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Biomedical subjects

H U Saragovi

Publications and source records attributed to H U Saragovi.

At least 19 recordsLinked to original sources
PubMed2006

Functional mimetics of neurotrophins and their receptors.

Neurotrophins regulate cell survival, death, differentiation and growth. Neurotrophins and their receptors have been validated for pathologies including neurodegenerative disorders of the central nervous system and the peripheral nervous system, certain types of cancers, asthma, inflammation and others. Development of neurotrophin-based therapeutics is important due to the limitations of using whole neurotrophins as pharmacological agents. The use of mimicry has proven to be an alternative. Mimetics can be developed through a number of different approaches. To develop receptor-binding agents, we have used anti-receptor antibody mimicry and neurotrophin mimicry. To develop ligand-binding agents, we have used antiligand antibody mimicry and receptor mimicry. High-throughput screening can be incorporated to complement any of these approaches. The end result is small molecule peptidomimetics with properties favourable over proteins. The present review will offer a general overview of these strategies with a few proven examples from our laboratory.

Animals↗
PubMed2001

p75 Co-receptors regulate ligand-dependent and ligand-independent Trk receptor activation, in part by altering Trk docking subdomains.

Neurotrophins signal via Trk tyrosine kinase receptors and a common receptor called p75. Nerve growth factor is the cognate ligand for TrkA, brain-derived neurotrophic factor for TrkB, and neurotrophin-3 (NT-3) for TrkC. NT-3 also binds TrkA and TrkB as a heterologous ligand. All neurotrophins bind p75, which regulates ligand affinity and Trk signals. Trk extracellular domain has five subdomains: a leucine-rich motif, two cysteine-rich clusters, and immunoglobulin-like subdomains IgG-C1 and IgG-C2. The IgG-C1 subdomain is surface exposed in the tertiary structure and regulates ligand-independent activation. The IgG-C2 subdomain is less exposed but regulates cognate ligand binding and Trk activation. NT-3 as a heterologous ligand of TrkA and TrkB optimally requires the IgG-C2 but also binds other subdomains of these receptors. When p75 is co-expressed, major changes are observed; NGF-TrkA activation can occur also via the cysteine 1 subdomain, and brain-derived neurotrophic factor-TrkB activation requires the TrkB leucine-rich motif and cysteine 2 subdomains. We propose a two-site model of Trk binding and activation, regulated conformationally by the IgG-C1 subdomain. Moreover, p75 affects Trk subdomain utilization in ligand-dependent activation, possibly by conformational or allosteric control.

Brain-Derived Neurotrophic Factor↗
PubMed2001

Taxane-antibody conjugates afford potent cytotoxicity, enhanced solubility, and tumor target selectivity.

Paclitaxel (Taxol) is a chemotherapeutic agent that prevents disassembly of microtubular polymers, causing a growth arrest in the G2-M phase of the cell cycle and leading to apoptotic death. Paclitaxel has remarkable efficacy against fast-growing tumors but possesses major drawbacks, such as poor solubility and lack of tumor selectivity. Conversely, monoclonal antibodies usually have low therapeutic efficacy but are highly soluble and selectively target tumor markers overexpressed in cancer cells. Therefore, to improve the therapeutic index of taxanes as chemotherapeutics, the high toxicity of paclitaxel was combined with the high selectivity and solubility of monoclonal antibodies as targeting agents. We report the chemical coupling and characterization of paclitaxel-antibody conjugates for treatment of neuroectoderm-derived tumors. Paclitaxel-antibody conjugates afforded selective toxicity toward cells expressing the target marker and were more cytotoxic in vitro than equimolar concentrations of free paclitaxel or free paclitaxel plus free antibody. In an in vivo model of xenografted tumors, systemic administration of paclitaxel-antibody conjugates prevented tumor growth and prolonged survival of mice better than free drugs. In addition, paclitaxel-antibody conjugates were highly soluble in water and stable at -20 degrees C for at least 3 months. These studies may lead to an increase or an improvement of the armamentarium and selectivity of cytotoxic agents.

3T3 Cells↗
PubMed2001

DiSSiMiL: Diverse Small Size Mini-Libraries applied to simple and rapid epitope mapping of a monoclonal antibody.

Methods for screening protein-protein interactions are useful in protein science and for the generation of drug leads. We set out to develop a simplified assay to rapidly test protein-protein interactions, with a library of 400 pentapeptides comprising the 20 natural amino acids at two variable positions followed by three glycines (NH2-X1X2GGG). The library was used to identify the epitope of monoclonal antibody (mAb) 10D11 directed against the HOXD4 protein. Three pentapeptide 'hits' were selected (VYGGG, PWGGG and WKGGG) from direct binding assays screening for pentapeptide-mAb interactions; and from assays using pentapeptides in solution to competitively block HOXD4-mAb interactions. Alignment of the three 'hit' pentapeptides to the HOXD4 sequence predicts the mAb 10D11 epitope as NH2-VYPWMK. Synthesis of NH2-VYPWMK hexapeptide confirmed this prediction; and an alanine scan of HOXD4 ablated binding by mAb 10D11 when amino acids in the putative epitope were mutated. We propose that these simplified but diverse libraries can be used for rapid epitope mapping of some mAbs, and for generating lead small peptide analogs that interfere with receptor-ligand or other protein-protein interactions, or with enzymatic activity.

Amino Acid Sequence↗
PubMed2000

Loss of nucleus basalis neurons containing trkA immunoreactivity in individuals with mild cognitive impairment and early Alzheimer's disease.

Recent studies indicate that there is a marked reduction in trkA-containing nucleus basalis neurons in end-stage Alzheimer's disease (AD). We used unbiased stereological counting procedures to determine whether these changes extend to individuals with mild cognitive impairment (MCI) without dementia from a cohort of people enrolled in the Religious Orders Study. Thirty people (average age 84.7 years) came to autopsy. All individuals were cognitively tested within 12 months of death (average MMSE 24.2). Clinically, 9 had no cognitive impairment (NCI), 12 were categorized with MCI, and 9 had probable AD The average number of trkA-immunoreactive neurons in persons with NCI was 196, 632 +/- 12,093 (n = 9), for those with MCI it was 106,110 +/- 14,565, and for those with AD it was 86,978 +/- 12,141. Multiple comparisons showed that both those with MCI and those with AD had significant loss in the number of trkA-containing neurons compared to those with NCI (46% decrease for MCI, 56% for AD). An analysis of variance revealed that the total number of neurons containing trkA immunoreactivity was related to diagnostic classification (P < 0.001), with a significant reduction in AD and MCI compared to NCI but without a significant difference between MCI and AD. Cell density was similarly related to diagnostic classification (P < 0.001). There was a significant correlation with the Boston Naming Test and with a global score measure of cognitive function. The number of trkA-immunoreactive neurons was not correlated with MMSE, age at death, education, apolipoprotein E allele status, gender, or Braak score. These data indicate that alterations in the number of nucleus basalis neurons containing trkA immunoreactivity occurs early and are not accelerated from the transition from MCI to mild AD.

Aged↗
PubMed2000

Design and solution structure of functional peptide mimetics of nerve growth factor.

The C-D loop in nerve growth factor (NGF) is involved in binding to the NGF receptor, TrkA. It is flexible and adopts several different types conformations in different NGF crystal forms. We have previously shown that a small cyclic peptide derived from the C-D loop of NGF binds to the TrkA receptor by mimicking the structure of this loop. To understand structure-function relationships in NGF C-D loop mimetics, we have produced a series of peptides predicted to form different types of beta-turns. The peptides were tested for their ability to promote cell survival in serum-free medium and to induce TrkA tyrosine phosphorylation. NMR structural studies were used to determined the backbone conformation and the spatial orientation of side chains involved in binding to the TrkA receptor. Peptides that form type I or type gammaL-alphaR beta-turns were the most active. The variety of active loop conformations suggests that the mimetics (and NGF) accommodate the binding site on TrkA by an 'induced fit' mechanism. In agreement with this hypothesis, NMR relaxation measurements detected both fast and slow motion in the peptides. We also characterized a retro-inverso peptide derived from the NGF C-D loop. This D-amino acid cyclic peptide did not adopt a conformation homologous to the NGF C-D loop and was inactive. This may be representative of difficulties in producing structural and functional mimetics by retro-inverso schemes.

Animals↗
PubMed2000

Genuine monovalent ligands of TrkA nerve growth factor receptors reveal a novel pharmacological mechanism of action.

Developing small molecule agonistic ligands for tyrosine kinase receptors has been difficult, and it is generally thought that such ligands require bivalency. Moreover, multisubunit receptors are difficult to target, because each subunit contributes to ligand affinity, and each subunit may have distinct and sometimes opposing functions. Here, the nerve growth factor receptor subunits p75 and the tyrosine kinase TrkA were studied using artificial ligands that bind specifically to their extracellular domain. Bivalent TrkA ligands afford robust signals. However, genuine monomeric and monovalent TrkA ligands afford partial agonism, activate the tyrosine kinase activity, cause receptor internalization, and induce survival and differentiation in cell lines and primary neurons. Monomeric and monovalent TrkA ligands can synergize with ligands that bind the p75 subunit. However, the p75 ligands used in this study must be bivalent, and monovalent p75 ligands have no effect. These findings will be useful in designing and developing screens of small molecules selective for tyrosine kinase receptors and indicate that strategies for designing agonists of multisubunit receptors require consideration of the role of each subunit. Last, the strategy of using anti-receptor mAbs and small molecule hormone mimics as receptor ligands could be applied to the study of many other heteromeric cell surface receptors.

Amino Acid Sequence↗
PubMed2000

Development of pharmacological agents for targeting neurotrophins and their receptors.

Neurotrophins comprise a family of protein growth factors that control the survival, growth, and/or differentiation of neurons and several other cell populations derived from the neuroectoderm. Neurotrophins and their receptors are important targets for the therapy of human disease, with potential applications ranging from the treatment of chronic or acute neurodegeneration to pain and cancer. Neurotrophins have been used clinically but are poor pharmacological agents. Consequently, approaches to develop pharmacological agents that target neurotrophins, their receptors or neurotrophin signaling pathways have been attempted.

Animals↗
PubMed2000

Reduction in TrkA-immunoreactive neurons is not associated with an overexpression of galaninergic fibers within the nucleus basalis in Down's syndrome.

Down's syndrome (DS) individuals develop neuropathological features similar to Alzheimer's disease (AD), including degeneration of cholinergic basal forebrain (CBF) neurons. In AD a reduction in CBF/trkA-containing neurons has been suggested to trigger a hyperexpression of galaninergic fibers within the nucleus basalis subfield of the basal forebrain. The present study examined the interrelationship between reductions in CBF/trkA-containing neurons and the overexpression of galaninergic fibers within the nucleus basalis in DS. Within the nucleus basalis stereologic evaluation revealed a 46% reduction in the number of trkA-immunopositive neurons, whereas optical density measurements displayed a nonsignificant 18% reduction in neuronal trkA immunoreactivity in DS as compared with age-matched controls. Western blot analysis also showed a significant reduction in cortical trkA protein levels in DS. A semiquantitative examination of galaninergic fibers in the nucleus basalis revealed only a modest hypertrophy of galaninergic fibers within the nucleus basalis in DS. The present findings indicate a significant reduction in trkA within the nucleus basalis and cortex with only a moderate hypertrophy of galaninergic fibers in DS. These observations suggest that DS may not be an exact genetic model for investigation of changes in the AD basal forebrain.

Aged↗
PubMed2000

A designed peptidomimetic agonistic ligand of TrkA nerve growth factor receptors.

A proteolytically stable small molecule beta-turn peptidomimetic, termed D3, was identified as an agonist of the TrkA neurotrophin receptor. D3 binds the Ig-like C2 region of the extracellular domain of TrkA, competes the binding of another TrkA agonist, affords selective trophic protection to TrkA-expressing cell lines and neuronal primary cultures, and induces the differentiation of primary neuronal cultures. These results indicate that a small beta-turn peptidomimetic can activate a tyrosine kinase neurotrophin receptor that normally binds a relatively large protein ligand. Agents such as D3 that bind the extracellular domain of Trk receptors will be useful pharmacological agents to address disorders where Trk receptors play a role, by targeting populations selectively.

Animals↗
PubMed2000

Ligand activation of nerve growth factor receptor TrkA protects monocytes from apoptosis.

Nerve growth factor (NGF) receptors are expressed in different cell types outside the nervous system, and increasing evidence indicates that NGF can act as a regulatory molecule during inflammatory and immune responses. In this study, we show that triggering of the high-affinity NGF receptor TrkA with agonists protects monocytes from apoptosis induced by gliotoxin or UVB radiation. TrkA stimulation up-regulates the expression of the anti-apoptotic Bcl-2 family members, Bcl-2, Bcl-XL, and Bfl-1. On the other hand, TrkA stimulation does not change the expression of MHC, CD80, CD86, CD40, and CD54 molecules, nor the antigen-presenting function of monocytes. In addition, during in vitro monocyte to dendritic cell differentiation TrkA expression is progressively lost, suggesting that NGF selectively affects monocyte but not dendritic cell survival.

Antibodies, Monoclonal↗
PubMed1999

A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat.

Cholinergic neurons respond to the administration of nerve growth factor (NGF) in vivo with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter-IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma.

Animals↗
PubMed1999

A G1 cell cycle arrest induced by ligands of the reovirus type 3 receptor is secondary to inactivation of p21ras and mitogen-activated protein kinase.

The reovirus type 3 S1 gene product (type 3 hemagglutinin; HA3) is the viral protein responsible for binding to a mammalian cell-surface receptor. It has been shown that HA3 binding to its receptor inhibits cell growth, even in the continuous presence of serum mitogens. Here, receptor-mediated signal transduction leading to growth arrest was studied after binding with synthetic or recombinant ligands in the absence of viral infection. Receptor ligation caused rapid inactivation of p21(ras), a decrease in Raf phosphorylation and in mitogen-activated protein kinase (MAPK) enzymatic activity, and G1 cell cycle arrest. Transfection and expression of constitutively active v-Has-ras prevented the G1 arrest, indicating that inactivation of p21(ras) is causative. Interestingly, v-Has-ras expression also decreased the efficiency of reoviridae replication, suggesting that inactivation of p21(ras) signals is required at some step of the viral cycle. This study may define new mechanisms regulating cell growth and support the approach of using viral proteins to identify and study cellular receptors. Synthetic receptor ligands with antiproliferative properties may be useful in drug development with the aim of blocking mitosis.

Animals↗
PubMed1999

PBX and MEIS as non-DNA-binding partners in trimeric complexes with HOX proteins.

HOX, PBX, and MEIS transcription factors bind DNA through a homeodomain. PBX proteins bind DNA cooperatively as heterodimers with MEIS family members and also with HOX proteins from paralog groups 1 to 10. MEIS proteins cooperatively bind DNA with ABD-B class HOX proteins of groups 9 and 10. Here, we examine aspects of dimeric and higher-order interactions between these three homeodomain classes. The most significant results can be summarized as follows. (i) Most of PBX N terminal to the homeodomain is required for efficient cooperative binding with HOXD4 and HOXD9. (ii) MEIS and PBX proteins form higher-order complexes on a heterodimeric binding site. (iii) Although MEIS does not cooperatively bind DNA with ANTP class HOX proteins, it does form a trimer as a non-DNA-binding partner with DNA-bound PBX-HOXD4. (iv) The N terminus of HOXD4 negatively regulates trimer formation. (v) MEIS forms a similar trimer with DNA-bound PBX-HOXD9. (vi) A related trimer (where MEIS is a non-DNA-binding partner) is formed on a transcriptional promoter within the cell. (vii) We observe an additional trimer class involving non-DNA-bound PBX and DNA-bound MEIS-HOXD9 or MEIS-HOXD10 heterodimers that is enhanced by mutation of the PBX homeodomain. (viii) In this latter trimer, PBX is likely to contact both MEIS and HOXD9/D10. (ix) The stability of DNA binding by all trimers is enhanced relative to the heterodimers. These findings suggest novel functions for PBX and MEIS in modulating the function of DNA-bound MEIS-HOX and PBX-HOX heterodimers, respectively.

Antennapedia Homeodomain Protein↗
PubMed1998

A TrkA-selective, fast internalizing nerve growth factor-antibody complex induces trophic but not neuritogenic signals.

Nerve growth factor (NGF) is a neurotrophin that induces neuritogenic and trophic signals by binding to TrkA and/or p75 receptors. We report a comparative study of the binding, internalization, and biological activity of NGF versus that of NGF in association with an anti-NGF monoclonal antibody (mAb NGF30), directed against the C termini of NGF. NGF.mAb complexes do not bind p75 effectively but bind TrkA with high affinity. After binding, NGF. mAb complexes stimulate internalization faster and to a larger degree than NGF. NGF.mAb-induced activation of TrkA, Shc, and MAPK is transient compared with NGF-induced activation; yet NGF and NGF. mAb afford identical trophic responses. In contrast, NGF induces Suc-1-associated neurotrophic activating protein phosphorylation and neuritogenic differentiation, but NGF.mAb does not. Thus, an absolute separation of trophic and neuritogenic function is seen for NGF.mAb, suggesting that biological response modifiers of neurotrophins can afford ligands with selected activities.

Antibodies, Monoclonal↗
PubMed1998

Functional and physical association of a cell surface phospholipid and interleukin-2 receptor p55(alpha) subunits.

A phosphatidylcholine-like phospholipid expressed in the outer leaflet of the cell membrane shortly after mitogenic activation of T-cells is described, based on the binding of monoclonal antibody 90. 60.3. Expression of the 90.60.3 phospholipid antigen in T-cells is activation-dependent. Once expressed, the 90.60.3 phospholipid is in direct physical association with the interleukin-2 (IL-2) binding domain of IL-2 receptor alpha subunits, but does not affect IL-2 binding. The association is specific, because the 90.60.3 phospholipid is not found in association with other domains of IL-2 receptor alpha subunits, or near IL-2 receptor beta or gamma subunits. Culturing cytokine-dependent cell lines in the presence of monoclonal antibody 90.60.3 potentiates IL-2-dependent cell survival and proliferation in a dose-dependent manner. In contrast, IL-4-dependent responses are not potentiated. Taken together, the data suggest that specific plasma membrane phospholipids expressed in the outer leaflet after T-cell activation associate with the IL-2 binding domain of IL-2 receptor alpha subunits (and perhaps other cytokine receptors), and may play a role in regulating receptor mobility or signal transduction.

Animals↗
PubMed1998

Solution structure and internal motion of a bioactive peptide derived from nerve growth factor.

The conformation and internal dynamics of a bioactive cyclic peptide, N-acetyl-YCTDEKQCY, derived from the C-D loop of beta-nerve growth factor (beta-NGF) were analyzed by solution NMR spectroscopy. NMR experimental data were used to calculate an ensemble of peptide structures. All of the structures had a beta-turn at residues Asp4-Gln7 but could be divided into two families according the presence or absence of a hydrogen bond at Gln7. Comparison of the calculated structures with the corresponding C-D loops from the x-ray structures of the NGF revealed striking similarity. The orientation of Glu5, Lys6, and Gln7 side chains in the NGF mimetic was very similar to the C-D loop of NGF. These residues are known to participate in interactions with the TrkA receptor. Relaxation measurements of the peptidomimetic alpha-carbons at 13C natural abundance and calculated dynamic parameters suggest that the loop region of peptide is well structured but that residues Thr3, Asp4, Glu5, and Lys6 undergo slow conformational exchange. These results suggest that conformational similarity and possibly peptide dynamics are responsible for the bioactivity of the peptide.

Amino Acid Sequence↗