Tony Giordano

New Therapeutic Strategies and Drug Candidates for Neurodegenerative Diseases: p53 and TNF‐α Inhibitors, and GLP‐1 Receptor Agonists

Abstract: Owing to improving preventative, diagnostic, and therapeutic measures for cardiovascular disease and a variety of cancers, the average ages of North Americans and Europeans continue to rise. Regrettably, accompanying this increase in life span, there has been an increase in the number of individuals afflicted with age‐related neurodegenerative disorders, such as Alzheimers disease, Parkinsons disease, and stroke. Although different cell types and brain areas are vulnerable among these, each disorder likely develops from activation of a common final cascade of biochemical and cellular events that eventually lead to neuronal dysfunction and death. In this regard, different triggers, including oxidative damage to DNA, the overactivation of glutamate receptors, and disruption of cellular calcium homeostasis, albeit initiated by different genetic and/or environmental factors, can instigate a cascade of intracellular events that induce apoptosis. To forestall the neurodegenerative process, we have chosen specific targets to inhibit that are at pivotal rate‐limiting steps within the pathological cascade. Such targets include TNF‐α, p53, and GLP‐1 receptor. The cytokine TNF‐α is elevated in Alzheimers disease, Parkinsons disease, stroke, and amyotrophic lateral sclerosis. Its synthesis can be reduced via posttranscriptional mechanisms with novel analogues of the classic drug, thalidomide. The intracellular protein and transcription factor, p53, is activated by the Alzheimers disease toxic peptide, Aβ, as well as by excess glutamate and hypoxia to trigger neural cell death. It is inactivated by novel tetrahydrobenzothiazole and ‐oxazole analogues to rescue cells from lethal insults. Stimulation of the glucagon‐like peptide‐1 receptor (GLP‐1R) in brain is associated with neurotrophic functions that, additionally, can protect cells against excess glutamate and other toxic insults.

RNA as a drug target: methods for biophysical characterization and screening.

RNA folds into complex structures that can interact specifically with effector proteins. These interactions are essential for various biological functions. In order to discover small molecules that can affect important RNA-protein complexes, a thorough analysis of the thermodynamics and kinetics of RNA-protein binding is required. This can facilitate the formulation of high-throughput screening strategies and the development of structure-activity relationships for compound leads. In addition to traditional methods, such as filter binding, gel mobility shift assay and various fluorescence techniques, newer methods such as surface plasmon resonance and mass spectrometry are being used for the study of RNA-protein interactions.

Alzheimer’s disease drug discovery targeted to the APP mRNA 5′Untranslated region

We performed a screen for drugs that specifically interact with the 5′ untranslated region of the mRNA coding for the Alzheimer’s Amyloid Precursor Protein (APP). Using a transfection based assay, in which APP 5′UTR sequences drive the translation of a downstream luciferase reporter gene, we have been screening for new therapeutic compounds that already have FDA approval and are pharmacologically and clinically well-characterized. Several classes of FDA-pre-approved drugs (16 hits) reduced APP 5′UTR-directed luciferase expression (>95% inhibition of translation). The classes of drugs include known blockers of receptor ligand interactions, bacterial antibiotics, drugs involed in lipid metabolism, and metal chelators. These APP 5′UTR directed drugs exemplify a new strategy to identify RNA-directed agents to lower APP translation and Aβ peptide output for Alzheimer’s disease therapeutics.

The integrated role of desferrioxamine and phenserine targeted to an iron-responsive element in the APP-mRNA 5'-untranslated region.

Abstract: The Alzheimers amyloid precursor protein (APP) is the metalloprotein that is cleaved to generate the pathogenic Aβ peptide. We showed that iron closely regulated the expression of APP by 5′‐untranslated region (5′‐UTR) sequences in APP mRNA. Iron modulated APP holoprotein expression by a pathway similar to iron control of the translation of the ferritin‐L and ‐H mRNAs by iron‐responsive elements in their 5′‐UTRs. APP gene transcription is also responsive to copper deficit where the Menkes protein depleted fibroblasts of copper to suppress transcription of APP through metal regulatory and copper regulatory sequences upstream of the APP 5′ cap site. APP is a copper‐zinc metalloprotein and chelation of Fe3+ by desferrioxamine and Cu2+ by clioquinol appeared to provide effective therapy for the treatment of AD in limited patient studies. We have introduced an RNA‐based screen for small APP 5′‐UTR binding molecules to identify leads that limit APP translation (but not APLP‐1 and APLP‐2) and amyloid Aβ peptide production. A library of 1200 drugs was screened to identify lead drugs that limited APP 5′‐UTR‐directed translation of a reporter gene. The efficacy of these leads was confirmed for specificity in a cell‐based secondary assay to measure the steady‐state levels of APP holoprotein relative to APLP‐1/APLP‐2 by Western blotting. Several chelators were identified among the APP 5′‐UTR directed leads consistent with the presence of an IRE stem‐loop in front of the start codon of the APP transcript. The APP 5′‐UTR‐directed drugs—desferrioxamine (Fe3+ chelator), tetrathiomolybdate (Cu2+ chelator), and dimercaptopropanol (Pb2+ and Hg2+ chelator)—each suppressed APP holoprotein expression (and lowered Aβ peptide secretion). The novel anticholinesterase phenserine also provided “proof of concept” for our strategy to target the APP 5′‐UTR sequence to identify “anti‐amyloid” drugs. We further defined the interaction between iron chelation and phenserine action to control APP 5′‐UTR‐directed translation in neuroblastoma (SY5Y) transfectants. Phenserine was most efficient to block translation under conditions of intracellular iron chelation with desferrioxamine suggesting that this anticholinesterase operated through an iron (metal)‐dependent pathway at the APP 5′‐UTR site.

Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs

A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250 nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible.

Design, synthesis and evaluation of novel 2-thiophen-5-yl-3H-quinazolin-4-one analogues as inhibitors of transcription factors NF-кB and AP-1 mediated transcriptional activation: Their possible utilization as anti-inflammatory and anti-cancer agents

In an attempt to discover novel inhibitors of NF-kappaB and AP-1 mediated transcriptional activation utilizing the concept of chemical lead based medicinal chemistry and bioisosterism a series of 2-(2,3-disubstituted-thiophen-5-yl)-3H-quinazolin-4-one analogs was designed. A facile and simple route for the synthesis of the designed molecules was developed. Synthesized molecules were evaluated for their activity as inhibitors towards NF-kappaB and AP-1 mediated transcriptional activation in a cell line report-based assay. This series provides us with a substantial number of compounds inhibiting the activity of NF-kappaB and/or AP-1 mediated transcriptional activation. These compounds also exhibit anti-inflammatory and anti-cancer activity in in vivo models of inflammation and cancer. The 4-pyridyl group is found to be the most important pharmacophore on the third position of thiophene ring for inhibiting NF-kappaB and AP-1 mediated transcriptional activation. The relationships between the activities shown by these compounds in the in vivo and in vitro models have been established by using FVB transgenic mice model. These results suggest the suitability of the designed molecular framework as a potential scaffold for the design of molecules with inhibitory activity towards NF-kappaB and AP-1 mediated transcriptional activation, which may also exhibit anti-inflammatory and anti-cancer activity. This series of molecules warrants further study to explore their potential as therapies for use in chronic inflammatory conditions and cancer. Development of the synthetic protocol for the synthesis of this series of molecules, biological activities and a structure-activity relationship (SAR) have been discussed herein.

Synthesis and evaluation of quinazolinone derivatives as inhibitors of NF-κB, AP-1 mediated transcription and eIF-4E mediated translational activation: Inhibitors of multi-pathways involve in cancer

In our effort to discover and develop small molecule multi-pathway inhibitors which may be useful as tools for treating cancerous conditions, we have synthesized a small library of 2-thiazole-5-yl-3H-quinazolin-4-one derivatives. Synthesized compounds were evaluated as inhibitors of NF-kappaB and AP-1 mediated transcriptional and eIF-4E mediated translational activation as these transcription and translation factors are known to play a pivotal role in initiation and progression of cancer. The results from the study suggest the utility of the 2-thiazole-5-yl-3H-quinazolin-4-one scaffold as a promising scaffold for the design of novel multi-pathway inhibitors, which can be explored as anti-cancer agents.

A surrogate-based approach for post-genomic partner identification

BackgroundModern drug discovery is concerned with identification and validation of novel protein targets from among the 30,000 genes or more postulated to be present in the human genome. While protein-protein interactions may be central to many disease indications, it has been difficult to identify new chemical entities capable of regulating these interactions as either agonists or antagonists.ResultsIn this paper, we show that peptide complements (or surrogates) derived from highly diverse random phage display libraries can be used for the identification of the expected natural biological partners for protein and non-protein targets. Our examples include surrogates isolated against both an extracellular secreted protein (TNFβ) and intracellular disease related mRNAs. In each case, surrogates binding to these targets were obtained and found to contain partner information embedded in their amino acid sequences. Furthermore, this information was able to identify the correct biological partners from large human genome databases by rapid and integrated computer based searches.ConclusionsModified versions of these surrogates should provide agents capable of modifying the activity of these targets and enable one to study their involvement in specific biological processes as a means of target validation for downstream drug discovery.