Molecular Medicine: Department Directory

Research in the Bohn laboratory is focused on understanding how G protein-coupled receptors function in an endogenous setting to control physiologically relevant processes. We are most interested in receptors that mediate neurological functions, particularly those of the opioid, serotonin and cannabinoid families. Ultimately, our goal is to refine therapeutics- to enhance the benefits and eliminate the side effects. In this manner, we hope to inspire new approaches in treating pain, addiction and mood disorders.

The Bohn laboratory is most widely known for our work in opioid receptors. Early work while in the laboratory of Marc Caron and in collaboration with Robert Lefkowitz at Duke University indicated that barrestin2 plays a critical role in determining the physiological role of the mu opioid receptor (MOR) in vivo. Our laboratory has shown that barrestin2 plays different roles in regulating the MOR depending upon the physiological function assessed. This is very important as activation of the MOR results in multiple physiological processes ranging from the highly desirable suppression of pain perception to the deadly effects of respiratory failure. By determining which barrestin2-mediated signaling pathways are associated with these different physiological outcomes, we aim to elucidate a means to develop potent opioid analgesics that circumvent the adverse side effects. The bulk of our work to date suggests that if we preserve MOR coupling to G proteins, but eliminate the interactions between the receptor and the scaffolding protein, barrestin2, then we may be able to separate analgesic potency from constipation, respiratory suppression, tolerance and physical dependence.

Our lab is now focused on developing tool compounds that will allow us to test these hypotheses. Our agonists are designed, in collaboration with Dr. Tom Bannister of TSRI, to activate MOR in a manner that preserves or improves G protein signaling while eliminating the recruitment of barrestins. In addition to generating potential therapeutic leads, we are very interested in using these tools to elucidate MOR function in vivo. As we refine the pathways underlying different physiological responses, we will then know the signaling mechanisms to preserve and the ones to avoid.

We are also taking a similar approach with the kappa opioid receptors (KOR). The KOR in the midbrain acts to regulate dopamine and serotonin levels and thereby serves as an attractive target for modulating mood and reward thresholds. KOR ligands that display bias towards or against recruiting barrestins are of interest as barrestin2 has been implicated in facilitating aversive KOR-mediated behaviors. In our work with Dr. Jeff Aubé of Kansas University, we have been developing and evaluating KOR biased agonists to determine which physiologies are preserved or disrupted in mouse models. Since the KOR is involved in diverse physiological functions, compounds generated in this project may serve as interesting candidates for the treatment of depressive disorders and addiction. Moreover, KOR agonism produces antinociception and blocks itch and may also represent potential therapeutic avenues.

Recently, we have begun evaluating ligands for biased agonism among cannabinoid receptor (CB1 and CB2) agonists in collaboration with Dr. Alex Makriyannis at Northeastern University. Given the emerging implications for using cannabinoids as therapeutics for a wide-range of disorders, there are many opportunities for new drug development. This collaboration has also involved working with Dr. Ray Stevens (USC) and Dr. James Liu (Shanghai Tech) to solve the first crystal structures of antagonist and agonist bound CB1 receptors. Additional efforts in the laboratory focus on evaluating how antipsychotic drugs and mood altering neurotransmitters such as serotonin act at serotonin receptors.

Since the receptors described above are involved in modulating mood, motivation, and sensory perception, it stands to reason that our laboratory is most interested in developing means to treat pain, whether due to injury, disease or mental state, in a manner that adequately manages the pain, without causing deabilitating side effects.

The Cameron Laboratory works on a variety of independent and collaborative projects centered on the metabolic fate of new chemical compounds. We explore the role of drug metabolism in chemical induced toxicity and drug-drug interactions. Current projects include evaluation of reactive metabolites and their role in drug induced toxicity, time and mechanism based inhibition of cytochrome P450, and the development of chemical tools to differentiate CYP3A4 and CYP3A5 activity in biological samples. The lab is also involved in several translational projects focused on the development of clinical candidates or molecular probes for the study of biological pathways. The lab offers Drug Metabolism and Pharmacokinetics (DMPK) expertise, collaborating with medicinal chemistry, biology and pharmacology groups. The lab evaluates such factors as chemical and metabolic stability, solubility, oral absorption, rat and mouse pharmacokinetics, tissue distribution, protein binding, P450 inhibition, reactive intermediate formation, and metabolite identification to help refine molecules. Current projects include optimization of neuropeptide Y Y2 receptor antagonists, orexin-1 receptor antagonists, (GABA) B receptor positive allosteric modulators, neurotensin 1 receptor agonists, a5* nicotinic acetylcholine receptors, positive allosteric modulators, kappa opioid receptor antagonists, and functionally biased mu opioid receptor agonists.

The Phinney lab combines basic and translational research and drug discovery to deliver highly efficacious cell-based and drug-based therapies for the treatment of skeletal-related pathologies and cancer. Currently the lab is pursuing several different areas of investigation to achieve these goals. One area of focus is directed at enhancing the potency and improving the efficacy of mesenchymal stem cell (MSC)-based therapies, which are widely used in regenerative medicine for the treatment of ischemic and immune-related diseases. These efforts have culminated in the development of a Clinical Indications Prediction (CLIP) Scale that may be used to develop MSC-based therapies tailored to specific disease indications. Ongoing work is directed at validating the CLIP scale and expanding its range of applications. Another area of focus is directed at understanding the molecular mechanism that drive skeletal pathology in response to diet-induced obesity, mechanical unloading (disuse), and chronological aging using mouse models. In these studies, emphasis is on evaluating how these conditions impact the frequency and function of nice resident skeletal stem cells in bone marrow and developing therapeutics to preserve bone integrity. Lastly, the laboratory is also pursuing development of small molecule therapeutics to augment the efficacy and reduce the toxicity of existing chemotherapeutics and immuno-therapies for treating breast cancer.

Organic/Medicinal Chemistry and Drug Discovery

The discovery of possible drug candidates is a highly collaborative endeavor, with medicinal chemistry as a core, problem-solving component. My major research efforts are joint projects with world experts in cancer biology and neuroscience, wherein my group provides the organic and medicinal chemistry expertise and drug design insights. In general, we strive to find novel ways to target poorly-treated, common, and devastating disorders that increasingly burden world health care systems.

Neuroscience studies include:

— Biased mu opioid agonists, aiming for a holy grail of sorts: to separate the robust pain relief –provided by opiates from their many unwanted side effects. This collaboration with Laura Bohn’s group has led to findings published in 2017 in Cell, with follow-up chemistry disclosure in the Journal of Medicinal Chemistry in late 2018 (featured on the cover). — NOP agonists, for post-traumatic stress disorder (PTSD) and alcohol addiction relapse therapy. — NAD-elevating neuroprotectants, for Alzheimer’s and Parkinson’s Diseases, and for ALS.

Cancer projects include:

— KLF5 inhibitors for colorectal cancer therapy. — TBK1 and IKKi dual kinase inhibitors, for hormone-refractory prostate cancer. — Inhibitors of kinases CK1delta, ASK1, and ULK1, for various cancers. — Modulators of the HIPPO-YAP pathway, for various cancers. — Other exploratory efforts include:

High-throughput screening-based “chemical probe development”, seeking first-in-class small molecules for investigating the therapeutic potential of new target proteins. Probe development efforts encompass multiple therapeutic areas, including treatments for cancers, glaucoma, amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), addiction, infectious diseases, and mood disorders. One chemical probe effort targets the orphan GPCR GPR151, a target that may be relevant for the development of treatments for addiction, depression, and schizophrenia.The discovery of possible drug candidates is a highly collaborative endeavor, with medicinal chemistry as a core, problem-solving component. Our major efforts are thus joint projects with world experts in cancer biology and neuroscience, wherein our group provides the organic and medicinal chemistry expertise.

Our cancer projects target unique metabolic phenotypes of tumor cells, identifying defining molecular characteristics to be exploited for the development of targeted therapies. Most tumor types have a shared reliance upon active transport of nutrients and building blocks to drive rapid cancer cell growth and to sustain survival. They also largely rely upon glycolysis for ATP production (the Warburg effect). As examples, we have created molecules to keep tumor cells from exporting lactate, the end product of glycolysis. We have also designed compounds to block amino acid transporters that are up-regulated by many tumors. We have a program targeting expression of a transcription factor that drives colon cancer progression. We also have a number of kinase inhibitor programs aimed the discovery of treatments for brain cancers, triple-negative breast cancer, hormone-resistant prostate cancer, and perhaps other forms as well. This are collaborative efforts with top TSRI cancer biologists including Derek Duckett, Joseph Kissil, Jun-li Luo, and also including John Cleveland from the Moffitt Cancer Center.

Many of our anticancer programs have a computational chemistry-directed focus, relying on molecular modeling based upon published coordinates, virtual screening, scoring, and validation of predicted hits through chemical synthesis. We use the Schrodinger suite of modeling software in these studies. For future work we may have a need to hire postdoctoral scientists with both computational and synthesis experience. Please contact me for further information.

In our neuroscience studies we are developing GPCR agonists that have targeted effects in the brain. We are exploring GPCR signaling bias in mu opioid receptor activation, aiming for a holy grail of sorts: to separate the robust pain relief provided by opiates from their many unwanted side effects. This collaboration with Laura Bohn’s group has led to pain relievers that seem to be devoid of many of the side effects of morphine and related opiates, such as respiratory suppression, heart rate effects, and GI effects (constipation). In a separate study we identified tool compound with promise in an animal model of post-traumatic stress disorder (PTSD).

Other exploratory efforts use medicinal chemistry in concert with high-throughput screening or following HTS campaigns, where we seek to discover and optimize “chemical probes”, or first-in-class small molecules that should prove useful for investigating the therapeutic potential of new target proteins. Such probe development efforts encompass multiple therapeutic areas, including treatments for ALS, Parkinson’s Disease, addiction, infectious diseases, cancers, glaucoma, and mood disorders.

One such chemical probe effort, a collaboration with Patsy McDonald, targets the orphan GPCR GPR151, a target that may be relevant for the development of treatments for addiction, depression, and schizophrenia. In a collaboration with Sathya Puthanveettil we are investigating the potential of facilitating the function of kinesin motor proteins as a novel approach to therapies for Alzheimer’s disease (AD) and frontal temporal dementia (FTD), which are poorly-treated, common, and devastating disorders that increasingly burden world health care systems. In a collaboration with Corinne Lasmezas, we are developing compounds that rescue neurons from toxicity of protein aggregates, relevant for developing new therapies for ALS and Parkinson’s Disease.

As you can tell, collaborative drug discovery research is order of the day in my lab!

Members of my group benefit from interactions not only with other chemists but with top biologists and pharmacologists, as they partake in project team meetings as well as in our weekly chemistry group meetings. My research is funded currently by 8 NIH grants on which I am a co-principal investigator and 8 others NIH grants where I am a named investigator or co-investigator.

On occasion I have openings for outstanding postdoctoral fellows in my labs. As mentioned above, an especially good fit would be a postdoc with lab synthesis experience and with prior expertise in using the Schrodinger suite of molecular modeling software, to aid our virtual screening-based efforts. Our postdoctoral scientists collaborate with a team of biological co-investigators, applying knowledge and experience in modern organic, heterocyclic, and/or medicinal chemistry toward an ongoing drug discovery effort. Excellent communication skills, good synthetic organic chemistry laboratory skills, ability to work in the US, and familiarity with modern synthetic techniques and instrumentation are required in this role. Contact me for further details.

Medicinal Chemistry and Drug Discovery

The research interest in my group is in the design, synthesis and evaluation of novel compounds of biological and therapeutic interest. Currently, we are involved in the design and synthesis of novel small molecule modulators of nuclear receptors, GPCR’s, ion channels, kinases and mitochondria for the therapeutic treatment of addiction, diabetes and obesity, multiple sclerosis, cognitive health and aging. Working closely with other departments such as molecular biology, pharmacology, and drug metabolism, we optimize lead compounds for potency, ADME (absorption, distribution, metabolism, and excretion), safety pharmacology, and toxicology in order to generate compounds suitable for preclinical development.

As a graduate student at the University of Virginia, I worked in Don Hunt’s lab alongside John Yates and together we performed ground-breaking studies in the use of mass spectrometry to determine the primary structure of proteins. I am first author on a paper describing the complete sequence of a protein using tandem mass spectrometry. Prior, only a few small proteins for which their amino sequence was known had been sequenced by tandem mass spectrometry. This work helped validate that it was possible to sequence proteins using tandem mass spectrometry. I am first author on one of the first papers describing the using low nano-LC coupled with ESI. I contributed to many other publications demonstrating the use of mass spectrometry for structural analysis of proteins.

While at Merck Research Laboratories, my lab made significant contributions to a wide range of projects including key contributions to the development of the DPP4 program which led to the approval and marketing of Januiva. The range of contributions to drug discovery and development are exemplified in manuscripts listed below. My efforts in drug discovery have continued in the academic setting.

In 2002 I became interested in the application of hydrogen/deuterium exchange mass spectrometry. My lab focused on the development of a fully automated system, advanced software to facilitate robust high precision analysis, and applications of HDX in protein-ligand interactions with an emphasis on nuclear receptors, enzymes, and GPCRs. The publications listed below include an extensive list of key contributions to the HDX field.

In addition to the advancement of structural proteomics, my research has focused on chemical biology approaches to better understand nuclear receptor signaling. Our group described the first synthetic ligand for the orphan nuclear receptor NR1F subfamily (ROR)s and subsequently showed the utility of advanced compounds as anti-inflammatory and anti-obesity agents as well as RORG agonists for enhancing protective immunity in the context of cancer therapy. Our lab has also contributed to a fundamentally new understanding of the mechanism by which the nuclear receptor PPARG impacts insulin sensitivity.

Highlights on grant funding related to drug development programs

• I was consortium PI of the “The Comprehensive Center for Chemical Probe Discovery and Optimization at Scripps,” I had both a leadership role and a scientific role on this large multi-center 6-year U54 MLPCN Roadmap initiative.

• I was consortium PI of a RC4 program in collaboration with Bruce Spiegelman at the Dana Faber, I was involved in the discovery and development of novel molecules for the treatment of obesity and diabetes. Work from our labs in this area led us to co-found Ember, a private biotech funded by Third Rock Ventures.

• I was Co-PI on a NIDA funded U19 NCDDDG focused on the discovery and development of novel positive allosteric modulators of GABAB receptor for treatment of nicotine addiction.

• I served on the Scripps-Pfizer collaboration Steering Committee for its 5 year term coordinating collaborative drug discovery programs.

• I am PI of a 13-year long collaboration between my lab and Eli Lilly. • I am PI on a collaborative grant with the private Biotech Synkine Therapeutics.

• I am Co-PI on a NIH Blueprint UH3 titled, “Developing nonmuscle myosin II inhibitors for substance use relapse.” Co-founder of Myosin Therapeutics. The Myosin Therapeutics’ clinical candidate emerged from the NIH Blueprint program.

Our laboratory studies the role of cholesterol in setting thresholds of anesthesia, mechanosensation, amyloid formation, and viral entry. In each area of research, we study how cholesterol controls the threshold that regulates biological function and the severity of disease. By understanding the thresholds, we aim to treat diseases caused by altered cholesterol levels and signaling lipids.

Anesthesia: For hundred years scientist believed that membrane lipids were involved in the anesthesia (reversible loss of consciousness). At the heart of the question was the following, How can disrupting a lipid membrane activate or inhibit and ion channel? We have shown that anesthetics disrupt compartmentalization of signaling molecules in cholesterol dependent lipid compartments. The anesthetics counteract cholesterol causing the proteins to escape and activate an ion channel. This established at least one clear molecular mechanism for the membrane as a target of inhaled anesthetics. We are studying this mechanism for additional ion channels that mediate anesthesia in people.

Pain threshold (mechanosensation): We have shown mechanical force disrupts cholesterol dependent compartmentalization of proteins, similar to anesthetics. In addition to defining a novel mechanosensation pathway, we are developing potential therapeutic compounds that will activate an analgesic channel downstream of mu opioid receptor. We aim to develop therapeutics that will have similar pain reducing benefits as opioids, but without the addiction.

Viral entry: We have recently proposed a model for cholesterol dependent SARS-COV-2 viral infectivity, based on our understanding of cholesterol mediated membrane protein translocation. As cholesterol increases the ability of the virus to enter the cell increases. Increased viral entry increases inflammation, which in turn increases cholesterol and more viral entry. We are currently studying cholesterol loading in lung tissues in aged animals.

See lab website: www.joazeirolab.com

Role of the ubiquitin-proteasome system in cellular regulation and disease

E3 ubiquitin ligases are the components that confer specificity to the ubiquitin system. Consistent with their critical role in cellular regulation, mutations affecting E3s can cause human disease—as illustrated by the cases of BRCA1 in breast cancer and of Parkin in Parkinsonism. Our laboratory is engaged in three main areas of research related to E3s:

1] We are interested in elucidating E3s’ function and mechanisms. For this purpose, we utilize cell and molecular biology approaches with mammalian cells, mice and yeast; our expertise is complemented by close collaborations on structural biology, bioinformatics, mass spectrometry, next-generation sequencing, and fly models;

2]We utilize the information we learn from the above studies to develop assays that are amenable to high-throughput screening, which are then used to identify small molecule inhibitors of E3 ligases. Such compounds can then be both turned into tools to interrogate biology, and developed as pharmaceutical drug candidates;

3] We collaborate with clinicians to translate our basic research findings into therapeutically-relevant discoveries.

The Miller Lab is working to develop new medications for the treatment of disorders marked by persistent, unwanted memories – specifically substance use disorder (SUD; addiction) and posttraumatic stress disorder (PTSD). Mechanistically, our efforts are large focused on epigenetic and synaptic structural players.

DEVELOPMENT OF THERAPEUTICS TO PREVENT ADDICTION RELAPSE

Dr. Miller has long been interested in ways to selectively disrupt drug-associated memories (Reconsolidation; Miller and Marshall, Neuron, 2005) and the mechanisms that contribute to the persistence of memory (DNA methylation; Miller et al, Nature Neuroscience, 2010). In bringing these two areas together, the Miller Lab made the surprising discovery that memories associated with the commonly abused stimulant, methamphetamine, employ a unique, actin-based storage mechanism in the brain’s emotional memory center, the amygdala, that allows for their selective disruption (Young et al, Biological Psychiatry, 2014). This was followed by identification of nonmuscle myosin II (NMII) as a viable therapeutic target (Young et al, Molecular Psychiatry, 2015) and we are now in the middle of a drug development program supported by the NIH’s Blueprint Neurotherapeutics Network to develop a clinically safe NMII inhibitor. The goal is to enter a Phase 1a safety trial in 2020.

REGULATION OF TRAUMATIC MEMORY RELATED TO PTSD

We recently developed an animal model with PTSD-like features, including differential susceptibility, which has been replicated by other groups. The stress susceptible subgroup displays persistent, stress-enhanced fear memory, hyperarousal, amygdala hyperactivation and differential expression of genes with known polymorphisms in human PTSD genomic studies (Sillivan et al, Biological Psychiatry, 2017). Importantly, the stress susceptible population can be identified by training behavior, removing the need for additional phenotyping that introduces a molecular confound. Because the model was developed in an inbred mouse line (c57’s), it’s an excellent opportunity to investigate epigenetic mechanisms. To date we have largely focused on noncoding RNAs, including miRNAs. Small RNA-sequencing integrated with quantitative proteomics revealed a panel of miRNAs persistently upregulated in the amygdala of stress susceptible mice. We recently completed a study demonstrating a functional role for one of these miRNAs, which is also expressed in the human amygdala. Working with our collaborators, we also discovered the miRNA’s passenger strand is elevated in serum of a Dutch military cohort diagnosed with PTSD six months after a combat deployment to Afghanistan, indicating this miRNA may be a therapeutic target and biomarker for PTSD. Efforts are now shifting to in vivo calcium imaging and manipulation of the activity-dependent neuronal ensembles regulating differential susceptibility to stress.

Progress in drug discovery is often coupled to parallel advancements in instrument technology. In our department, High Throughput Screening (HTS) robotics is employed to accelerate the drug discovery process thorough full automation of large scale screening experiments. HTS technology can be used to execute and analyze hundreds of thousand of experiments against large compound libraries to identify a few select compounds of therapeutic value. These “therapeutic leads” provide important insight and basis for the medicinal development of novel drugs for clinical application.

Research efforts are currently focused on:

— Working with collaborators to develop biological assays for HTS compatibility.

— Performing exploratory HTS campaigns for drug lead discovery.

— Technology development and instrumentation for the advancement of HTS robotics.

Collaborative efforts are coordinated with a large community of scientists and include TSRI and UF faculty, Florida researchers, and academia often funded through the National Institutes of Health, Department of Defense and private foundations.

The focus of our research involves enabling technologies for High Throughput Screening (HTS) of any target of unmet therapeutic need. Our facility supports scientist and faculty locally as well as those in the US and throughout the world. We do this by implementing their biological applications into high density plates for HTS. The breadth of biology we deal with is virtually unlimited due to the diversity of skill sets of the scientist and engineers within our group as well as the expanded capability of the readers associated to our system. We operate a fully automated 1536 well compatible platform and perform HTS on large compound libraries such as the Scripps Drug Discovery Library (645K) or small focused collections (FDA approved drugs). We have expertise in microfluidics, low nanoliter volume liquid handling, ultra-sensitive plate reader technologies and informatics set-up to handle large volume data sets. Key components are high-content readers, imaging detectors such as the ViewLux and FLIPR Tetra, as well as pintool and acoustic transfer devices. Through collaborative efforts we have successfully translated multiple small molecules into the clinics. We are open to any pharmacology desired (agonist, PAMs, NAMs, inverse agonist, etc.) vs. kinases, GPCR, NHRs, proteases, phenotypic assays, etc. which can be applied to any therapeutic area; i.e. CNS, infectious diseases, cardiovascular, oncology, metabolic diseases. In addition, we are currently funded (R33CA206949) to develop 3D spheroid based uHTS assays for the purpose of testing molecules in a more phenotypically relevant format to cancer biology.