Focused ultrasound delivery of a selective TrkA agonist rescues cholinergic function in a mouse model of Alzheimer's disease.

The degeneration of cholinergic neurons is a prominent feature of Alzheimer's disease (AD). In animal models of injury and aging, nerve growth factor (NGF) enhances cholinergic cell survival and function, contributing to improved memory. In the presence of AD pathology, however, NGF-related therapeutics have yet to fulfill their regenerative potential. We propose that stimulating the TrkA receptor, without p75NTR activation, is key for therapeutic efficacy. Supporting this hypothesis, the selective TrkA agonist D3 rescued neurotrophin signaling in TgCRND8 mice, whereas NGF, interacting with both TrkA and p75NTR, did not. D3, delivered intravenously and noninvasively to the basal forebrain using MRI-guided focused ultrasound (MRIgFUS)-mediated blood-brain barrier (BBB) permeability activated TrkA-related signaling cascades and enhanced cholinergic neurotransmission. Recent clinical trials support the safety and feasibility of MRIgFUS BBB modulation in AD patients. Neuroprotective agents targeting TrkA, combined with MRIgFUS BBB modulation, represent a promising strategy to counter neurodegeneration in AD.

VRQ397 (CRAVKY): a novel noncompetitive V2 receptor antagonist.

Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC(50) = 0.69 +/- 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-L-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI(2) generation but not that of cAMP or recruitment of beta-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.

Design and synthesis of a mimetic from an antibody complementarity-determining region.

A technique for producing non-peptide compounds (mimetics) of designed specificities was developed that permitted the synthesis of a conformationally restricted molecule that mimicked the binding and functional properties of monoclonal antibody (MAb) 87.92.6, which recognizes the reovirus type 3 cellular receptor. Binding of either MAb 87.92.6, peptide analogs, or 87.1-mimetic to the cellular receptor inhibited cellular proliferation. The mimetic was a synthetic beta-loop structure that mimics the second complementarity-determining region of the MAb. These studies may lead to strategies for the synthetic design of antibody complementarity regions, ligands, and other pharmacologically active agents that are water soluble, resistant to proteolysis, and nonimmunogenic.

Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.

An enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques. Chemical cross-linking studies with a biotin-labeled P-ODN (bP-ODN), revealed the existence of five major cell surface P-ODN binding protein groups ranging in size from approximately 20-143 kD. Binding to these proteins was competitively inhibited with unlabeled P-ODN, but not free biotin, suggesting specificity of the interactions. Additional experiments suggested that binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding. Uptake studies with 35S-labeled P-ODN revealed that endocytosis, mediated by a receptor-like mechanism, predominated at P-ODN concentrations < 1 microM, whereas fluid-phase endocytosis prevailed at higher concentrations. Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes. Labeled ODN were also found in significant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER). Since nuclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import process is suggested. These data imply that antisense DNA may exert their effect in the nucleus. They also suggest rational ways to design ODN which might increase their efficiency.

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.

Small molecule nerve growth factor analogs image receptors in vivo.

The in vivo targeting efficacy of small molecule analogs of nerve growth factor (NGF) that bind the NGF receptor p140 TrkA was evaluated and compared with that of a high-affinity anti-TrkA monoclonal antibody (Mab 5C3). Nuclear imaging studies were done after the injection of 99mTc-labeled compounds in nude mice bearing tumors. Kinetics of tumor targetting, blood clearance, and bioavailability of NGF mimics were equivalent or better than Mab 5C3. Tumors that do not express TrkA were not targeted, demonstrating the specificity of NGF mimics in vivo. This comparative biodistribution study demonstrates that receptor-specific small molecule analogs designed from large polypeptides may be more useful than antibodies and may be effective agents for the detection, diagnosis, and possible treatment of neoplasias involving overexpressed oncogenic receptors such as TrkA.

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.

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.

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.

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.

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.

Monoclonal antibody to human Trk-A: diagnostic and therapeutic potential in neuroblastoma.

5C3 is a murine IgG1 antibody specific for the nerve growth factor (NGF) docking site of the human p140 trk-A receptor, with no cross-reactivity with human trk-B. In vitro, 5C3 and its Fab mimic the effects of NGF, a neurotrophin mediating growth and differentiation of neural crest-derived cells. When labelled with radioisotope, 5C3 images human trk-A positive tumours in vivo. More importantly, 5C3 induces regression of human trk-A positive tumours in rodents. We therefore investigated the value of 5C3 in detecting trk-A expression in human neuroblastoma by immunohistochemistry. 5C3 reactivity was detected in 73 of 113 neuroblastoma specimens and correlated strongly with localised/4s disease (55/60) with either a homogeneous or mixed pattern. Among stage 4 neuroblastoma, only 18/53 had homogeneous or mixed trk-A expression. 5C3 did not react with 46/48 other human malignancies, but was positive in 1 melanoma and 1 Wilms' tumour specimen. The prognostic, imaging and NGF-mimetic properties of antibody 5C3 and its derivatives may offer alternatives for the diagnosis and treatment of neuroblastoma.

Correlation of MYCN amplification, Trk-A and CD44 expression with clinical stage in 250 patients with neuroblastoma.

In contrast to MYCN amplification, expression of either trk-A or CD44 in neuroblastoma is a favourable prognostic factor and were therefore investigated in tumours from 250 patients. One hundred and eleven localised/4s (Group 1) and 139 stage 4 (Group 2) tumours were analysed. MYCN copy number was obtained by Southern blotting or PCR amplification and was detected in 28 stage 4 tumours. Trk-A and CD44 expression was detected by immunoperoxidase staining using murine monoclonal antibodies 5C3 and L178, respectively. Expression was scored as positive (homogeneous), mixed (heterogeneous) or non-reactive (negative). Trk-A expression was found in 95% of Group 1 tumours and 49% of Group 2 tumours. CD44 expression was found in 100% of Group 1 tumours, the majority of which had a strong homogeneous expression. Lack of CD44 expression occurred in 25% of tumours, all stage 4 neuroblastoma. Of the 28 MYCN amplified tumours, 27/28 (96%) were trk-A negative, and 13/28 (46%) CD44 negative. We conclude that (1) a significant percentage of stage 4 neuroblastoma express either or both trk-A and CD44, (2) the absence of CD44 expression is highly restricted to stage 4 neuroblastoma and is associated with the lack of trk-A expression, (3) trk-A negativity among CD44-positive tumours is associated with stage 4 neuroblastoma and (4) the absence of trk-A expression is highly correlated with MYCN amplification.

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.

A cell cycle regulating receptor is localized on cell surface and in nuclei of mitotically and meiotically dividing cells.

We previously showed that a heterodimeric surface receptor of molecular weight 65,000 (p65) and 95,000 (p95) is expressed on the surface of proliferating cells such as activated T lymphocytes and neural precursors. This p65/p95 receptor is recognized by a monoclonal antibody and by type 3 reovirus hemagglutinin. Binding of the surface p65/p95 receptor leads to a growth arrest of mitotic cells and a consequent inhibition of proliferation. The p65/p95 receptor was demonstrated to be associated with kinase activity. Because p65/p95 is involved in the regulation of mitotic cell division, we sought to study the cellular distribution of the receptor and its possible role in meiotic cell division. Immunohistochemical labeling and flow cytometry studies were done using adult rat testes and cell lines. All cells undergoing mitotic or meiotic division in the rat testis expressed the p65/p95 receptor; cells that do not divide did not express receptors. Dividing cells had two receptor pools. As previously reported for several mitotically active tissues, a pool of receptors was localized on the cell surface. Interestingly, a pool of receptors was also seen intracellularly over the nucleus of labeled cells. The nuclear label seemed to be associated with chromosomes during specific stages of the mitotic and the two meiotic divisions, suggesting a role in the regulation of nuclear events. Further studies on this receptor and the function of the nuclear pool should provide a better understanding of the control of cell division.

A receptor that subserves reovirus binding can inhibit lymphocyte proliferation triggered by mitogenic signals.

A novel surface receptor complex involved in inhibition of T-cell proliferation is described. Biochemical isolation revealed two non-covalently associated proteins of about M(r) 65,000 (p65) and 95,000 (p95). These polypeptides may be related. The p65 form is expressed after cellular activation and replication and is recognized by monoclonal antibody (mAb) 87.92.6 or reovirus hemagglutinin as unnatural ligands. The p95 species is associated with tyrosine kinase enzymatic activity. Receptor ligation results in rapid alteration of the phosphotyrosine content of cellular substrates, and this activity correlates with antiproliferative effects. The inhibition of proliferation is a time-dependent reversible arrest at the G1-S phase of the cell cycle. Activation through the T-cell receptor, protein kinase C, or addition of cytokines does not reverse the antiproliferative effect. This receptor complex may define novel features of T-cell proliferation.

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.

TrkA antagonists decrease NGF-induced ChAT activity in vitro and modulate cholinergic synaptic number in vivo.

Cholinergic neurons are known to respond in vivo to the administration of nerve growth factor (NGF) by a prominent and selective increase of choline acetyl transferase activity and by cholinergic synaptogenesis in the rat brain. By using a synthetic TrkA antagonist we demonstrated that endogenously produced NGF is involved in the continual re-modeling of cholinergic neuronal connections during adulthood, acting through TrkA receptors.

Loops and secondary structure mimetics: development and applications in basic science and rational drug design.

One goal of protein design and structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high resolution analysis and rapid modification. We have developed a variety of small molecules which biochemically and biologically mimic the combining sites of proteins of the immunoglobulin superfamily. The chemical and biological properties of peptide mimetics suggest that these analogs can be used as indicators for new pharmaceutical agents. Mimetics are powerful tools for the study of molecular recognition since they are small in size, maintain solubility in physiologic fluids and are amenable to detailed structural studies. As such, they represent a step toward the rational design of low molecular weight non-peptide pharmaceutical agents devoid of some of the shortcomings of conventional peptides. Here we discuss the rationale and approaches for the development of these molecules, and their current and future applications.