is a promising approach to eliminate spirochetes in rodent reservoir hosts and tick vectors. The bacterial second messengers, c-di-GMP and c-di-AMP, have emerged as central regulators for bacterial physiology and are potential drug targets. Many bacteria encode multiple copies of cyclases for the synthesis of c-di-GMP and c-di-AMP, which make it difficult to target the pathways. Borrelia burgdorferi only has a single diguanylate cyclase Rrp1 and a single diadenylate cyclase CdaA for c-di-AMP synthesis, which makes them attractive drug targets. As shown in the preliminary data, we found that these two cyclic dinucleotide, one controls spirochetes? survival in each of the two hosts in B. burgdorferi enzootic cycle, ticks and mammals: while c-di-GMP is essential for tick colonization, c-di-AMP is indispensable for mammalian infection. The hypothesis of this proposal is that small molecule inhibitors targeting c-di-GMP and c-di- AMP cyclases, Rrp1 and CdaA, would eliminate B. burgdorferi in ticks and mammalian reservoirs, which can be exploited to reduce Lyme disease incidence. The co-PI of this proposal, Dr. Herman Sintim, a Drug Discovery Professor of Chemistry, pioneered developing inhibitors for bacterial diguanylate cyclase and diadenylate cyclase, and his group has already reported several potent inhibitors against these cyclases of other bacteria. Accordingly, we propose to develop a strategy to target CdaA of B. burgdorferi to eliminate in spirochetes rodent reservoirs (Aim 1), and a strategy to target Rrp1 of B. burgdorferi to eliminate spirochetes in ticks (Aim 2). We will also test a combination of inhibitors targeting both pathways to eliminate B. burgdorferi in its enzootic cycle. The underlying mechanisms of how c-di-AMP and c-di-GMP are employed by B. burgdorferi to survive in mammals and ticks will also be investigate. This proposal address one of the specific Focus Areas listed in this RFA: reservoir-targeted approaches to interrupt the natural history of infection. Upon accomplishing the proposed work, further field experiments will be conducted using animal baits containing a combination of both inhibitors to evaluate their effects on reducing spirochete burden in ticks and animals in nature. Such host-targeted strategy will have significant impact on combating Lyme disease.

PROJECT SUMMARY Targeting MYC promoter G-quadruplex for MYC inhibition by Indenoisoquinolines G-quadruplex (G4) DNA is a globular DNA secondary structure and considered as a new class of molecular targets for anticancer drugs. MYC, one of the most commonly deregulated genes in human cancers, has a DNA G4 motif in its promoter that functions as a transcriptional silencer. Compounds that bind to and stabilize the G-quadruplex formed in the MYC promoter have been shown to significantly lower MYC levels in cancer cells. Thus, the MYC promoter G-quadruplex (MycG4) represents a novel target for MYC inhibition by small molecules. However, little is known about how MycG4 is regulated by proteins and development of MycG4- targeting drugs has been focused solely on G4 DNA. Whereas drug-DNA interactions may be insufficient for MYC inhibition, the effective mechanism of drug action could involve protein-DNA interactions, which is analogous to topoisomerase inhibitors. Very recently, we have discovered that indenoisoquinolines, a clinically tested scaffold with excellent drug-like properties, are strong MycG4 binders and potent MYC inhibitors. We have also discovered that the DDX5 helicase actively unfolds MycG4 and is critically involved in MYC gene transcriptional activation. These results provide new and critical insights to effectively downregulate MYC transcription by targeting MycG4 and its interactions with DDX5. Our central hypothesis is that indenoisoquinolines effectively suppress MYC transcription by binding to the MYC promoter G-quadruplex and disrupting DDX5-MycG4 interactions. The overall objective is to determine the molecular mechanism of effective MYC inhibition by indenoisoquinolines, establish the structure?activity relationships (SAR), and discover lead indenoisoquinolines for preclinical testing. The long-term research goal is to develop potent indenoisoquinoline MYC inhibitors as new anticancer drugs. The specific aims are: 1) Structural characterization of the MycG4-indenoisoquinoline complexes. 2) Establishing a compound library to determine indenoisoquinolines that bind MycG4 and inhibit MYC. 3) Determining the effect of MycG4-interactive indenoisoquinolines on DDX5 unfolding of the MYC promoter G4 and how this correlates with MYC suppression. 4) Designing and synthesizing optimized indenoisoquinolines for MYC suppression using structure-based rational approach; establishing SAR for MycG4-binding and inhibition of DDX5 unfolding. The expected outcome of this work is a determination of the SAR of indenoisoquinolines for MycG4-targeting, demonstration of the effective MYC suppression by inhibiting DDX5-MycG4 interaction, and discovery of lead compounds for future preclinical testing. The results will have an important positive impact because they lay the groundwork to develop new indenoisoquinoline anticancer drugs with MYC-targeted activity.