The Zoltowski Research Group is focused on using a combination of biophysical, computational, and cellular approaches to understand how organisms sense and adapt to the environment. Currently, specific focus is on understanding how environmental factors regulate our natural 24-hour biological rhythms (circadian clocks) and how organisms sense and navigate the earth’s magnetic field during seasonal migration. These projects interface with drug discovery in three fundamental ways:
Small Molecule Regulators of Cryptochrome
Circadian clocks couple environmental sensing to regulation of physiological processes through a core transcription/translation feedback loop. Misregulation of clock function is implicated in the onset and progression of obesity, diabetes, heart disease, psychological disorders, and cancer. Recently, two isoforms of the protein Cryptochrome (CRY1 and CRY2) have been identified as potential targets for therapeutic intervention, however the two isoform often function in divergent antagonistic roles. Further, despite progress in understanding CRY function, our knowledge of redundant and independent roles of CRY1 and CRY2 in disease is limited. The limitations in part stem from a lack of isoform selective small molecule regulators of CRY function. Therapeutic strategies have targeted two regulatory regions within CRY proteins. Pockets termed primary, and secondary in CRYs regulate interactions with Fbxl3 (primary), CLOCK (secondary), and PER (primary and secondary). Both regulatory pockets have near 100% sequence identify, limiting viable metrics to design isoform selective compounds. Recently, high-throughput screening methods, in combination with structural and biochemical characterization of CRY regulatory compounds indicate that isoform selectivity and function can be imparted via an isoform specific dynamic C-terminal tail (CTT). Currently, high-specificity ligands for CRY2, and selective compounds for the secondary pocket remain elusive. The primary limitation is a lack of structures containing intact CTTs, and understanding of CRY CTT regulation. Here we leverage biophysical techniques to decipher the structure of the CTT enabling the design of isoform specific CRY regulators via both rational and computational drug discovery.
Engineered Optogenetic Tools
Optogenetic tools leverage natural mechanisms of photoreceptors to engineer proteins enabling light-dependent control of cellular processes. The advent of optogenetics has transformed our ability to interrogate complex signaling networks, has greatly expanded our understanding of neurobiology, and has led to new methods to restore sight in blind individuals. Leveraging our expertise in understanding the structure and mechanism of blue-light photoreceptors we are actively involved in developing new optogentic tools that control gene expression and cellular localization in response to light. Further, we leverage our understanding of magnetoreception in migratory organisms to engineer first-in-class magentogenetic tools that alter cellular signaling in response to external magnetic fields. These reagents allow direct control of cellular processes, but also the development of complex engineered biological circuits and structural assemblies to aid in drug discovery and delivery.
Small molecule regulators of ACE2-Spike as novel COVID-19 Treatments
SARS-CoV2 the causative agent of COVID-19 has devastated global health and the economy. In the past 20-years, coronaviruses have emerged with increasing frequency, as such, it is imperative that we develop generalized strategies to treat and prevent SARS-like virus infections. For both SARS-CoV-2 and SARS-CoV cell entry is mediated by binding of the surface spike protein to the ACE2 receptor in human cells, thereby identifying the ACE2 receptor as a putative drug target for a generalized therapy for SARS-like infections. Development of ACE2 based therapeutics are currently limited by two primary factors: 1) The ACE2 receptor plays protective roles within the Renin-Angiotensin-Aldosterone-System (RAAS) to control inflammation, blood pressure, and immune responses. Currently, existing ACE2 inhibitors have been abandoned in the pharmaceutical industry due to deleterious side effects to the RAAS axis. Thus, inhibitors of ACE2 mediated viral entry must be selective to disrupting the ACE2-Spike complex. 2) The ACE2 protein exists in both open and closed conformations, and undergoes large scale conformational changes essential to its activity. Currently, little is known about ACE2 allosteric transitions and its role in viral infectivity and its diverse roles in human physiology. Thus, we leverage a unified computational, structural, and cellular analysis of ACE2 allostery and function. Our research platform leverages the expertise of the Kraka, Tao and Zoltowski groups with expertise in computational drug design, protein allostery, and cellular and structural biology. Our specific approach will consist of three aims designed towards a primary objective of developing allosteric regulators of SARS-CoV2 viral entry. Aim 1 will employ machine learning based approaches to screen molecular libraries in excess of 1-billion compounds to identify putative molecules that may impact ACE2 function. In Aim 2 these molecules will be tested in cellular contexts to examine their effect on ACE2 activity, and ACE2-Spike complex formation to identify compounds selective to disrupting cellular entry. Aim 3 will guide all efforts by using molecular dynamics and machine learning algorithms to identify allosteric transitions within the ACE2 protein that gate function and Spike recognition. The innovative multi-disciplinary approach will allow unprecedented insight into the allosteric mechanisms gating ACE2 function, while enabling the design and identification of small molecule regulators of viral entry. The resulting compounds will afford new modalities to explore the mechanism of ACE2 in diverse human pathologies, and new therapeutics targeting SARS-like viral infections.
Recent related publications:
Verma, Niraj, Qu, Xingming, Trozzi, Francesco, Elsaied, Mohamed, Karki, Nischal, Tao, Peng, Zoltowski, Brian D., Larson, Eric, Kraka, Elfi, “SSnet: A Deep Learning Approach for Protein-Ligand Interaction Prediction,” Int. J. Mol. Sci., 2021, 22, 1392; doi:10.3390/ijms22031392.
Karki, Nischal, Verma, Niraj, Trozzi, Francesco, Tao, Peng, Kraka, Elfi, Zoltowski, Brian D., “Predicting Potential SARS-COV2 Drugs - In Depth Drug Database Screening Using Deep Neural Network Framework SSnet, Classical Virtual Screening and Docking,” Int. J. Mol. Sci, 2021, 22, 1573; doi: 10.3390/ijms22041573.
Lara, Julia., Zoltowski, Brian D. “A tail of CRY selectivity” Nature Chemical Biology, 2020, 16, 6, 608-609.
Zoltowski, Brian D., Chelliah, Yogarany, Wickramaratne, Anushka, Jarocha, Lauren, Karki, Nischal, Xu, Wei, Mouritsen, Henrik, Hore, Peter J., Hibbs, Ryan E., Green, Carla B., Takahashi, Joseph S., “Chemical and structural analysis of a photoactive vertebrate cryptochrome from pigeon” PNAS, 2019, 39, 19449-19457; doi:10.1073/pnas.1907875116.
Pudasaini, Ashutosh, Shim, Jae-Sung, Song, Young-hun, Shi, Hua, Somers, David E., Imaizumi, Takato, Zoltowski, Brian D., “Kinetics of the LOV domain of ZEITLUPE determine its circadian function in Arabidopsis” eLIFE, 2017; doi:10.7554/eLife.21646.
Taslimi, Amir, Zoltowski, Brian D., Miranda, Jose G., Pathak, Gopal, Hughes, Robert M., Tucker, Chandra L., “Optimized second generation CRY2/CIB optical dimerizers and photoactivatable Cre recombinase,” Nat. Chem. Biol., 2016, doi:10.1038/nchembio.2063
Zoltowski, Brian D., “Resolving cryptic aspects of Cryptochrome signaling.” Proc. Natl. Acad. Sci., 2015, doi:10.1073/pnas.1511092112
Computational Drug Discovery
Agilent 8453 UV-vis spectrophotometer (in lab)
GE Aktapurifier 10 FPLC (in lab)
Biotek Cytation V plate reader (in lab)
Innova 43R refrigerated incubator shaker (in lab)
2X Innova 44R refrigerated incubator shakers (in lab)
Two fully-equipped tissue culture facilities (in lab)
Illumination and imaging system to monitor circadian rhythms in real time. (in lab)
Bruker EMX-6/1 (EPR in Biology Department)
Hitachi F7000 Spectrophotometer (in department)
Beckman Coulter DU800 UV/Vis (in department)
EVOS-fl fluorescent microscope (in department)
JEOL 500 MHz NMR (in chemistry department)
Bruker 400 MHz NMR (in chemistry department)
Agilent GC/MS (in chemistry department)
Agilent HPLC (in chemistry department)
Biotek multi-well plate reader (CD4)
3-axis CNC machine (Deason Innovation Gymnasium)
3D Printer (Deason Innovation Gymnasium)
Laser cutter Deason Innovation Gymnasium)