If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Merging cultures and disciplines to create a drug discovery ecosystem at Virginia commonwealth university: Medicinal chemistry, structural biology, molecular and behavioral pharmacology and computational chemistry
The Department of Medicinal Chemistry, together with the Institute for Structural Biology, Drug Discovery and Development, at Virginia Commonwealth University (VCU) has evolved, organically with quite a bit of bootstrapping, into a unique drug discovery ecosystem in response to the environment and culture of the university and the wider research enterprise. Each faculty member that joined the department and/or institute added a layer of expertise, technology and most importantly, innovation, that fertilized numerous collaborations within the University and with outside partners. Despite moderate institutional support with respect to a typical drug discovery enterprise, the VCU drug discovery ecosystem has built and maintained an impressive array of facilities and instrumentation for drug synthesis, drug characterization, biomolecular structural analysis and biophysical analysis, and pharmacological studies. Altogether, this ecosystem has had major impacts on numerous therapeutic areas, such as neurology, psychiatry, drugs of abuse, cancer, sickle cell disease, coagulopathy, inflammation, aging disorders and others. Novel tools and strategies for drug discovery, design and development have been developed at VCU in the last five decades; e.g., fundamental rational structure-activity relationship (SAR)-based drug design, structure-based drug design, orthosteric and allosteric drug design, design of multi-functional agents towards polypharmacy outcomes, principles on designing glycosaminoglycans as drugs, and computational tools and algorithms for quantitative SAR (QSAR) and understanding the roles of water and the hydrophobic effect.
According to Wikipedia an ecosystem is defined as “consisting of all the organisms and the physical environment with which they interact. These … components are linked together through … energy flows. Energy enters the system through photosynthesis …”. It goes on to define the roles of animals, decomposers and other energy transfer mechanisms. By definition, ecosystems change very slowly over time unless changes are triggered by a cataclysmic event. Economic (or business) ecosystems are a concept put forth by James F. Moore [
], using ecological metaphors, such as firms being embedded in a (business) environment, needing to coevolve with others, and that their niches in the ecosystem are challenged by newly arriving species (competition), and that they need to be proactive in developing symbiotic relationships with customers, suppliers, etc.
] has been in use for several years, this is a loose extension of the above definitions. It is difficult to envisage that a robust drug discovery ecosystem would change slowly. Significant and regular inputs and outputs are crucial for a drug discovery ecosystem to function optimally and respond to the environment. Inputs would almost certainly include funding (money) and new technologies, while outputs would include new chemical probes and new chemical entities but may also include new mechanisms and new targets. Most importantly, change is very important to drug discovery and new tools and technologies are very often the catalysts to new drugs. Perhaps the key aspect of a robust drug discovery ecosystem is the diversity of ideas and concepts as its drivers. With regard to an institution, the big idea is to connect and synergize diverse individuals, disciplines and technologies that result in products that are greater than the sum of their parts.
1.2 Defining moments in drug discovery
As most academic medicinal chemistry departments, Virginia Commonwealth University's (VCU) major initial role was in teaching and service to the School of Pharmacy. The recruitment of Richard A. Glennon in 1975 heralded an additional major commitment to research by the School. His co-interests in both neuropharmacology and state-of-the-art medicinal chemistry synthesis were soon focused on the rapidly evolving field of G-coupled protein receptors (GCPRs), in particular, efforts to pharmacologically illuminate the serotonin (5-HT) receptor(s). Glennon catalyzed the establishment of an extensive in-house program of behavioral pharmacology including drug discrimination, and probed – at an unprecedented in-depth level – structure-activity-relationships with custom synthesis of hundreds of serotonin analogues [
] and for building one of the first models in which molecular orbital theory was applied successfully to drug design and development. Kier was also a proponent for, and a significant contributor to, the early development of quantitative structure-activity relationships (QSAR) with the topological indices he co-invented with Lowell Hall of Eastern Nazarene College [
], which is probably the key principle in modern drug discovery and design.
Donald J. Abraham joined the Department in 1988. His passion was finding a cure for sickle cell disease using multidisciplinary tools. By the mid-1970s, Abraham was already thinking about the intersection between medicinal chemistry and X-ray crystallography, and had published a landmark paper in 1986 describing the design of small molecules effectors of hemoglobin with the Nobel Laureate Max Perutz, who had solved the first structure of hemoglobin [
]. His infectious enthusiasm quickly paid off with the largely unprecedented integration of structural biology and medicinal chemistry in one unit – embodied in the VCU Institute for Structural Biology and Drug Discovery (ISBDD). Over the past five decades, numerous research-focused faculty members were recruited to the VCU Department of Medicinal Chemistry in diverse areas of medicinal chemistry and drug discovery including William H. Soine, Richard B. Westkaemper, Glen E. Kellogg, Martin K. Safo, Kevin A. Reynolds, Malgorzata Dukat, Jason P. Rife, Umesh R. Desai, Yan Zhang, Shijun Zhang, John C. Hackett, Keith C. Ellis, Rong Huang, Aaron E. May, Youzhong Guo, Jiong Li, and Yana Cen. These 50+ years have been a period of robust growth in terms of influx of ideas from a diversity of fields and individuals that offer a model on the growth and evolution of an academic drug discovery ecosystem (Figure 1). The VCU drug discovery ecosystem is a rather fluid ecosystem that has attempted to address the research and teaching needs and challenges of the School, University, and healthcare environment while constantly driving to impact the clinical needs of the nation.
Fig. 1Cartoon representation of the VCU Medicinal Chemistry Drug Discovery Ecosystem. The various components – synthetic chemistry, protein production, assays and screening, behavioral pharmacology, structural biology, computational chemistry, etc. – are illustrated in the context of an environmental system. VCU faculty members contributing to these components are indicated.
2. Vision and resources in an urban public institution
Virginia Commonwealth University is a quite atypical public institution. It was created through the merger of two independent colleges (the Richmond Professional Institute and the Medical College of Virginia) in 1968, both located in urban Richmond. The former focused on the arts and social work, while at the time, the latter included a medical school, nursing school and the only dental and pharmacy programs in Virginia. These unique characteristics define the VCU brand and have driven the palpable entrepreneurial spirit amongst faculty, administration and citizenry of the Department. Despite the funding challenges of the past decades, a conscious effort of bootstrapping resources has led to the current VCU drug discovery ecosystem, including unique collection of instrumentation (Fig. 2). In the sections below, we describe the evolution of our ecosystem while also describing the logic, facilities and instrumentation in the process.
Fig. 2Selected instrumentation in the VCU Department of Medicinal Chemistry and Institute for Structural Biology, Drug Discovery and Development. a) 35 liter sterilizable New Brunswick 510 fermenter with an associated Beckman flow centrifuge and AKTA Pure fast protein liquid chromatography (FPLC) system; b) Biotek Cytation 5 cell imaging multimode reader system; c) Labcyte Echo 550 liquid handler for nL-level dispensing; d) Molecular Devices FlexStation 3 Multi-Mode Microplate Reader; e) Nanotemper NT-Automated for microscale thermophoresis (MST) to measure interactions and affinity; f) Thermo Scientific Orbitrap Fusion Lumos Mass Spectrometer; g) Malvern Microcal PEAQ Isothermal Calorimeter (ITC); h) ARI Crystal Gryphon robot for crystallization fitted with Minstrel/Gallery imaging system; and i) Rigaku MicroMax-007HF X-ray Generator with VariMax-HF Arc Optics/Hybrid Photon Counter, Eiger R 4M Detector and AFC11 Goniometer.
Making novel molecules is, and always has been, a key focus of the VCU Department of Medicinal Chemistry. Prior to the mid-1970s, the number of faculty was relatively small and teaching loads were quite high. Due to the nature of chemistry's role in pharmacy education at the time, department faculty members carried a broader research profile, e.g., analytical chemistry, physical chemistry, synthetic chemistry, medicinal chemistry, etc. The recruitment of Richard Glennon, as mentioned above, signaled a substantive change in the Department's direction. Since joining the faculty, Glennon (who later served as the Department Chair) strived for state-of-the-art synthetic capabilities in his laboratory and the department; he attracted well over 50 postdoctoral fellows and visiting scientists from around the world, many of whom were previously trained in synthetic chemistry and wished to apply those skills by learning about medicinal chemistry and drug design. His group trained dozens of scientists in synthetic and medicinal chemistry skills in the area of structure-activity relationships (SAR) of serotonin receptors. The group adopted and optimized the newest synthetic methods and techniques, which they shared at seminars and informal meetings, thereby encouraging synthetic innovation. The trend continues today with current department faculty recruiting highly skilled chemists and biologists as postdoctoral scientists. The major synthetic medicinal efforts in the department today relate to serotoninergic ligands, natural products, glycosaminoglycan mimetics, and multi-functional agents. These are described in brief below.
2.1.1 Serotoninergic and related ligands and targets
In the Glennon and Dukat laboratories, the focus has been on psychiatric disorders and drugs of abuse, particularly those involving serotonin and serotonin receptors. Synthetically, in addition to investigating the mechanisms of action of these drugs, they examined the chemistry and chirality of a vast variety of these agents [
[125I]-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane: an iodinated radioligand that specifically labels the agonist high-affinity state of 5-HT2 serotonin receptors.
Toward selective drug development for the human 5-hydroxytryptamine 1E receptor: a comparison of 5-hydroxytryptamine 1E and 1F receptor structure-affinity relationships.
des-Formylflustrabromine (dFBr): a structure-activity study on its ability to potentiate the action of acetylcholine at α4β2 nicotinic acetylcholine receptors.
Total synthesis of natural products has been a hallmark of organic chemistry training for a number of decades. Besides the raw challenge of reproducing a complex chemical using current laboratory methods (and inventing new ones), there is a strong correlation between molecules found in nature and biological activity thereof. Further, natural products inspire design and/or discovery of new drugs. Yan Zhang and Keith Ellis have used concepts from total synthesis in their projects in targeting G-protein coupled receptors and cytosolic enzymes as antibacterial and oncology molecular targets, respectively. Yan Zhang has developed a program largely focusing on the opioid receptor subtypes and discovered a number of new potent and selective analogs with properties and activities suggestive of new treatments for addiction and pain (vide infra) [
Design, Synthesis and Biological Evaluation of 6α- and 6β-N-Heterocyclic Substituted Naltrexamine Derivatives as Mu Opioid Receptor Selective Antagonists.
Design, Synthesis, and Biological Evaluation of 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4’-pyridyl)carboxamido]morphinan Derivatives as Peripheral Selective Mu Opioid Receptor Antagonists.
Structure activity relationship studies of 6β- and 6α-indolylacetamidonaltrexamine derivatives as bitopic mu opioid receptor modulators and elaboration of ‘message-address concept’ to comprehend their functional conversion.
Design, Synthesis and Biological Evaluation of NAP Isosteres: a Switch from Peripheral to Central Nervous System Acting Mu-Opioid Receptor Antagonists.
]. Even though the epoxymorphinan skeleton is commonly found in natural products derived from opium, e.g., morphine and codeine, and has been studied for decades, utilizing this skeleton to develop novel opioid ligands in order to treat opioid use disorders remains advantageous [
Characterization of 6α- and 6β-N-Heterocyclic Substituted Naltrexamine Derivatives as Novel Leads to Development of Mu Opioid Receptor Selective Antagonists.
Behavioral and Cellular Pharmacology Characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan (NAQ) as a Mu Opioid Receptor Selective Ligand.
Comparison of Pharmacological Properties between the Kappa Opioid Receptor Agonist Nalfurafine and 42B, its 3-Dehydroxy Analogue: Disconnect between in Vitro Agonist Bias and in Vivo Pharmacological Effects.
]. He also has investigated AIDS and cancer with the C-C chemokine receptor type 5 (CCR5) receptor that plays a role in both diseases, a study begun with the natural product anibamine [
Structure-Activity-Relationship Studies of Natural Product Chemokine Receptor CCR5 Antagonist Anibamine toward the Development of Anti Prostate Cancer Agents.
]. Anibamine is the first natural product binding to the chemokine receptor CCR5 at a micromolar level. Its analogs were also studied for their anti-malaria activities in the Zhang lab [
Keith Ellis has used natural products with novel mechanisms of action as the starting point to develop new chemical probes and pharmacophores for potential therapeutics in the antibacterials and oncology areas. He explored the natural product simocyclinone D8, which displays a novel mechanism of action against bacterial type-II topoisomerase enzymes and is active against drug resistant DNA gyrase mutants [
] as an inhibitor of bacterial DNA gyrase and Topoisomerase IV (TopoIV), as well and synthesizing and testing new analogues as potential antibacterial compounds [
The Ellis Lab also used the scaffold and novel irreversible mechanism of action of the natural product kalafungin to explore new inhibitors and define new pharmacophores for the oncogenic serine-threonine protein kinase Akt [
Design, synthesis, and in vitro evaluation of a fluorescently labeled irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKACα).
], a single point mutation that causes adrenocorticotropic hormone (ACTH)-independent Cushing's Disease.
2.1.3 Synthetic analogs of glycosaminoglycan biopolymers
Although drugs are thought to be hydrophobic molecules with few polar atoms, an entire group of natural biopolymers exist that can theoretically serve as leads for highly polar drugs. These are glycosaminoglycans (GAGs), which include biopolymers heparin (Hp), heparan sulfate (HS), chondroitin sulfate and others. The synthesis of these GAG biopolymers is phenomenally challenging. To address this, Umesh Desai's laboratory is interested in chemical mimicry of these GAG biopolymers. Hp/HS contain a large number of sulfate groups and no organic molecule has been developed to date containing as many negative charges. In fact, introducing multiple sulfate groups with a high level of efficacy and purity on a small organic scaffold is a major challenge because of repulsive forces disfavoring sulfate moiety addition. To address this, the Desai group has developed a very novel microwave-based sulfation route [
]. Collectively, this technology has led to the synthesis and characterization of a library of more than 150 synthetic, highly sulfated aromatic molecules (Fig. 3), which have been protected by the VCU Innovation Gateway, including some that have been licensed to biotechnology incubators for further development.
Fig. 3The library of unique sulfated non-saccharide glycosaminoglycan mimetics synthesized in the Desai laboratory. The characteristic feature of these molecules is their three-dimensional sulfated aromatic scaffold, which induces target selectivity as well as high aqueous solubility. The library includes mono-sulfated to poly-sulfated molecules belonging to the flavonoid, benzofuran, isoquinoline, quinazolinone, glucoside and inositol scaffolds. Distinct members of this library have been found to exhibit distinct biological activities including antithrombotic, anti-cancer, anti-inflammatory, and anti-viral. These have been documented in numerous papers from the group in collaboration with biologists.
]. Bivalent ligands contain two pharmacophores attached by a linker. This concept has been applied in numerous ways recently, particularly in both the Yan Zhang and Shijun Zhang research groups. The former has performed several studies of homo and hetero G protein-coupled receptors (GPCR) dimerization [
]. Novel ligands that could perform such dimerizations would reveal new pharmacology and biology that defines new targets and drug discovery paradigms to treat a wide variety of diseases [
Shijun Zhang's approach has been somewhat different: his novel bivalent (and multivalent) compounds have been designed around multiple hypotheses concerning the causes and potential treatments for Alzheimer's disease. Such compounds are not only synthetically challenging, but their development also introduces complexity into the associated SAR studies as they are intended to target two or more molecular targets. His group has developed unique chemistry and insights in their efforts to provide these compounds as chemical tools and potential therapeutics [
Keith Ellis’ interest in Pd-catalyzed chemistries in the synthesis of pharmaceutically relevant drug molecules led to his discovery of C-H activation chemistries catalyzed by solid-supported Pd(II) catalysts in collaboration with the Gupton Lab in the VCU Chemical Engineering Department [
]. This project formed the foundation for the Ellis Lab to join the Medicines for All (M4A) institute at VCU and begin bringing synthesis automation, continuous flow chemistry, and other high-throughput synthesis methods to bear on his projects, particularly in the realms of fluorination chemistry and kinase inhibitor synthesis. The M4A Institute has developed into a sizeable enterprise linking multiple units of VCU with respect to drug discovery, development and supply [
], medicinal chemistry and drug discovery have increasingly embraced computational methods. The very simple notion behind QSAR belies its power and influence on the generations of computational medicinal chemists that today may not even be aware of it.
In QSAR's first implementation, very basic and very empirical parameters, e.g., from partition coefficients, reaction rates and molar refractivity, were used as independent variables in a regression against some measure of biological activity as the dependent variable. Later, the limitations of relying on only measurable quantities from physical samples were overcome with methods such as CLOG-P to predict logPo/w – the partition coefficient for solute transfer between 1-octanol and water – from structure (connection matrices) [
] and by descriptors derived from graph theory. Lemont Kier was a leader in this latter area and the ensuing Kier-Hall indices, designed and implemented with Lowell Hall [
]. Kier's influence elevated the department faculty's interest and expertise in computational tools for drug discovery and design. These methods became a fundamental element of modern drug discovery at VCU by the mid-to-late 1970s, and eventually in most medicinal chemistry departments – both academic and industrial – around the world.
These “classical” QSAR descriptors, which are one-dimensional or pseudo-two dimensional, enabled ligand-based drug discovery. Kier's graph theory-based research is, interestingly, totally non-empirical: the input is simply atom type and connections, which makes the approach applicable to any molecule. He, with Lowell Hall, developed the Electrotopological State model that allowed atomistic non-empirical QSAR [
] and bolstered traditional 1D and 2D QSAR into the 1990s and beyond. Kier also began a series of investigations into cellular automata as a low-cost simulation approach to a number of solution phenomena [
Donald Abraham's arrival in 1988 brought an X-ray diffractometer and an interest in what was called “molecular modeling” to the department. Molecular modeling added the third dimension and the capability to visualize molecules and interactions. With his training in X-ray crystallography, Abraham had a native interest in molecular modeling, and in particular structure-based drug discovery (SBDD), wherein the structure of the target receptor, enzyme, etc. informs the discovery/design of active agents. His research on allosteric modifiers of hemoglobin was inspired by the search for treatments or cures for sickle cell disease, which was one of the first disorders fully understood on a structural level.
A significant success factor for Abraham's research on hemoglobin allosteric effectors, e.g., RSR-13 (vide infra), was the embedded molecular modeling that illustrated the design principles for the most potent molecules. An idea of Abraham, that hydrophobicity (or hydropathy) carried more information than just solubility, was the genesis of the HINT (Hydropathic INTeractions) program written by Glen Kellogg and Abraham, and was first featured in this hemoglobin modulators research [
Computational Methodology for Estimating Changes in Free Energies of Biomolecular Association Upon Mutation. The Importance of Bound Water in Dimer-Tetramer Assembly of beta 37 Mutant Hemoglobins.
]. In brief, HINT is based on extracting the thermodynamic and interaction propensity information from logPo/w, which at its core is a free energy for the solute transfer. Kellogg continued the development of HINT and expanded its applications to 3D QSAR (Comparative Molecular Field Analysis, CoMFA) [
Simple, Intuitive Calculations of Free Energy of Binding for Protein-Ligand Complexes. 3. The Free Energy Contribution of Structural Waters Molecules in HIV-1 Protease Complexes.
Frontiers in Drug Design and Discovery: Structure-Based Drug Design in the 21st Century. 3. Bentham Science Publishers, Ltd,
2007: 317-379https://doi.org/10.2174/000000007780960562
], and in protein structure analysis and prediction (vide infra). Kellogg has developed a suite of computational tools for examining the roles of water in liganded and unliganded protein structures [
Amadasi A., Surface J.A., Spyrakis F., Cozzini P., Mozzarelli A., Kellogg G.E. Robust Classification of “Relevant” Water Molecules in Putative Protein Binding Sites. J Med Chem 200;:51:1063-67. doi: 10.1021/jm701023h.
Spyrakis F., Dellafiora L., Da C., Kellogg G.E., Cozzini P. Correct Protonation States and Relevant Waters = Better Computational Simulations? In: De Benedetti, P.G., editor. Protonation States and Conformational Dynamics in Ligand-Target Recognition and Binding, Curr Pharm Des 2013;19:4291-309. doi: 10.2174/1381612811319230011.
3-D Interaction Homology. The Structurally Known Rotamers of Tyrosine Derive from a Surprisingly Limited Set of Information-Rich Hydropathic Interaction Environments Described by Maps.
The increasing availability of protein crystal structures, computational/visualization power and innovative molecular modeling programs probably made structure-based drug discovery inevitable, but Abraham's contribution and foresight should be highlighted. When he arrived as Department Chair in 1988, the drug discovery enterprise was in the midst of a major paradigm shift towards SBDD, and he quickly established it as a major department strength, with the department's first diffractometer and dedicated molecular modeling hardware and software. Nearly every faculty member to join VCU Medicinal Chemistry since then has arrived with an interest in computational techniques and/or developed one, often with collaborations amongst their colleagues.
2.2.3 Computational glycomics
Upon his arrival in 1998, Umesh Desai totally embraced the computational/structural biology/medicinal chemistry ecosystem that Abraham had established and developed a unique and internationally recognized program in designing/discovering glycosaminoglycan (GAG) and synthetic GAG mimetics for therapeutic applications, especially in the treatment of thrombosis, cancer, inflammation, microbial infection and other diseases/disorders. His team has a significant computational emphasis that over the past two decades has focused on highly translational projects that are rooted in the fundamentals of the GAGs binding to proteins [
]. Clinically relevant agents developed on the basis of either structure-based computational design or phenotypic screening-based strategies have emerged, e.g., for thrombin [
Allosterism-based simultaneous, dual anticoagulant and antiplatelet action: allosteric inhibitor targeting the glycoprotein Ibα-binding and heparin-binding site of thrombin.
Designing allosteric regulators of thrombin. Exosite 2 features multiple subsites that can be targeted by sulfated small molecules for inducing inhibition.
Sulfated dehydropolymer of caffeic acid: In vitro anti-lung cell death activity and in vivo intervention in emphysema induced by VEGF receptor blockade.
]. Computational tools developed in the Desai lab play a key role in the GAG research. The primary tool, called combinatorial virtual library screening (CVLS), is a highly novel computational screening protocol (dual filter, genetic algorithm) applied against 103 to 105 GAG sequences to identify those that are high-affinity/high-specificity [
Finding a needle in a haystack: development of a combinatorial virtual screening approach for identifying high specificity heparin/heparan sulfate sequence(s).
]. The CVLS algorithm relies on computational infrastructure available through VCU's computational core facility and within the Department. A server for non-profit users has been developed by his group and is available [
]. Recently, this tool was used to explore the dynamics of a library of GAG hexasaccharides to provide key insights into how GAGs fold, especially around residues containing the 3-O-sulfate group [
]. GAGs are seen to occupy unique topologies in a sequence-specific manner, alternately referred to as ‘dynamic sulfation codes’ that are selectively recognized by proteins. In fact, much of the fundamental computational work performed in the Desai laboratory has been translated into the development of synthetic, highly sulfated, aromatic molecules that selectively inhibit cancer stem cells [
A question often asked is “What is the difference between chemistry and medicinal chemistry?”. Obviously, the answer is “medicinal”; that is, the latter involves instruction and, to a varying degree, hands-on experience with problems of a biological nature that require an understanding of the basic principles of, for example, pharmacology, biochemistry, and physiology. Early progress in the field was, for the most part, limited to enzyme inhibition or various relatively simple in vitro assays because they were well understood at the time.
2.3.1 Drug discrimination
Glennon introduced the concept of evaluating novel agents using in vivo studies, such as drug discrimination using rodents at VCU [
]. Prior to that time, drug discrimination experiments were the domain of, primarily, psychology programs to investigate learning behavior (i.e., various training conditions and then drugs were employed to alter an animal's learning) and later, to some extent, in pharmacology departments to understand the differences in action of various established agents. For a medicinal chemistry department to collect the expertise to synthesize novel compounds and the expertise to test them and characterize them in higher order pharmacology assays such as drug discrimination was very unique and clearly paradigm shifting. Much of the VCU Medicinal Chemistry “brand” from the mid-1970s until 2010 was founded on these unique and important innovations, especially with respect to drugs of abuse and the so-called designer drugs (see Fig. 4).
Fig. 4Figure 4. Drug Discrimination or Stimulus Generalization. In these studies. animals (rats) are trained, using a two-lever operant apparatus, to discriminate (i.e., recognize) a specific training drug / drug- dose stimulus (by responding on Lever 1) from a non-drug condition (by responding on Lever 2). Animals are then administered a novel test drug under non-reinforcement conditions (i.e., without reward) to determine how they will respond; (that is, does the test drug produce a stimulus effect similar, and in a dose-responsive manner, to the training drug (i.e., by responding on Lever 1)? Or, is the response different than that of the training drug (as indicated by their responding on Lever 2, not responding, or disruption of behavior). Animals tested during test sessions are free to select either lever (as indicated by the animal and its shadow), and respond on one of the two levers; that is, the animals “decide” (as indicated by “?”) whether the test drug is perceived to produce a stimulus effect similar to or distinct from the training drug condition. See Glennon and Young
Tests of stimulus antagonism can also be conducted using known neurotransmitter antagonists to investigate novel agents employed as training drugs whose mechanism of action are unknown, or to develop new antagonists for training drugs whose mechanism of action has been already established. With the synthetic chemistry and behavioral pharmacology combination, Glennon and colleagues identified a class of agents they termed classical hallucinogens [
] (currently referred to as serotonergic psychedelics, some of which are now being examined clinically for the treatment of several neuropsychiatric disorders) [
], formulated structure-activity relationships, and provided the first evidence that the behavioral potency for an extended series of such agents in animals including humans was correlated with a specific neurotransmitter (5-HT2) mechanism. The Glennon and Dukat laboratories also investigated agents such as central stimulants [
N-Methyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA) and N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) produce non-identical discriminative stimuli in rats.
Representative examples of other types of in vivo or behavioral studies involving synthesis of novel agents conducted in-house include the mouse tail suspension test for identifying novel antidepressants [
]. In vivo assays have been indispensable for many of the studies conducted in the department and have resulted in well over one hundred publications.
The drug epidemic of the last decade has been clearly drawn around the abuse of opioids, from prescription (e.g., oxycontin), to extralegal (e.g., fentanyl), to illegal natural products and synthetics, e.g., heroin, new synthetic opioids (NSOs) [
]. Yan Zhang's opioid receptor antagonist research program includes a significant component of pharmacological testing, in vitro with cloned receptors and with animal studies. Application of mouse models in antinociception and rat models in self-administration identify novel and potent mu opioid receptor modulators for targeted development of opioid use disorder treatments [
]. His research group has an extensive array of collaborators, many in the VCU Department of Pharmacology and Toxicology, but also has built his own extensive and unique capabilities within the department including in vivo animal models to characterize pain, addiction, dependence, and respiratory depression.
With the increase of the aging population in the US and worldwide, and the lack of effective treatment for Alzheimer's disease, the most common type of dementia, there is clearly an urgent need to develop cures or effective treatments for this devastating disease. Shijun Zhang's drug discovery program has, rather than treading over the same Aβ/plaque ground as many Alzheimer's disease researchers, been developing effective neuroprotectants and disease-modifying agents by targeting neuroinflammation and mitochondrial dysfunctions. One important component of his research is the use of transgenic animal models to demonstrate in vivo efficacy of improved cognitive functions. Through extensive collaborations at VCU and other institutions, a battery of behavioral tests are applied including contextual fear conditioning, novel object recognition and Morris Water Maze assessments [
The three major structural biology experimental techniques, X-ray crystallography, nuclear magnetic resonance (NMR) and cryo-electron microscopy (Cryo-EM) have become crucial tools for atomic-level understanding of macromolecule functions, as well as for rational or structure-based drug discovery (SBDD). Another, rapidly evolving, approach is computational prediction of protein structure. To date, X-ray crystallography has by far played the most significant role in structural biology related research, and it is no surprise that the field of structural biology and structure-based drug discovery took off after Max Perutz and John Kendrew used X-ray crystallography to solve the structures of hemoglobin and myoglobin in the late 1950s and early 1960s [
], respectively, earning them the Nobel Prize in Chemistry in 1962. Due to several limitations of X-ray crystallography, e.g., the need for pure, stable, and crystallizable protein in sufficient amounts, and NMR, for pure protein and its inherent unsuitability for large macromolecules, there is now an increasing reliance on Cryo-EM, especially with improvements in hardware and software that have made it easier to solve structures very fast and at high resolutions. In 2017, the Nobel Prize in Physics was awarded to Jacques Dubochet, Richard Henderson and Joachim Frank for their work in Cryo-EM technique. Currently, about 170,000 structures have been determined using X-ray crystallography, 14,000 with NMR, and 13,000 with Cryo-EM and are available to the community [
After Max Perutz published the structure of hemoglobin (Hb) in the 1960s (see Fig. 5), there was a flurry of activity using structural biology not only to study the function of Hb but also to target it with antisickling agents to treat sickle cell disease, an inherent genetic disorder caused by a single mutation (βGlu6 →βVal6) in normal hemoglobin, forming sickle Hb (HbS) [
]. Unlike normal Hb, when HbS offloads oxygen to tissue and becomes deoxygenated, it forms a polymer due to the formation of long, rigid and insoluble 14-stranded fibers from hydrophobic interactions between HbS molecules; thus, resulting in sickle-shaped red blood cells [
]. Adverse downstream pathophysiological events include adhesion of sickled cells to tissue endothelium to prevent blood flow, cell brittleness and hemolysis with consequent anemia, oxidative stress, decreased vascular nitric oxide bioavailability, and inflammation, all of which lead to morbidity, poor quality of life and premature mortality [
]. Sickle cell disease affects about 100,000 people in the U.S., and about 20 million individuals worldwide.
Fig. 5The X-ray crystal structure of deoxygenated hemoglobin. Abraham and Safo have exploited this structure to design numerous allosteric effectors and anti-sickling agents.
In the 1970s, Don Abraham realized that the structure of hemoglobin holds promise for developing antisickling drugs that would prevent the primary pathophysiology – hypoxia-induced HbS polymerization and red blood cell sickling – and subsequent downstream adverse effects. In collaboration with Max Perutz and others, Don Abraham used X-ray crystallography and structure-based techniques to start a systematic drug discovery program for sickle cell disease to find compounds that not only target pockets on the surface of Hb, but also the central water cavity of Hb; the former to directly prevent sickle hemoglobin molecules from contacting each other and forming the pathological polymer, and the latter to bind to hemoglobin's central water cavity to increase the oxygen affinity of sickle hemoglobin and prevent hypoxia-induced HbS polymerization [
Design, synthesis, and testing of potential antisickling agents. 5. Disubstituted benzoic acids designed for the donor site and proline salicylates designed for the acceptor site.
]. Since only deoxygenated sickle hemoglobin forms the polymer, increasing the concentration of the oxygenated state should prevent sickling. Abraham discovered a number of moderately active compounds, including aromatic aldehydes, proline derivatives, substituted benzoic acids, substituted alkanoic acid, ethacrynic acid and derivatives, and others [
Design, synthesis, and testing of potential antisickling agents. 5. Disubstituted benzoic acids designed for the donor site and proline salicylates designed for the acceptor site.
]. As noted below, Abraham's research using X-ray crystallography also played a pivotal role in the discovery of compounds improving potency and pharmacokinetics of next generation antisickling drug candidates, as well as compounds to treat underlying hypoxic diseases.
While Abraham started his sickle cell disease drug discovery program at the University of Pittsburgh, his arrival at Virginia Commonwealth University in 1988 really launched his drug discovery program using X-ray crystallography. The Rigaku (Molecular Structure, The Woodlands, TX) AFC5R rotating anode diffractometer operating at 9 kW power and equipped with a 60-cm-long evacuated beam tunnel was state-of-the art, yet took days to measure even 3.0 Ǻ X-ray diffraction data. Nonetheless, the data was good enough to allow computation of difference electron density maps between liganded and native oxy or deoxy hemoglobin for hundreds of co-crystallized potential antisickling agents [
], which allowed determination of the binding modes of the compounds with respect to Hb. This classic research contributed to understanding of how these compounds elicit activities (or lack thereof), and in a medicinal chemistry sense enabled targeted modifications of the compounds for improved pharmacologic activities. His work also was key in developing a detailed understanding of hemoglobin allostery [
]. This approach had numerous experimental limitations compared to current methodology, but nevertheless, Abraham solved the difference maps for several Hb-drug complex structures monthly. Tours of his laboratory complete with computer graphics visualization – often in stereo – of his solved structures were a regular occurrence for visitors.
2.4.2 Allosteric effectors of hemoglobin
The advent of CCD and image plate detectors and high intensity X-ray beams, modern computers and highly automated software led to significant reduction in data X-ray diffraction collection time, quality and resolution. Hemoglobin-ligand complex structures could be fully refined, allowing for determination of the precise atomic interactions between the protein and the compounds (Fig. 2h-i). Collaborating with Martin Safo, who began his VCU career as a postdoctoral with Abraham, several novel findings in both Hb allostery and drug discovery were made. Although Abraham passed away in 2021, Safo has continued many of their projects and has made significant impact in sickle cell disease drug discovery. Their work with aromatic aldehydes set the stage for the targeted discovery of several potent antisickling agents [
Design, Synthesis, and Investigation of Novel Nitric Oxide (NO)-Releasing Aromatic Aldehydes as Drug Candidates for the Treatment of Sickle Cell Disease.
]. Safo and his team have reported several other novel aromatic aldehydes that exhibit dual antisickling activities. These compounds not only prevent the hypoxia-induced sickle Hb polymerization but also directly prevent sickle Hb from interacting with each other [
Design, Synthesis, and Investigation of Novel Nitric Oxide (NO)-Releasing Aromatic Aldehydes as Drug Candidates for the Treatment of Sickle Cell Disease.
] potentially making these compounds superior to existing options to combat sickle cell disease pathophysiology. Several of these compounds have been licensed to Illexcor therapeutics and are undergoing IND-enabling studies for the treatment of sickle cell disease. Lastly, Abraham's pioneering structure-based work also included the invention of Hb allosteric effectors, which unlike antisickling agents, decrease the protein's affinity for oxygen. These rationally designed compounds, resulting from his earlier work with antisickling agents, notably RSR13, can increase tissue oxygenation and have potential to treat hypoxic diseases such as angina, stroke, trauma, and aid in transplant or pulmonary bypass surgery, blood storage and enhancing radiation treatment of hypoxic tumors [
Allosteric modifiers of hemoglobin: 2-[4-[[(3,5-disubstituted anilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid derivatives that lower the oxygen affinity of hemoglobin in red cell suspensions, in whole blood, and in vivo in rats.
]. Abraham formed a company, Allos Therapeutics, to further develop RSR13, which was studied in the clinic for treatment of brain metastases originating from breast carcinoma [
Survival of patients with newly diagnosed glioblastoma multiforme treated with RSR13 and radiotherapy: results of a phase II new approaches to brain tumor therapy CNS consortium safety and efficacy study.
A phase I study of RSR13, a radiation-enhancing hemoglobin modifier: tolerance of repeated intravenous doses and correlation of pharmacokinetics with pharmacodynamics.
As noted above, Abraham's vision of structure-based and rational drug discovery, especially for hemoglobin-based conditions, and as carried forward by Martin Safo, was a catalyst for the VCU drug discovery ecosystem. Below, we briefly describe a few other discovery projects that have taken advantage of Abraham's linkage of structural biology and medicinal chemistry.
Yan Zhang and his group – in their high-profile work on opioid receptors and agonists/antagonists – have taken a somewhat different approach: he has adopted and extended the “Message-Address” concept of Portoghese [
]. Their approach of atom-level SAR (supported by pharmacological results) exploits the GPCR structures with homology modeling and docking studies/MD simulations, and has identified the novel mu opioid receptor-selective antagonists, NAP and NAQ [
In vitro and in vivo functional profile characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ) as a low efficacy mu opioid receptor modulator.
Characterization of 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(indole-7-carboxamido)morphinan (NAN) as a Novel Opioid Receptor Modulator for Opioid Use Disorder Treatment.
Fig. 6Portoghese's "message-address" concept. Yan Zhang's research group uses this concept to design selective ligands for different type opioid receptor types by installing unique structural features that recognize the "address" domain (in green) of the receptor. For the ligands, they carry the same “message” portions (in red) to recognize the common “message” domains in the opioid receptors (in brown) and modulate their function, while the different “address” portions of the ligands bind to differentiated “address” domains of the receptors to achieve high selectivity. This is further demonstrated by the delta-selective opioid receptor ligand NTI and the kappa-selective opioid receptor ligand GNTI from Portoghese's lab.
The Ellis Lab has used crystallography and rational design approaches to new, selective anti-tumor compounds that inhibit the oncogenic transcriptional co-regulator C-Terminal Binding Protein (CtBP). [
CtBP- an emerging oncogene and novel small molecule drug target: Advances in the understanding of its oncogenic action and identification of therapeutic inhibitors.
] Once activated by NADH binding, CtBP acts as a key protein in transcriptional complexes that turn off tumor-suppressor genes and turn on various oncogenes. [
Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities.
] This multifaceted epigenetic co-regulation effect of CtBP which drives tumor growth and metastasis, makes CtBP a promising therapeutic target. The Ellis Lab began their work designing mechanism-based inhibitors using CtBP crystal structures, and were soon identified 2-hydroxy-imino-phenyl pyruvate (HIPP) as an initial lead compound. [
] HIPP and subsequent generations of analogues have been shown to inhibit tumor cell grown and induce apoptosis in several cancer types including breast, colon, ovarian, and pancreatic cell lines. [
Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities.
Using Cryo-EM, structures can be determined with nearly comparable atomic resolution to X-ray crystallography, and the fact that Cryo-EM structures can be determined closer to their native state gives structure-based drug design a great deal of hope. In addition, the method has vastly improved, permitting imaging or visualization of macromolecular complexes in their functional cellular context. Although cryo-EM has come close to achieving the high resolution we have come to associate with X-ray crystallography, the majority of structures determined are still of relatively low resolution, making it difficult to see atomic-level interactions between the macromolecule and bound ligand, which makes it suboptimal for drug discovery. Cryo-EM will likely become routine for generating high-resolution models, including the atomic-level interactions required for drug discovery, in the near future.
In determining the structures of membrane, or non-water-soluble, proteins, however, cryo-EM has distinct advantages. These proteins are essential macromolecules with diverse physiological functions, are implicated in numerous human diseases and pathological conditions, and serve as drug targets for over sixty percent of commercially available drugs. The majority of these drug targets are G-protein coupled receptors. The Nobel Prize awarded to Brian Kobilka and Robert Lefkowitz for their work on the X-ray crystallographic structure elucidation of the β2 Adrenergic Receptor/G protein complex demonstrates the significance of membrane protein structure [
]. The research group of Youzhong Guo focuses on membrane protein structural biology and the advancement of related technologies. Cryo-EM innovations such as single-particle cryo-EM and cryo-EM tomography are currently used to determine the structures of numerous membrane proteins for which crystallization is either difficult or impossible. Single-particle cryo-EM can determine the structures of membrane proteins with high resolution, but only if the single-particle samples are of high quality. Detergents have been used for decades to extract membrane proteins from the cell membrane, but they have significant drawbacks because they frequently result in membrane proteins being over-delipidated [
], without preserving their native structures and functions. As a platform for membrane protein structural biology and related drug discovery, Guo has been developing a novel detergent-free native cell membrane nanoparticles system [
]. His team has developed more than thirty distinct membrane-active polymers and application protocols for the production of nanoparticles. They determined the first high-resolution single-particle cryo-EM structure of the E. coli multidrug efflux transporter AcrB associated with a native, genuine patch of lipid bilayer from the cell membrane using this technique [
The Guo group has structure-based drug discovery projects in a variety of therapeutic areas, including neurology, anti-infectives, and others. For instance, the human mitochondrial TSPO is a known target for the development of positron emission tomography (PET) imaging probes (or tracers) for monitoring brain inflammation typically observed in neurodegenerative diseases such as Alzheimer's or Parkinson's. While the (first generation) [11C]PK-11195-type tracers have been extensively utilized in preclinical and clinical research. Several second and third generation tracers, including [18F]GE-180, (S)-[18F]GE-387, and [11C]ER176, have been developed; to evaluate the TSPO selectivity and insensitivity to TSPO polymorphism of these new tracers, however, additional research is required [
The team environment in the VCU ecosystem, which possesses extensive expertise in drug discovery and design, has been made available to facilitate the connection between structure and drug discovery. The promise of being able to characterize membrane proteins with their extremely crucial structure- and function-supporting lipids is a bright ray of hope for tackling increasingly complex therapeutic problems with ever-more-complex proteins and protein assemblies. With its rapidly advancing capabilities, cryo-EM will likely have a significant impact in the future. Notably, VCU has acquired a 100 keV Tundra Cryo-TEM [
], from Thermo-Fisher Scientific. This is the very first Tundra Cryo-TEM to be installed in the United States.
2.4.5 Computational protein structure prediction
Computational techniques have for many years served as a surrogate for determining the structures of macromolecules when the three-dimensional structures cannot be determined experimentally, usually due to lack of pure and/or crystallizable protein. Some of the traditional computational techniques to predict macromolecule structures include homology modeling, threading and ab Initio. In recent years, macromolecule structures determined by computational methods have gained a lot of attention, with the advent of deep-learning/artificial intelligence approaches, with one such method AlphaFold [
] accounting for over 1,000,000 predicted structures so far. This number compares to about 200,000 structures determined experimentally. While this vast dump of predicted structural data is certainly enabling, it is not foretelling the end of experimental structure determination. Many subtle features of structure are exactly those that differentiate between drugs and just compounds that bind.
Glen Kellogg and his group have been exploring the structure prediction problem starting from a residue-level perspective. The hypothesis is that a thorough understanding of the types, strengths and location of residue-residue (and residue-water, etc.) interactions is the framework of homology – not just sequence. These interactions are codified with 3D HINT interaction maps that, in turn, can be clustered into limited sets of interaction motifs representative of the “hydropathic valence” for each residue type, categorized by its backbone angles. Several publications describing this approach include: exploration of aromatic residues [
3D interaction homology: Hydropathic interaction environments of serine and cysteine are strikingly different and their roles adapt in membrane proteins.
AL Mughram M.H., Catalano C., Herrington N.B., Safo M.K., Kellogg G.E. 3D Interaction Homology: the Hydrophobic Residues Alanine, Isoleucine, Leucine, Proline and Valine Play Different Structural Roles in Soluble and Membrane Proteins. Front Mol Biosci (submitted).
]. The latter two reports also examined the different structural roles of these residues in soluble proteins and membrane proteins. Each of these studies revealed interesting, yet subtle, clues about protein structure that will be invaluable in predicting it.
Fig. 7Protein structure analysis and prediction. (Left) 3D HINT maps showing interactions between aspartate residues and their local environments. The contours represent favorable polar interactions (blue), unfavorable polar interactions (red), unfavorable hydrophobic interactions (purple). The large numbers, 32, 58, 394 and 396, are specific cluster names referenced to the cluster's exemplar (residue closest to its centroid) from an ordered list
. The fraction of each numbered residue compared to all with the same backbone angles is indicated. (Right) The computed titration curve of all ∼40,000 aspartates in the data set.
The wide diversity of projects and targets under investigation in the department has necessitated establishment of a robust and extensive collection of instrumentation to evaluate molecular properties, but more importantly, their biomolecular associations. Following Donald Abraham's retirement, Umesh Desai took over the leadership of the ISBDD [with a slight rebranding as the Institute for Structural Biology, Drug Discovery and Development (ISB3D)] and began a measured program to develop the biophysical and biochemical instrumentation infrastructure in 2007. Within a decade, this effort led to the procurement of multiple instruments for biophysical studies. This includes a high-end Photon Technology Interational (PTI) spectrofluorometer, a NanoTemper Microscale Thermophoresis (MST) instrument (Fig. 2e), a Nicoya Surface Plasmon Resonance (SPR) instrument, a Waters UPLC coupled to TQD-ESI-MS, a Malvern MicroCal PEAQ-Isothermal Calorimeter (Fig. 2g), and a ThermoFisher Fusion Lumos High Resolution Mass Spectrometer (Fig. 2f). To analyze the effects of these biomolecular associations, the Department and Institute have made a sizeable investment in high-throughput screening (HTS) instrumentation, from plate preparation robotics (Echo 550 Liquid Handler using acoustic droplet ejection (ADE) technology, Fig. 2c) to screening platforms, i.e., a BMG Labtech Clariostar for HTS kinetic and endpoint assays, a Cytation 5 imaging plate reader (Fig. 2b), a Molecular Devices Flexstation 3 (Fig. 2d), and a Microarray Printer and a Sensospot microarray slide reader. In addition, a substantial investment has been made in infrastructure for protein production (Fig. 2a) for both structural and assay experiments. Following are a few snapshots describing faculty research projects with successful outcomes that have been largely, if not wholly, supported by the unique and diverse biophysics, screening and other enabling tools available in the Department and the ISB3D at VCU.
2.5.1 Imaging and screening
The focus of Shujun Zhang's lab on small molecule discovery and development for neurodegenerative disorders, particularly Alzheimer's disease and other inflammatory diseases, relies on cutting-edge synthetic chemistry, molecular biology, etc. and takes advantage of the VCU ecosystem for drug screening, design and optimization. The molecular targets that Shijun Zhang's laboratory has been focused on include the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, an essential component of innate immunity, and mitochondrial complex I [
]. NLRP3 senses both exogenous microbial products and endogenous dangers associated with cellular stress and damage, and activation of the NLRP3 inflammasome leads to the production of proinflammatory cytokines interleukin (IL)-1β and IL-18, and promotes inflammatory responses and cell death. In the past several years, his lab has successfully developed small molecule inhibitors from novel chemical scaffolds. This research has provided new insights on the roles of the innate immune responses and mitochondrial dysfunction in the development of Alzheimer's disease and other neurodegenerative disorders through chemical biology and medicinal chemistry research. The shared-use NanoTemper MST, flow cytometer and gel imaging systems in the Department and Institute collections have proven indispensable and enabling for this research. Their studies also provided promising candidate compounds through preclinical studies to fill the drug discovery pipeline for development of Alzheimer's disease therapeutics.
Yan Zhang's lab has established calcium flux assays for both opioid receptors and chemokine receptors in order to understand the signaling of relevant antagonists and defining novel opioid, anti-HIV and anti-cancer agents. Such internal capacity not only enhances his group's and the department's screening platform for drug discovery and development, but also provides an invaluable training tool for graduate students and postdoctoral fellows by allowing them to participate in drug screening scenarios very similar to those in the pharmaceutical industry.
2.5.2 New therapeutic targets
Yana Cen's laboratory is interested in the group of NAD+-dependent protein deacylases known as sirtuins. These proteins have been implicated in aging processes and aging-related diseases and are considered attractive targets for the treatment of cancer, neurodegenerative disorders, metabolic dysfunctions, and inflammatory diseases. Her group takes a highly integrative chemical biology approach to study physiological functions and regulation of sirtuins as well as their pharmacological modulation with small molecules. They have been developing chemical probes that can directly confer the functional state of a specific sirtuin isoform in complex biological samples [
]. Such activity-based probes selectively “highlight” the active sirtuin content in a complicated cellular context. Side-by-side comparison of functional sirtuin profiles under different physiological and pathological conditions, combined with proteomics analysis, should unwind the intricate interaction relationships between human sirtuins and various cellular pathways and empower the better manipulation of these epigenetic enzymes for therapeutic purposes.
In particular, SIRT6 is a chromatin-associated epigenetic modification enzyme that has been implicated in the regulation of gene silencing, genome stability, DNA repair, and glucose homeostasis [
]. Cen's findings indicate that it plays a previously uncharacterized role as a DNA damage senor and possibly a DNA damage response pathway facilitator. Their tools, largely available in the shared instrumentation at VCU, are a unique combination of HPLC-based enzyme activity assays, isothermal titration calorimetry (ITC), electrophoretic mobility shift assays (EMSA), and photoaffinity labeling of SIRT6 with novel chemical probes [
] as well as site-directed mutagenesis and truncation analysis to investigate the DNA structure preferences for activation and to identify the allosteric binding site of SIRT6.
SIRT5, a mitochondrial sirtuin, has emerged as a critical player in maintaining cardiac health and neuronal viability upon stress, and functions as a tumor suppressor in a context-specific manner. Cen's group has identified the first SIRT5-selective allosteric activator, nicotinamide riboside, which can increase SIRT5 deacetylation efficiency with different synthetic peptide substrates as well as its endogenous cognate substrate. These continuing studies also involve significant collaboration within VCU, e.g., molecular modeling (Glen Kellogg), X-ray crystallography (Martin Safo), and solution state NMR studies (Brian Fuglestad, VCU Chemistry Department), in addition to activity-based protein profiling, cellular target engagement and activation analyses to elucidate unprecedented molecular details of SIRT5 allosteric regulation.
Jiong Li's group, in its aims to identify new therapeutic targets and develop novel strategies for eradicating colorectal cancer, has taken full advantage of the drug discovery ecosystem. Li is a cancer biologist, but has established a rich collaboration with Yan Zhang for drug discovery, design and synthesis. It is known that the abnormal activation of Wnt/β-catenin signaling can lead to initiation and development of colorectal cancer, and targeting Wnt signaling has been demonstrated to be an effective therapeutic target for the treatment of colorectal cancer [
]. Li's group is identifying new and viable targets and alternative strategies to overcome the current barriers to Wnt/β-catenin-dependent therapies. His group has identified several novel key components that control Wnt target gene transcription and Wnt-induced colorectal tumorigenesis, such as the KDM3A and KDM3B histone demethylases [
]. Further, his group has identified a promising KDM3 inhibitor, IOX1, which can suppress Wnt target gene transcription and Wnt-induced colorectal oncogenesis [
] and is currently developing IOX1 derivatives that are more potent and selective. Another result from the group, which promotes an alternative strategy, is the recent development of small molecule inhibitors that target β-catenin/TCF-dependent transcription, the key step in mediating nuclear Wnt/β-catenin signaling [
]. Computational studies suggest that naphthoquinone compounds bind within the DNA binding HMG-box domain of TCF4 to elicit their inhibitory action. The new compounds inhibited Wnt signaling in a dose-dependent manner, suppressed Wnt direct target genes and demonstrated unexpected degradation of the TCF4 protein. This is a potentially novel mechanism of action for chloro-naphthoquinone as a multi-targeting scaffold with therapeutic promise.
2.5.3 Microarrays and high-throughput screening (HTS)
The Desai lab has recently developed the GAG microarray technology for studying the range of proteins that these biopolymers modulate in vivo. This technology affords study of hundreds of proteins and/or ligands at nanoliter scales. Glass slides that are few cm long can be used to print a number of proteins and study their interaction with GAGs. One example of such a study was the revelation that a receptor tyrosine kinase called IGF-1R was modulated a small hexasaccharide unit of heparan sulfate [
]. Although this instrumentation was developed for study of GAGs, it is applicable to any library of biomolecules and/or synthetic agents.
A theme of ISB3D bacterial research is the discovery and use of natural products to prevent pathogens from causing infection. A common mechanism by which Gram-negative pathogens cause infection is by the use of a virulence apparatus called the type III secretion system (T3SS) [
]. The T3SS acts as a molecular syringe to secrete virulence proteins into host cells to subvert the immune response and allow colonization. The May lab has developed an assay capable of measuring T3SS activity and inhibition by small molecules [
]. Tannic acid is a plant-derived natural product, adding to the growing body of knowledge that plants use a T3SS inhibition strategy to protect themselves against infection [
A revolution in the field of liquid handling and HTS has been the use of acoustic droplet ejection (ADE) to transfer liquids from one well to another in individual droplet increments [
]. This process can be performed on an extremely small scale (increments of 2.5 nanoliters) and the quantized nature of these transfers makes them immune to causing changes in coefficient of variation. The May lab has used ADE to study the S. aureus essential protease named phage-related ribosomal protease, or Prp [
Phage-Related Ribosomal Protease (Prp) of Staphylococcus aureus: in Vitro Michaelis-Menten Kinetics, Screening for Inhibitors, and Crystal Structure of a Covalent Inhibition Product Complex.
]. Studies of this enzyme were initially performed on a 200 microliter scale using traditional pipetting techniques, giving a coefficient of variation of ∼2% in a peptide cleavage assay. ADE allowed this assay volume to be reduced to 6 microliters, compatible with 1536-well plates, and kept the coefficient of variation at ∼2%.
The focus of the Medicinal Chemistry Department and the ISB3D has always been on drug discovery, which is well known to be an extremely expensive endeavor. Our efforts to capture as much of the process as possible in-house has produced the capabilities and research successes mentioned above. Another point to be highlighted is that we possess the ability to miniaturize assays while keeping good statistics. Drug discovery is expensive, and challenging in academic environments. Our small and efficient, but powerful, instrumentation portfolio, allowing more innovations in-house, has been a key factor in the Department's success. Furthermore, making assays smaller while keeping them reliable is an emerging way to either lower costs or more importantly to be able to screen more compounds to discover potential leads.
3. Summary and assessment
The VCU Medicinal Chemistry Department and the Institute for Structural Biology, Drug Discovery [and Development] have accumulated and deployed an impressive collection of expertise and instrumentation. This was accomplished brick-by-brick through the vision of the previous and current leadership. The institution has provided funding for the purchase of many of these instruments largely through the State of Virginia Higher Education Equipment Trust Fund, but there never has been a bolus, and funding for technical staff to maintain, manage and train users on major instruments has been a continuing, and often unmet, challenge.
Nonetheless, one measure of success, and evidence that VCU has created a successful drug discovery ecosystem is the large number of collaborations developed inside this small group. It is fair to say that more than a quarter of the Department of Medicinal Chemistry's annual publications involve more than one PI from the group. Thus, while each faculty member seeks his or her own research niche, the environment – both people and facilities – draws broader lines that encourage, facilitate and maintain robust collaborations. It is clear in this case that the whole is greater than the sum of its parts!
Declaration of Competing Interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors acknowledge the School of Pharmacy of Virginia Commonwealth University for support when needed over the past five decades. The Commonwealth of Virginia Higher Education Equipment Trust Fund (HEETF) made substantial investments in many of the instruments available to us. The current X-ray diffractometer was supported in part by an NIH Shared Instrument grant in 2017. Numerous extramural grants have supported the research of the department over the past five decades. The faculty, both current and past, are grateful for the beating heart of our program and its successes from our numerous graduate and postdoctoral students, undergraduate and pharmacy students, visiting scientists, staff scientists, technicians and others. Photography for this report was performed by Connor O'Hara, with assistance by Savannah Biby, Akua Donkor, Rawan Fayyad and Faik Musayev.
[125I]-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane: an iodinated radioligand that specifically labels the agonist high-affinity state of 5-HT2 serotonin receptors.
Toward selective drug development for the human 5-hydroxytryptamine 1E receptor: a comparison of 5-hydroxytryptamine 1E and 1F receptor structure-affinity relationships.
des-Formylflustrabromine (dFBr): a structure-activity study on its ability to potentiate the action of acetylcholine at α4β2 nicotinic acetylcholine receptors.
Design, Synthesis and Biological Evaluation of 6α- and 6β-N-Heterocyclic Substituted Naltrexamine Derivatives as Mu Opioid Receptor Selective Antagonists.
Design, Synthesis, and Biological Evaluation of 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4’-pyridyl)carboxamido]morphinan Derivatives as Peripheral Selective Mu Opioid Receptor Antagonists.
Structure activity relationship studies of 6β- and 6α-indolylacetamidonaltrexamine derivatives as bitopic mu opioid receptor modulators and elaboration of ‘message-address concept’ to comprehend their functional conversion.
Design, Synthesis and Biological Evaluation of NAP Isosteres: a Switch from Peripheral to Central Nervous System Acting Mu-Opioid Receptor Antagonists.
Characterization of 6α- and 6β-N-Heterocyclic Substituted Naltrexamine Derivatives as Novel Leads to Development of Mu Opioid Receptor Selective Antagonists.
Behavioral and Cellular Pharmacology Characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan (NAQ) as a Mu Opioid Receptor Selective Ligand.
Comparison of Pharmacological Properties between the Kappa Opioid Receptor Agonist Nalfurafine and 42B, its 3-Dehydroxy Analogue: Disconnect between in Vitro Agonist Bias and in Vivo Pharmacological Effects.
Structure-Activity-Relationship Studies of Natural Product Chemokine Receptor CCR5 Antagonist Anibamine toward the Development of Anti Prostate Cancer Agents.
Design, synthesis, and in vitro evaluation of a fluorescently labeled irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKACα).
00017-5&id=gr1.jpg){kind=link}
00017-5&id=gr2.jpg){kind=link}
00017-5&id=gr3.jpg){kind=link}
00017-5&id=gr4.jpg){kind=link}
00017-5&id=gr5.jpg){kind=link}
00017-5&id=gr6.jpg){kind=link}
00017-5&id=gr7.jpg){kind=link}