Inter- and intraspecies polymorphisms in the cholecystokinin-B/gastrin receptor alter drug efficacy

The brain cholecystokinin-B/gastrin receptor (CCK-BR) is a major target for drug development because of its postulated role in modulating anxiety, memory, and the perception of pain. Drug discovery efforts have resulted in the identification of small synthetic molecules that can selectively activate this receptor subtype. These drugs include the peptide-derived compound PD135,158 as well as the nonpeptide benzodiazepine-based ligand, L-740,093 (S enantiomer). We now report that the maximal level of receptor-mediated second messenger signaling that can be achieved by these compounds (drug efficacy) markedly differs among species homologs of the CCK-BR. Further analysis reveals that the observed differences in drug efficacy are in large part explained by single or double aliphatic amino acid substitutions between respective species homologs. This interspecies variability in ligand efficacy introduces the possibility of species differences in receptor-mediated function, an important consideration when selecting animal models for preclinical drug testing. The finding that even single amino acid substitutions can significantly affect drug efficacy prompted us to examine ligand-induced signaling by a known naturally occurring human CCK-BR variant (glutamic acid replaced by lysine in position 288; 288E → K). When examined using the 288E → K receptor, the efficacies of both PD135,158 and L-740,093 (S) were markedly increased compared with values obtained with the wild-type human protein. These observations suggest that functional variability resulting from human receptor polymorphisms may contribute to interindividual differences in drug effects.

Selective effects of methylphenidate in attention deficit hyperactivity disorder: A functional magnetic resonance study

Functional MRI revealed differences between children with Attention Deficit Hyperactivity Disorder (ADHD) and healthy controls in their frontal–striatal function and its modulation by methylphenidate during response inhibition. Children performed two go/no-go tasks with and without drug. ADHD children had impaired inhibitory control on both tasks. Off-drug frontal–striatal activation during response inhibition differed between ADHD and healthy children: ADHD children had greater frontal activation on one task and reduced striatal activation on the other task. Drug effects differed between ADHD and healthy children: The drug improved response inhibition in both groups on one task and only in ADHD children on the other task. The drug modulated brain activation during response inhibition on only one task: It increased frontal activation to an equal extent in both groups. In contrast, it increased striatal activation in ADHD children but reduced it in healthy children. These results suggest that ADHD is characterized by atypical frontal–striatal function and that methylphenidate affects striatal activation differently in ADHD than in healthy children.

A model-based approach for assessing in vivo combination therapy interactions

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach compares in vivo tumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

A genetic method for dissecting the mechanism of transcriptional activator synergy by identical activators

Pairs of transcriptional activators in prokaryotes have been shown to activate transcription synergistically from promoters with two activator binding sites. In some cases, such synergistic effects result from cooperative binding, but in other cases each DNA-bound activator plays a direct role in the activation process by interacting simultaneously with separate surfaces of RNA polymerase. In such cases, each DNA-bound activator must possess a functional activating region, the surface that mediates the interaction with RNA polymerase. When transcriptional activation depends on two or more identical activators, it is not straightforward to test the requirement of each activator for a functional activating region. Here we describe a method for directing a mutationally altered activator to either one or the other binding site, and we demonstrate the use of this method to examine the mechanism of transcriptional activator synergy by the Escherichia coli cyclic AMP receptor protein (CRP) working at an artificial promoter bearing two CRP-binding sites.

Genetic Separation of FK506 Susceptibility and Drug Transport in the Yeast Pdr5 ATP-binding Cassette Multidrug Resistance Transporter

Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.

RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans

Introduction of exogenous double-stranded RNA (dsRNA) into Caenorhabditis elegans has been shown to specifically and potently disrupt the activity of genes containing homologous sequences. In this study we present evidence that the primary interference effects of dsRNA are post-transcriptional. First, we examined the primary DNA sequence after dsRNA-mediated interference and found no evidence for alterations. Second, we found that dsRNA-mediated interference with the upstream gene in a polar operon had no effect on the activity of the downstream gene; this finding argues against an effect on initiation or elongation of transcription. Third, we observed by in situ hybridization that dsRNA-mediated interference produced a substantial, although not complete, reduction in accumulation of nascent transcripts in the nucleus, while cytoplasmic accumulation of transcripts was virtually eliminated. These results indicate that the endogenous mRNA is the target for interference and suggest a mechanism that degrades the targeted RNA before translation can occur. This mechanism is not dependent on the SMG system, an mRNA surveillance system in C. elegans responsible for targeting and destroying aberrant messages. We suggest a model of how dsRNA might function in a catalytic mechanism to target homologous mRNAs for degradation.

Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains.

p53 is a multifunctional tumor suppressor protein involved in the negative control of cell growth. Mutations in p53 cause alterations in cellular phenotype, including immortalization, neoplastic transformation, and resistance to DNA-damaging drugs. To help dissect distinct functions of p53, a set of genetic suppressor elements (GSEs) capable of inducing different p53-related phenotypes in rodent embryo fibroblasts was isolated from a retroviral library of random rat p53 cDNA fragments. All the GSEs were 100-300 nucleotides long and were in the sense orientation. They fell into four classes, corresponding to the transactivator (class I), DNA-binding (class II), and C-terminal (class III) domains of the protein and the 3'-untranslated region of the mRNA (class IV). GSEs in all four classes promoted immortalization of primary cells, but only members of classes I and III cooperated with activated ras to transform cells, and only members of class III conferred resistance to etoposide and strongly inhibited transcriptional transactivation by p53. These observations suggest that processes related to control of senescence, response to DNA damage, and transformation involve different functions of the p53 protein and furthermore indicate a regulatory role for the 3'-untranslated region of p53 mRNA.

Genetic and molecular analysis of the gypsy chromatin insulator of Drosophila.

Boundary or insulator elements set up independent territories of gene activity by establishing higher order domains of chromatin structure. The gypsy retrotransposon of Drosophila contains an insulator element that represses enhancer-promoter interactions and is responsible for the mutant phenotypes caused by insertion of this element. The gypsy insulator inhibits the interaction of promoter-distal enhancers with the transcription complex without affecting the functionality of promoter-proximal enhancers; in addition, these sequences can buffer a transgene from chromosomal position effects. Two proteins have been identified that bind gypsy insulator sequences and are responsible for their effects on transcription. The suppressor of Hairy-wing [su(Hw)] protein affects enhancer function both upstream and downstream of its binding site by causing a silencing effect similar to that of heterochromatin. The modifier of mdg4 [mod(mdg4)] protein interacts with su(Hw) to transform this bi-directional repression into the polar effect characteristic of insulators. These effects seem to be modulated by changes in chromatin structure.

Genetic variation in vulnerability to the behavioral effects of neonatal hippocampal damage in rats.

We explored how two independent variables, one genetic (i.e., specific rat strains) and another environmental (i.e., a developmental excitotoxic hippocampal lesion), contribute to phenotypic variation. Sprague-Dawley (SD), Fischer 344 (F344), and Lewis rats underwent two grades of neonatal excitotoxic damage: small and large ventral hippocampal (SVH and LVH) lesions. Locomotion was tested before puberty [postnatal day 35 (P35)] and after puberty (P56) following exposure to a novel environment or administration of amphetamine. The behavioral effects were strain- and lesion-specific. As shown previously, SD rats with LVH lesions displayed enhanced spontaneous and amphetamine-induced locomotion as compared with controls at P56, but not at P35. SVH lesions in SD rats had no effect at any age. In F344 rats with LVH lesions, enhanced spontaneous and amphetamine-induced locomotion appeared early (P35) and was exaggerated at P56. SVH lesions in F344 rats resulted in a pattern of effects analogous to LVH lesions in SD rats--i.e., postpubertal onset of hyperlocomotion (P56). In Lewis rats, LVH lesions had no significant effect on novelty- or amphetamine-induced locomotion at any age. These data show that the degree of genetic predisposition and the extent of early induced hippocampal defect contribute to the particular pattern of behavioral outcome. These results may have implications for modeling interactions of genetic and environmental factors involved in schizophrenia, a disorder characterized by phenotypic heterogeneity, genetic predisposition, a developmental hippocampal abnormality, and vulnerability to environmental stress.

A general genetic approach in Escherichia coli for determining the mechanism(s) of action of tumoricidal agents: application to DMP 840, a tumoricidal agent.

We describe here a simple and easily manipulatable Escherichia coli-based genetic system that permits us to identify bacterial gene products that modulate the sensitivity of bacteria to tumoricidal agents, such as DMP 840, a bisnaphthalimide drug. To the extent that the action of these agents is conserved, these studies may expand our understanding agents is conserved, these studies may expand our understanding of how the agents work in mammalian cells. The approach briefly is to use a library of E. coli genes that are overexpressed in a high copy number vector to select bacterial clones that are resistant to the cytotoxic effects of drugs. AtolC bacterial mutant is used to maximize permeability of cells to hydrophobic organic molecules. By using DMP 840 to model the system, we have identified two genes, designated mdaA and mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located between the parE and parC genes, and encodes a protein of 22 kDa. mdaA maps to E. coli map coordinate 18, is located adjacent to the glutaredoxin (grx) gene, and encodes a protein of 24 kDa. Specific and regulatable overproduction of both of these proteins correlates with DMP 840 resistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. In contrast, overproduction of the MdaA protein produces resistance only to Adriamycin. Based on its drug-resistance properties and its location between genes that encode the two subunits of the bacterial topoisomerase IV, we suggest that mdaB acts by modulating topoisomerase IV activity. The location of the mdaA gene adjacent to grx suggests it acts by a drug detoxification mechanism.