Wayne C Guida

University of South Florida, Chemistry, Faculty Member

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I am a full professor in the Chemistry Department at the University of South Florida in Tampa. In our lab, we are interested in discovering molecules that can be used as drugs to treat cancer and autoimmune diseases. We have a long-standing interest in inhibitors of enzymes in signal transduction pathways and in the biosynthetic pathways of polyamines but our latest research project involves the discovery of compounds that can modulate a protein called STING (STimulator of INterferon Genes). STING is a protein involved in the innate immune response and STING-mediated production of Beta-Interferon (IFN-beta) within a tumor’s microenvironment can result in activation of tumor antigen-specific (CD8 ) T-cell immunity that can lead to solid tumor regression. Prior studies have demonstrated that STING activation should result in T-cell mediated anti-tumor activity, which has significant potential as a therapeutic strategy for the treatment of patients with advanced solid tumors. On the other hand, inhibition of STING (by STING antagonists) would lead to a decreased production of IFN-beta which could have implications in the treatment of autoimmune diseases.
Address: 4202 East Fowler Avenue
Tampa, FL 33620

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Abstract 6: Computationally assisted target screening of STING agonist for immunologic therapy

Cancer Research, Jul 1, 2019

Stimulator of interferon genes (STING) is a receptor protein involved in the propagation of innat... more Stimulator of interferon genes (STING) is a receptor protein involved in the propagation of innate immune sensing of cytosolic DNA through the production of IFN-β. Mechanistic studies have shown IFN-β production within the tumor microenvironment can result in activation of tumor antigen-specific CD8+ T-cell immunity that can lead to tumor regression [1, 2]. STING activation by STING agonists should result in innate T-cell mediated anti-tumor immunity in the tumor microenvironment and have significant potential as a cancer therapeutic. Conversely, inhibition of STING would lead to a decreased production of IFN-β which could have implications in the treatment of autoimmune disease such as lupus erythematosus. MD equilibrated crystal structures for human HAQ, REF, and WT alleles were clustered to find optimal conformations for diverse chemical library screening. Novel consensus docking protocols utilizing rigid receptor, induced fit, and quantum polarized ligand docking were applied for quantifying and refining proposed binding mechanisms of STING isoforms. Models for STING agonists and antagonists were developed. From directed virtual screening, a novel low-molecular-weight organic molecule (GF3-002) that is not based on a cyclic dinucleotide was found as a potential STING activator and is currently under investigation. GF3-002 and analogs were synthesized and structures were confirmed with LCMS and proton NMR. Both the HAQ and WT alleles, representing 78.3% of the human population were tested against compounds and controls. 2,3-cGAMP and DMSO were used as positive and negative controls, respectively. Microscale thermophoresis and SPR confirmed binding of GF3-002 to WT STING CTD with a kD of 3.2 ± 1.7 μM. IFN-dependent luciferase expression was measured by luminescence in THP-1 monocytic leukemia cells and found an EC50 of 29 ± 1.6 μM, compared to the native ligand 2,3-cGAMP EC50 of 42 ± 4.1 μM. Finally, qPCR was used quantify production of IFN-β via treatment of dendritic cells with GF3-002. 1. Sali, Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses. 2017. 2. Corrales, L., et al., Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. Cell reports, 2015. 11(7): p. 1018-1030. Citation Format: Grace A. Binder, Christine S. Gambino, Anna Kharitonova, Rainer S. Metcalf, Kenyon G. Daniel, Wayne C. Guida. Computationally assisted target screening of STING agonist for immunologic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 6.

Virtual and experimental high throughput screening identifies a proteasome inhibitor highly selective for inducing apoptosis of cancer cells over normal cells

Cancer Research, Apr 15, 2006

The fact that many proapoptotic proteins are degraded by the proteasome prompted the development ... more

ChemInform Abstract: Structure-Based Design of Inhibitors of Purine Nucleoside Phosphorylase. Part 3. 9-Arylmethyl Derivatives of 9-Deazaguanine Substituted on the Methylene Group

ChemInform, Aug 19, 2010

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Structure-based design of inhibitors of purine nucleoside phosphorylase. 3. 9-Arylmethyl derivatives of 9-deazaguanine substituted on the arylmethyl group. [Erratum to document cited in CA119(25):271601v]

Journal of Medicinal Chemistry, Apr 1, 1994

Page 1. J. Med. Chem. 1993,36, 55-69 66 Structure-Based Design of Inhibitors of Purine Nucleoside... more

A Small-Molecule E2F Inhibitor Blocks Growth in a Melanoma Culture Model

Cancer Research, Aug 1, 2008

HLM006474 was identified using a computer-based virtual screen and the known crystal structure of... more HLM006474 was identified using a computer-based virtual screen and the known crystal structure of the DNA-bound E2F4/DP2 heterodimer. Treatment of multiple cell lines with HLM006474 resulted in the loss of intracellular E2F4 DNAbinding activity as measured by electrophoretic mobility shift assay within hours. Overnight exposure to HLM006474 resulted in down-regulation of total E2F4 protein as well as known E2F targets. The effects of HLM006474 treatment on different cell lines varied but included a reduction in cell proliferation and an increase in apoptosis. HLM006474 induced apoptosis in a manner distinct from cisplatin and doxorubicin. E2F4-null mouse embryonic fibroblasts were less sensitive than wild-type counterparts to the apoptosis-inducing activity of the compound, revealing its biological specificity. A375 cells were extremely sensitive to the apoptosis-inducing activity of the compound in twodimensional culture, and HLM006474 was a potent inhibitor of melanocytes proliferation and subsequent invasion in a three-dimensional tissue culture model system. Together, these results suggest that interference with E2F activity using small molecules may have clinical application in cancer therapy.

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Structure-Based Design of Inhibitors of Purine Nucleoside Phosphorylase. 4. A Study of Phosphate Mimics

Journal of Medicinal Chemistry, Apr 1, 1994

Introduction Acyclovir diphosphate (ACVdiP, 1) is a potent inhibitor of purine nucleoside phospho... more Introduction Acyclovir diphosphate (ACVdiP, 1) is a potent inhibitor of purine nucleoside phosphorylase (PNP),&amp;amp;amp;amp;#x27; and its binding to the active site of the enzyme has been studied by X-ray crystallography.2 This study showed that, as predicted,l ACVdiP is a bisubstrate inhibitor ...

Structure-based design of inhibitors of purine nucleoside phosphorylase. 2. 9-Alicyclic and 9-heteroalicyclic derivatives of 9-deazaguanine

Journal of Medicinal Chemistry, Jun 1, 1993

Alicyclic and heteroalicyclic derivatives of 9-deazaguanine (2-amino-1,5-dihydro-4H-pyrrolo[3,2-d... more Alicyclic and heteroalicyclic derivatives of 9-deazaguanine (2-amino-1,5-dihydro-4H-pyrrolo[3,2-d] [pyrimidin-4-one) are, with one exception, potent inhibitors of purine nucleoside phosphorylase (PNP) equaling the corresponding 9-arylmethyl derivatives previously investigated. The mode of binding of these compounds to PNP was determined by X-ray crystallography.

Structure-based design of inhibitors of purine nucleoside phosphorylase. 3. 9-Arylmethyl derivatives of 9-deazaguanine substituted on the arylmethyl group

Journal of Medicinal Chemistry, Nov 1, 1993

Introduction Previous studies using X-ray crystallography and com-puter-assisted molecular modeli... more

Application of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors

Proceedings of the National Academy of Sciences of the United States of America, Dec 15, 1991

Competitive inhibitors of the salvage path- way enzyme purine-nucleoside phosphorylase (purine- n... more Competitive inhibitors of the salvage path- way enzyme purine-nucleoside phosphorylase (purine- nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) have been designed by using the three-dimensional strucre of the enzyme as determined by x-ray crystallography. The process was an Iterative one that utilized interactive omputer graphics, Monte Carlo-based orational s ing, en- ergy minimization, and x-ray crystallography. The proposed

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Structure-based design of inhibitors of purine nucleoside phosphorylase. 1. 9-(Arylmethyl) derivatives of 9-deazaguanine

Journal of Medicinal Chemistry, 1993

Page 1. J. Med. Chem. 1993,36, 55-69 66 Structure-Based Design of Inhibitors of Purine Nucleoside... more

Data from A Small-Molecule E2F Inhibitor Blocks Growth in a Melanoma Culture Model

HLM006474 was identified using a computer-based virtual screen and the known crystal structure of... more HLM006474 was identified using a computer-based virtual screen and the known crystal structure of the DNA-bound E2F4/DP2 heterodimer. Treatment of multiple cell lines with HLM006474 resulted in the loss of intracellular E2F4 DNA-binding activity as measured by electrophoretic mobility shift assay within hours. Overnight exposure to HLM006474 resulted in down-regulation of total E2F4 protein as well as known E2F targets. The effects of HLM006474 treatment on different cell lines varied but included a reduction in cell proliferation and an increase in apoptosis. HLM006474 induced apoptosis in a manner distinct from cisplatin and doxorubicin. E2F4-null mouse embryonic fibroblasts were less sensitive than wild-type counterparts to the apoptosis-inducing activity of the compound, revealing its biological specificity. A375 cells were extremely sensitive to the apoptosis-inducing activity of the compound in two-dimensional culture, and HLM006474 was a potent inhibitor of melanocytes proliferation and subsequent invasion in a three-dimensional tissue culture model system. Together, these results suggest that interference with E2F activity using small molecules may have clinical application in cancer therapy. [Cancer Res 2008;68(15):6292–9]

Abstract 3904: Pyridylthiazole-based ureas as inhibitors of Rho-associated protein kinases

Cancer Research, Apr 1, 2012

Rho GTPase is a small G-protein which plays a critical role in signaling pathways and controls ce... more Rho GTPase is a small G-protein which plays a critical role in signaling pathways and controls cell growth and division. Rho-associated protein kinase (known as ROCK or Rho kinase) is a Ser/Thr protein kinase activated by GTP-bound Rho and phosphorylates target proteins involved in various signal transduction pathways. Rho and the Rho-kinase signalling pathways are implicated in cell morphology, motility, smooth muscle contraction, formation of stress fibres, focal adhesion and cell transformation. ROCKs have been subject to growing attention, having been implicated in a range of therapeutic areas including cardiovascular diseases, CNS disorders, and cancer. Further, the pharmacological inhibition of ROCKs has been suggested as a promising strategy in the prevention of cell invasion, a central event in the process of metastasis. Potent ROCK inhibitors of a new class of 1-benzyl-3-(4-pyridylthiazol-2-yl)ureas have been identified. Remarkable differences in activity were observed for ureas bearing a benzylic stereogenic center. Derivatives with hydroxy, methoxy and amino groups at the meta position of the phenyl ring give rise to the most potent inhibitors (low nM). Substitutions at the para position result in substantial loss of potency. Changes at the benzylic position are tolerated resulting in significant potency in the case of methyl and methylenehydroxy groups. X-ray crystallography was used to establish the binding mode of this class of inhibitors and provides an explanation for the observed differences of the enantiomer series. Potent inhibition of ROCK in human lung cancer cells was shown by suppression of the phosphorylation levels of the ROCK substrate MYPT-1. One of the series, RKI-1447 inhibits migration, invasion and anchorage-independent tumor growth of breast cancer cells and is discussed in an accompanying poster. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3904. doi:1538-7445.AM2012-3904

Structure-based design of inhibitors of purine nucleoside phosphorylase

Acta Crystallographica Section D-biological Crystallography, Jul 1, 1995

Inhibitors of purine nucleoside phosphorylase may have therapeutic value in the treatment of T-ce... more

Design of inhibitors of the protein tyrosine phosphatase Shp2 as novel anticancer agents

Cancer Research, May 1, 2008

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 1307 Shp2, encoded by PTPN11 gene, is a prot... more AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 1307 Shp2, encoded by PTPN11 gene, is a protein tyrosine phosphatase (PTP) that mediates growth factor- and cytokine-stimulated cellular activities essential for oncogenesis. It is a positive regulator of Src family kinases (SFKs), Ras, and Erk MAP kinases. Shp2 mutations have been found in juvenile myelomonocytic leukemia (JMML), myelodysplastic syndrome, acute myeloid leukemia, acute lymphoblastic leukemia, and solid tumors such as neuroblastoma, lung cancer, colon cancer, and melanoma. In particular, PTPN11 is the most frequently mutated gene in JMML, accounting for 35-50% of cases. All Shp2 mutations found in human malignancies are gain-of-function mutations, resulting in constitutively active Shp2. Thus, Shp2 PTP is an important target for new cancer therapy. The structure of the Shp2 phosphatase has been determined by X-ray crystallography at 2.0 A resolution. Shp2 contains two Src homology-2 (SH2) domains (N-SH2, C-SH2) at its N-terminal region. In the absence of a tyrosine-phosphorylated binding partner, the N-SH2 domain blocks the catalytic domain until the upstream signaling effectors bind to Shp2. Consequently, Shp2 is basally inactive due to autoinhibition and inhibition of Shp2 by a small molecule is not predicted to affect resting cells, making it an attractive target for drug design. A challenge of this approach is the design of a small molecule that does not bind to the phosphotyrosine binding sites that control the dynamic (on/off) binding of the N-SH2 domain to the PTP pocket. Screening of a small library has led to the identification of an inhibitor of the signaling protein tyrosine phosphatase (PTP) Shp2. A focused library was designed incorporating the isatin scaffold was screened for Shp2 and Shp1 activity. Several compounds were identified that selectively inhibit Shp2 with low to sub-micromolar activity. A model for the selective binding of the active compounds will be presented.

Supplementary Figure 1 from A Novel Inhibitor of STAT3 Homodimerization Selectively Suppresses STAT3 Activity and Malignant Transformation

PDF file - 264K, Figure S1: STAT3-C rescues from S3I-1757 down regulation of STAT3-regulated gene... more PDF file - 264K, Figure S1: STAT3-C rescues from S3I-1757 down regulation of STAT3-regulated genes and inhibition of proliferation/survival. MDA-MB-468 cells were transiently transfected with vector or STAT3C then were treated with S3I-1757 as described in Methods. The cells were then processed for western immuno-blotting (A) and for MTT assays (B) as described in Methods.

Supplementary Figures 1-5, Methods and Materials from A Small-Molecule E2F Inhibitor Blocks Growth in a Melanoma Culture Model

Supplementary Figures 1-5, Methods and Materials from A Small-Molecule E2F Inhibitor Blocks Growt... more

Intracellular protein degradation of BRD4 by dBET1 reveals conserved in vivo cereblon function in human and mouse T-cells

The Journal of Immunology

Protein degraders have been developed that can aide in understanding gene function. These chemica... more Protein degraders have been developed that can aide in understanding gene function. These chemicals contain complementary bifunctional components that 1) interact with specific proteins, and 2) engage an E3 ligase which leads to polyubiquitination and intracellular degradation by the proteasome. Thalidomide, infamously known for its teratogenic effects, is central to this new technology since it binds cereblon (CRBN) which promiscuously recruits the DDB1/Cul4A/Rbx1 complex to trigger target destruction. Thalidomide, and especially its immunomodulatory derivatives lenalidomide and pomalidomide, induce NK and T-cell activation by degrading IKZF1 which is a transcription factor that suppresses IL-2. Currently, it is debated whether mouse cereblon has conserved function due to a single non-conserved amino acid in mouse CRBN, Ile390 (equivalent to Val388 in human CRBN). First, our work confirms that lenalidomide induces IL-2 and the ubiquitin-dependent degradation of IKZF1 only in human ...

p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis

ABSTRACTSince deregulation of intracellular Ca2+ can lead to intracellular trypsin activation and... more ABSTRACTSince deregulation of intracellular Ca2+ can lead to intracellular trypsin activation and STIM1 (stromal interaction molecule-1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) interactions and enhanced SERCA pump activity leading to increased Store Operated Calcium Entry (SOCE). In the pancreatic AR42J cells expressing t...

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Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon

The Journal of biological chemistry, Jan 15, 2018

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor ce... more Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteasomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3-ubiquitin ligase in human cells but not in mouse cells suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBNs activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBNs non-primate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug binding properties to both classical immunomodulatory drugs and to dBET1, a chemical compound and targeting ligand designed to deg...

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Molecular Dynamics Simulations of Membrane-Bound STIM1 to Investigate Conformational Changes during STIM1 Activation upon Calcium Release

Journal of chemical information and modeling, Feb 27, 2017

Calcium is involved in important intracellular processes, such as intracellular signaling from ce... more Calcium is involved in important intracellular processes, such as intracellular signaling from cell membrane receptors to the nucleus. Typically, calcium levels are kept at less than 100 nM in the nucleus and cytosol, but some calcium is stored in the endoplasmic reticulum (ER) lumen for rapid release to activate intracellular calcium-dependent functions. Stromal interacting molecule 1 (STIM1) plays a critical role in early sensing of changes in the ER's calcium level, especially when there is a sudden release of stored calcium from the ER. Inactive STIM1, which has a bound calcium ion, is activated upon ion release. Following activation of STIM1, there is STIM1-assisted initiation of extracellular calcium entry through channels in the cell membrane. This extracellular calcium entering the cell then amplifies intracellular calcium-dependent actions. At the end of the process, ER levels of stored calcium are reestablished. The main focus of this work was to study the conformation...

Wayne C Guida

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