|Drug Design Employing Neuropeptides of Multiple Actions: Potential Novel Treatments for Alzheimer's Disease, Cancer and Impotence |
Prof. Illana Gozes has been studying brain-specific molecules encompassing aspects from genetic engineering to behavior and memory. Major studies have been devoted to the neuropeptide VIP (vasoactive intestinal peptide, a small protein found in the brain).(http://www.acnp.org/citations/GN401000057/Default.htm); http://pharmrev.aspetjournals.org/cgi/content/full/50/2/265).
Originally discovered as a potent dilator of blood vessels, its activity leads to increases in blood flow and improvement of bodily functions. The laboratory of Prof. Gozes was the first to isolate and purify (clone) the gene (http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=192320) that contains the genetic information for the synthesis of VIP. It was further shown that its expression is reduced in the aging brain. Inhibition of VIP gene activity resulted in loss of memory, decreased learning abilities and inhibition of sexual function.
VIP-based drug design
The studies concerning VIP gene functions prompted the synthesis of novel neuropeptide-based drugs (in collaboration with Prof. Mati Fridkin, Weizmann Institute of Science). These molecules of chosen architecture are designed to penetrate barriers, and overcome the hindrance of entry to the brain and impediments of entry through the skin. The experimental use of these compounds allows better understanding of VIP's actions in the body and indicates that it is essential for embryonic brain development, with its inhibition resulting in severe microcephaly (in collaboration with the laboratory of Dr. Douglas E. Brenneman, NIH, USA and Dr. Pierre Gressens, Robert-Debre Hospital, France). Furthermore, design of specific VIP inhibiting molecules has ramifications for cancer therapy, such as breast cancer, lung cancer, neuroblastoma and colon cancer - all very prominent, life threatening malignancies (in collaboration with Drs. T.W. Moody, NIH, USA, Z. Dreznik and Y. Wollman, Tel-Aviv University) (http://www3.interscience.wiley.com/cgi-bin/issuetoc?ID=85515582); (http://humanapress.com)
Skin application of the vasodilator VIP-based neuropeptide is aimed towards developing a non-invasive anti-impotence therapeutics. Finally, chemically altered neuropeptides are designed for intranasal application for the treatment of memory loss in neurodegenerative diseases (http://www.pnas.org/cgi/reprint/93/1/427); (http://www.pnas.org/cgi/content/full/96/7/4143). A key invention in those studies is the use of hybrid molecules, such as hybrids between proteins and fatty substances allowing better biological availability. (http://www.jci.org/cgi/content/full/100/2/390)
Prof. Illana Gozes in collaboration with Dr. Douglas E. Brenneman (National Institutes of Health, USA) found a new chemical in the brain that has remarkable protective properties from substances that can kill nerve cells. The new protective protein is called Activity-Dependent Neurotrophic Factor (ADNF), a name coined to reflect its relationship to electrical activity in the brain. This new protein was isolated from cells in the brain that nurture and protect nerve cells as a part of its normal function. These cells are called astrocytes. ADNF was discovered in the fluid surrounding astrocytes after they were treated with a small amount of VIP (http://www.jci.org/cgi/content/full/97/10/2299); (http://www.jci.org/cgi/content/full/99/12/2837).
A key finding: ADNF acts in extremely low concentrations (10-100 molecules/cell) making it an attractive candidate for drug design. A short fragment derived from ADNF, a peptide ADNF-14, prevented neuronal cell death from the beta amyloid peptide, a substance implicated in the death of nerve cells in Alzheimer's disease. Another promising study indicated that nerve cells grown in culture were protected by ADNF-14 from a toxic protein (gp120) shed by the human immunodeficiency virus, the causative agent in AIDS. These two studies serve not only as examples of the protective action of ADNF-14, but they also represent the first step in a long series of investigations which will be needed to determine if ADNF-14 has potential as a drug to alleviate the symptoms of patients with either AIDS or Alzheimer's disease. Furthermore, ADNF is associated with VIP-mediated nerve-cell connectivity (synapse formation) during brain development
From a basic science point of view, the novel protein and the unraveling of its mechanisms of action will open new horizons in the study of the processes leading to nerve cell survival and death. These mechanisms are of importance to our understanding of brain development and the acquisition of learning abilities and of aging and the loss of memory capacities.
From a clinical point of view, new small compounds will be developed and tested for their therapeutic potential in Alzheimer's patients and/or patients suffering from any neurodegenerative diseases (diseases affecting the brain cells leading to their destruction and death which is then manifested by loss of functions).
- ADNF activity is essential for neuronal survival as assayed by specific antibodies.
- VIP's neuroprotective and neurotrophic activities are mediated by ADNF.
- VIP -derived compounds can provide prophylactic neuroprotection.
Key findings, 1999-2005, the discovery of a new VIP-responsive gene coding for Activity-Dependent Neuroprotective Protein (ADNP)
The complete coding sequence of a novel protein derived from mouse neuro-glial cells, was revealed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=6752989&dopt=GenBank); (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=4406072&dopt=GenBank).
The sequence contained: 1) an eight amino acid neuroprotective peptide NAP, sharing structural homologies with the previously reported, ADNF; 2) a glutaredoxin active site; and 3) zinc binding domains. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide, VIP, in cerebral cortical astrocytes. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of VIP, an established neuroprotective substance, imply a role for the cloned protein in brain function. Daily injections of the new, eight-amino-acid, peptide to newborn animals (retarded in their development), accelerated the acquisition of developmental reflexes and prevented short-term memory deficits. Inhalation of the peptide provided protection against cognitive loss associated with Alzheimer's disease related deficiencies (http://jpet.aspetjournals.org/cgi/content/full/293/3/1091"). Single peptide injections protected against the devastating consequences of head trauma in relevant model systems (http://jpet.aspetjournals.org/cgi/content/full/296/1/57). Comparative studies suggested that this new peptide was very efficacious compared to other neuroprotective peptides. A potential basis for rational drug design against neurodegeneration is suggested.
In 2001, the human ADNP gene was cloned and published (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=AF068198.1);(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=12229216&dopt=GenBank); (http://www.jbc.org/cgi/content/full/276/1/708)
Comparative sequence analysis of the human and mouse ADNP genes indicated 90% identity at the mRNA level. Several single nucleotide polymorphic sites were noticed on the human gene. The deduced protein structure contained nine zinc fingers, a proline-rich region, a nuclear bipartite localization signal, and a homeobox domain profile, suggesting a transcription factor function. Further comparative analysis identified an ADNP-like gene (33% identity and 46% similarity), indicating that these genes belong to a novel protein family with a nine-zinc finger motif followed by a homeobox domain. However, the new member does not contain the active peptide, NAP. The human ADNP gene was mapped to chromosome 20q12-13.2, a region associated with aggressive tumor growth, frequently amplified in many neoplasias, including breast, bladder, ovarian, pancreatic, and colon cancers. The human ADNP mRNA is abundantly expressed in distinct normal tissues, and high expression levels were encountered in malignant cells. Down-regulation of ADNP by antisense oligodeoxynucleotides (a potential form of gene therapy) up-regulated the tumor suppressor p53 and reduced the viability of intestinal cancer cells by 90% (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=13170291&dopt=GenBank). Thus, ADNP is implicated in maintaining cell survival, on the crossroads between cell protection and cell division
Gozes, I. and Brenneman, D.E. A new concept in the pharmacology of neuroprotection. J. Mol. Neurosci. 14, 61-64, 2000 (http://humanapress.com)
Bassan, M., Zamostiano, R., Davidson, A., Pinhasov, A., Giladi, E., Perl, O., Bassan, H., Blatt, C., Gibney, G., Glazner, G., Brenneman, D.E., and Gozes, I. (1999) "Complete cDNA sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide." J. Neurochem. 72,1283-1293.
Zamostiano, R., Pinhasov, A., Gleber, E., Steingart, R.A., Seroussi, E., Giladi, E., Bassan, M., Wollman, Y., Mulley, J.C., Brenneman, D.E., and Gozes, I. (2001) "Cloning and characterization of the human activity-dependent neuroprotective protein." J. Biol. Chem. 276, 708-714.
Recent results showed that ADNP is vital for brain formation and have identified the NAP binding target as the microtubules subunit, tubulin. As tubulin is a major subunit of the neuronal and glial cytoskeleton, this finding explains the breadth of neuroprotective activity of NAP and ADNP.
Pinhasov A, Mandel S, Torchinsky A, Giladi E, Pittel Z, Goldsweig AM, Servoss SJ, Brenneman DE, Gozes I. (2003) Activity-dependent neuroprotective protein: a novel gene essential for brain formation. Brain Res Dev Brain Res. 144, 83-90
Divinski I, Mittelman L, Gozes I. A femtomolar acting octapeptide interacts with tubulin and protects astrocytes against zinc intoxication. J Biol Chem. 2004;279(27):28531-8.
Furman S, Steingart RA, Mandel S, Hauser JM, Brenneman DE and Gozes I. (2004) Subcellular localization and secretion of activity-dependent neuroprotective protein in astrocytes. Neuron Glia Biology, 1,193-199.
Additional results have indicated that NAP stimulates neurite outgrowth, similar to known neurotrphins.
Visochek L, Steingart RA, Vulih-Shultzman I, Klein R, Priel E, Gozes I, Cohen-Armon M. (2005) PolyADP-ribosylation is involved in neurotrophic activity.
J Neurosci. 25(32):7420-8.
Smith-Swintosky VL, Gozes I, Brenneman DE, D'Andrea MR, Plata-Salaman CR. (2005) Activity-dependent neurotrophic factor-9 and NAP promote neurite outgrowth in rat hippocampal and cortical cultures. J Mol Neurosci. 25(3):225-38.
Lagreze WA, Pielen A, Steingart R, Schlunck G, Hofmann HD, Gozes I, Kirsch M. (2005) The peptides ADNF-9 and NAP increase survival and neurite outgrowth of rat retinal ganglion cells in vitro. Invest Ophthalmol Vis Sci. 46(3):933-8.
NAP is now in a clinical development program at Allon Therapeutics, Inc (www.allontherapeutics.com)