Inhibitors of protein geranylgeranyltransferase I and Rab geranylgeranyltransferase identified from a library of allenoate-derived compounds

Protein geranylgeranylation is critical for the function of a number of proteins such as RhoA, Rac, and Rab. Protein geranylgeranyltransferase I (GGTase-I) and Rab geranylgeranyltransferase (RabGGTase) catalyze these modifications. In this work, we first describe the identification and characterization of small molecule inhibitors of GGTase-I (GGTI) with two novel scaffolds from a library consisting of allenoate-derived compounds. These compounds exhibit specific inhibition of GGTase-I and act by competing with a substrate protein. Derivatization of a carboxylic acid emanating from the core ring of one of the GGTI compounds dramatically improves their cellular activity. The improved GGTI compounds inhibit proliferation of a variety of human cancer cell lines and cause G(1) cell cycle arrest and induction of p21(CIP1/WAF1). We also report the identification of novel small molecule inhibitors of RabGGTase. These compounds were identified first by screening our GGTI compounds for those that also exhibited RabGGTase inhibition. This led to the discovery of a common structural feature for RabGGTase inhibitors: the presence of a characteristic six-atom aliphatic tail attached to the penta-substituted pyrrolidine core. Further screening led to the identification of compounds with preferential inhibition of RabGGTase. These compounds inhibit RabGGTase activity by competing with the substrate protein. These novel compounds may provide valuable reagents to study protein geranylgeranylation.     

A new class of type I protein geranylgeranyltransferase (GGTase I) inhibitor

Replacement of the thiol groups in 1, a potent and highly selective Candida albicans GGTase I inhibitor discovered through screening, with an imidazole ring was achieved by using solid phase synthesis. A non-thiol compound, 7, was found as a representative of a new class of potent C. albicans GGTase I inhibitor with high selectivity against human GGTase I.     

Signaling mechanisms mediating synapse formation

Recent experiments have begun to decipher the molecular dialog that mediates differentiation at sites of synaptic between neurons and their targets. It had been hypothesized that the protein agrin is released by axon terminals at embryonic neuromuscular junctions and binds to a receptor on the myofiber surface to trigger postsynaptic differentiation. Now a genetic ‘Knockout’ experiment has confirmed the essential role of agrin in signaling between developing nerve and muscle⁽¹⁾. A second ‘knockout’ has shown that the muscle-specific receptor tyrosine kinase MuSK is a critical element in the agrin-induced signaling cascade⁽²⁾. Additional results suggest that MuSK may comprise a portion of the agrin receptor⁽³⁾.     

WNTs in synapse formation and neuronal circuitry

Wnt proteins play important roles in wiring neural circuits. Wnts regulate many aspects of neural circuit generation through their receptors and distinct signalling pathways. In this review, we discuss recent findings on the functions of Wnts in various aspects of neural circuit formation, including neuronal polarity, axon guidance, synapse formation, and synaptic plasticity in vertebrate and invertebrate nervous systems.     

An unconventional role of neurotransmission in synapse formation

How do presynaptic inputs regulate synapse formation? In this issue of Neuron, Lin et al. show that the neurotransmitter acetylcholine decreases the stability of AChR clusters. This dispersing activity, which requires the serine/threonine kinase Cdk5, cooperates with positive signals from motoneurons to ensure high concentration of AChRs at the neuromuscular junction.     

Role of cholesterol in synapse formation and function

Cholesterol is a multifaceted molecule, which serves as essential membrane component, as cofactor for signaling molecules and as precursor for steroid hormones. Consequently, defects in cholesterol metabolism cause devastating diseases. So far, the role of cholesterol in the nervous system is less well understood. Recent studies showed that cultured neurons from the mammalian central nervous system (CNS) require glia-derived cholesterol to form numerous and efficient synapses. This suggests that the availability of cholesterol in neurons limits the extent of synaptogenesis. Here, I will summarize the experimental evidence for this hypothesis, describe what is known about the structural and functional role of cholesterol at synapses, and discuss how cholesterol may influence synapse development and stability.     

Role of cholesterol in synapse formation and function

Cholesterol is a multifaceted molecule, which serves as essential membrane component, as cofactor for signaling molecules and as precursor for steroid hormones. Consequently, defects in cholesterol metabolism cause devastating diseases. So far, the role of cholesterol in the nervous system is less well understood. Recent studies showed that cultured neurons from the mammalian central nervous system (CNS) require glia-derived cholesterol to form numerous and efficient synapses. This suggests that the availability of cholesterol in neurons limits the extent of synaptogenesis. Here, I will summarize the experimental evidence for this hypothesis, describe what is known about the structural and functional role of cholesterol at synapses, and discuss how cholesterol may influence synapse development and stability.     

Lysine beta311 of protein geranylgeranyltransferase type I partially replaces magnesium

Protein geranylgeranyltransferase type I (GGTase I) catalyzes the attachment of a geranylgeranyl lipid group near the carboxyl terminus of protein substrates. Unlike protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type II, which require both Zn(II) and Mg(II) for maximal turnover, GGTase I turnover is dependent only on Zn(II). In FTase, the magnesium ion is coordinated by aspartate beta352 and the diphosphate of farnesyl diphosphate to stabilize the developing charge in the transition state (Pickett, J. S., Bowers, K. E., and Fierke, C. A. (2003) J. Biol. Chem. 278, 51243-51250). In GGTase I, lysine beta311 is substituted for this aspartate and is proposed to replace the catalytic function of Mg(II) (Taylor, J. S., Reid, T. S., Terry, K. L., Casey, P. J., and Beese, L. S. (2003) EMBO J. 22, 5963-5974). Here we demonstrate that the prenylation rate constant catalyzed by wild type GGTase I (k(chem) = 0.18 +/- 0.02 s(-1)) is not dependent on Mg(II), is approximately 20-fold slower than the maximal rate constant catalyzed by FTase, and has a single pKa of 6.4 +/- 0.1, likely reflecting deprotonation of the peptide thiol. Mutation of lysine beta311 in GGTase I to alanine (Kbeta311A) or aspartate (Kbeta311D) decreases the k(chem) in the absence of magnesium 9-41-fold without significantly affecting the binding affinity of either substrate. Furthermore, the geranylgeranylation rate constant is enhanced by the addition of Mg(II) for Kbeta311A and Kbeta311D GGTase I 2-5-fold compared with wild type GGTase I with K(Mg) of 140 +/- 10 mm and 6.4 +/- 0.8 mm, respectively. These results demonstrate that lysine beta311 of GGTase I partially replaces the catalytic function of Mg(II) observed in FTase.     

Possible mechanisms of synapse formation during ontogenesis

Electron microscopic investigation of synaptogenesis in the sensomotor cortex and in the caudate nucleus has been performed in the prenatal ontogenesis (16-22 days) and in newborn rats. The first immature synapses are demonstrated to appear beginning on the 16th day of embryogenesis. At the end of the prenatal development and especially in newborn animals desmosome-like, asymmetric and symmetric, mixed and complex forms of the synaptic contacts are revealed. As a result of the analysis performed on the ultrastructural organization of the contacts, a hypothesis explaining mechanisms of development of various elements of the synapses has been suggested. A part of the synaptic contacts of the asymmetric and symmetric types is supposed to be genetically programmed and membrane specialization of these contacts is formed earlier than synaptic vesicles appear. Other part of the synapses undergoes certain stages of differentiation before the functionally mature contact is formed. The initial stage in the synapses formation in formation of the desmosome-like junction. The second stage is appearance of synaptic vesicles in the area of this contact. The third stage includes development of pre- and postsynaptic membranous specialization and owing to this the contact acquires either asymmetric or symmetric appearance. For the ontogenetic periods investigated establishment of complex forms of the intercellular junctions (tangent, reciprocal, etc.) is specific; this evidently demonstrates certain plastic rearrangements in the synapses during the process of development.     

Activity-dependent switch from synapse formation to synapse elimination during development of neuromuscular junctions

The embryonic development of neuromuscular junctions consists of two successive epochs, an early period marked by exuberant synapse formation and a later period marked by synapse elimination. In the frog muscles we have studied, myogenesis is protracted and overlaps the periods of synapse formation and elimination. Thus, the formative and regressive events of synaptic development do not occur in synchrony across different fibers in the muscle. We propose that local activity orchestrates a shift from synaptogenesis to synapse elimination at the level of single muscle fibers. We also present evidence that perisynaptic Schwann cells and the expression of ion channels in the sarcolemma play important roles in the development of neuromuscular junctions. Questions for future study are outlined.     

N-Cadherin signaling in synapse formation and neuronal physiology

Neural cadherin (N-cadherin) is an adhesion receptor that is localized in abundance at neuron-to-neuron synapses. N-cadherin contains an extracellular domain that binds to other cadherins on juxtaposed cell membranes, a single-pass transmembrane region, and a cytoplasmic tail that interacts with various proteins, including catenins, kinases, phosphatases, and presenilin 1. N-cadherin contributes to the structural and functional organization of the synaptic complex by ensuring the adhesion between synaptic membranes and organizing the underlying actin cytoskeleton. Additionally, recent findings have shown that N-cadherin may participate in synaptic physiology by regulating calcium influx through voltage-activated calcium currents. The diverse activities of N-cadherin stem from its ability to operate as both an adhesion molecule that links cytoskeletons across cell membranes and a ligand-activated homophilic receptor capable of initiating intracellular signaling. An important mechanism of cadherin signaling is the regulation of small Rho guanosine triphosphatase activity that affects cytoskeleton dynamics and calcium influx. Because both the regulation of cadherin adhesive activity and cadherin-mediated signaling are affected by the binding of molecules to the intracellular domain, changes in the composition of the N-cadherin complex are central to the regulation of cadherin-mediated functions. This article focuses on the roles that N-cadherin might play at the level of the synapse through its effect on adhesion and signaling in the proximity of the synaptic junction.     

A model of selective synapse formation in sympathetic ganglia

In the sympathetic system, neurons from several spinal segments are mapped onto targets in the periphery in a topographically ordered way by means of selective synaptic connections in the superior cervical ganglion. Experimental evidence points to a crucial role for chemoaffinity in establishing this topographic map. Furthermore, rearrangements of synapses after surgical manipulations indicate that this chemoaffinity is not based on rigid “key-and-lock” markers. Our model is used to study how such nonrigid markers may interact with other regulatory factors, including growth-regulating signals and the growth potential of individual nerons. In the model, these latter factors are limiting, so that an increasing number of synaptic contacts decreases the likelihood of further synapse formation. These factors are combined with chemoaffinity using a linear threshold model. The model is robust to parameter changes and reproduces experimental observations with reasonable detail. Simulation results are used to discuss characteristic experimental results, such as the substantial plasticity of the connections seen after partial denervation. A surprisingly small effect of transient hyperinnervation in the model may help explain why final connectivities are similar in two real situations with high and low degrees of transient hyperinnervation (development and adult reinnervation). It is shown that spatial restrictions on post-synaptic neurons (dendrites) may contribute significantly to the segmentally broad innervation of each ganglion cell. Finally, we discuss potential effects of presynaptic neuronal death in systems with a high degree of plasticity. © 1993 John Wiley & Sons, Inc.