John A. Arnott

The influence of lipophilicity in drug discovery and design

Introduction: The role of lipophilicity in drug discovery and design is a critical one. Lipophilicity is a key physicochemical property that plays a crucial role in determining ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties and the overall suitability of drug candidates. There is increasing evidence to suggest that control of physicochemical properties such as lipophilicity, within a defined optimal range, can improve compound quality and the likelihood of therapeutic success. Areas covered: This review focuses on understanding lipophilicity, techniques used to measure lipophilicity, and summarizes the importance of lipophilicity in drug discovery and development, including a discussion of its impact on individual ADMET parameters as well as its overall influence on the drug discovery and design process, specifically within the past 15 years. Expert opinion: A current review of the literature reveals a continued reliance on the synthesis of novel structures with increased potency, rather than a focus on maintaining optimal physicochemical properties associated with ADMET throughout drug optimization. Particular attention to the optimum region of lipophilicity, as well as monitoring of lipophilic efficiency indices, may contribute significantly to the overall quality of candidate drugs at different stages of discovery.

Selective estrogen receptor modulators: tissue specificity and clinical utility

Selective estrogen receptor modulators (SERMs) are a diverse group of nonsteroidal compounds that function as agonists or antagonists for estrogen receptors (ERs) in a target gene-specific and tissue-specific fashion. SERM specificity involves tissue-specific expression of ER subtypes, differential expression of co-regulatory proteins in various tissues, and varying ER conformational changes induced by ligand binding. To date, the major clinical applications of SERMs are their use in the prevention and treatment of breast cancer, the prevention of osteoporosis, and the maintenance of beneficial serum lipid profiles in postmenopausal women. However, SERMs have also been found to promote adverse effects, including thromboembolic events and, in some cases, carcinogenesis, that have proven to be obstacles in their clinical utility. In this review, we discuss the mechanisms of SERM tissue specificity and highlight the therapeutic application of well-known and emergent SERMs.

Connective tissue growth factor (CTGF/CCN2) is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts.

Connective tissue growth factor (CTGF/CCN2) is a cysteine‐rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor‐β1 (TGF‐β1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF‐β1‐induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF‐β1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF‐β1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF‐specific siRNA was used to prevent TGF‐β1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the requirement of CTGF for their up‐regulation. To examine the effects of TGF‐β1 on osteoblast cell growth, cultures were treated with TGF‐β1 during the proliferative stage. Cell number was significantly reduced and the cells exhibited a decrease in G1 cyclin expression, consistent with TGF‐β1‐induced cell‐cycle arrest. Cultures transfected with CTGF siRNA prior to TGF‐β1 treatment showed an even greater reduction in cell number, suggesting that TGF‐β1‐induced growth arrest is independent of CTGF in osteoblasts. Collectively, these data demonstrate for the first time that CTGF is an essential downstream mediator for TGF‐β1‐induced ECM production in osteoblasts, but these two growth factors function independently regarding their opposing effects on osteoblast proliferation. J. Cell. Physiol. 210: 843–852, 2007.

Molecular Requirements for Induction of CTGF Expression by TGF-β1 in Primary Osteoblasts

Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by TGF-beta1 where it acts as a downstream mediator of TGF-beta1 induced matrix production. The molecular mechanisms that control CTGF induction by TGF-beta1 in osteoblasts are not known. To assess the role of individual Smads in mediating the induction of CTGF by TGF-beta1, we used specific Smad siRNAs to block Smad expression. These studies demonstrated that Smads 3 and 4, but not Smad 2, are required for TGF-beta1 induced CTGF promoter activity and expression in osteoblasts. Since the activation of MAPKs (Erk, Jnk and p38) by TGF-beta1 is cell type specific, we were interested in determining the role of individual MAPKs in TGF-beta1 induction of CTGF promoter activity and expression. Using dominant negative (DN) mutants for Erk, Jnk and p38, we demonstrated that the expression of DN-Erk caused a significant inhibition of TGF-beta1 induced CTGF promoter activity. In contrast, the expression of DN-p38 or DN-Jnk failed to inhibit activation of CTGF promoter activity. To confirm the vital role of Erk, we used the Erk inhibitor (PD98059) to block its activation, demonstrating that it prevented TGF-beta1 activation of the CTGF promoter and up-regulation of CTGF expression in osteoblasts. Since Src can also act as a downstream signaling effector for TGF-beta in some cell types, we determined its role in TGF-beta1 induction of CTGF in osteoblasts. Treatment of osteoblasts with a Src family kinase inhibitor, PP2, or the expression of two independent kinase-dead Src mutant constructs caused significant inhibition of TGF-beta1 induced CTGF promoter activity and expression. Additionally, blocking Src activation prevented Erk activation by TGF-beta1 demonstrating a role for Src as an upstream mediator of Erk in regulating CTGF expression in osteoblasts. To investigate the involvement of the TGF-beta1 response element (TRE) and the SMAD binding element (SBE) in CTGF induction, we cloned the rat CTGF proximal promoter (-787 to +1) containing the TRE and SBE motifs into a pGL3-Luciferase reporter construct. Using a combination of CTGF promoter deletion constructs and site-directed mutants, we demonstrated the unique requirement of both the TRE and SBE for CTGF induction by TGF-beta1 in osteoblasts. Electro-mobility shift assays using specific probes containing the TRE, SBE or both showed TGF-beta1 inducible complexes that can be ablated by mutation of the respective motif, confirming their requirement for TGF-beta1 induced CTGF promoter activity. In conclusion, these studies demonstrate that CTGF induction by TGF-beta1 in osteoblasts involves Smads 3 and 4, the Erk and Src signaling pathways, and requires both the TRE and SBE motifs in the CTGF proximal promoter.

Src is a major signaling component for CTGF induction by TGF-β1 in osteoblasts†

Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor β1 (TGF‐β1) where it acts as a downstream mediator of TGF‐β1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk, and Smad signaling for CTGF induction by TGF‐β1 in osteoblasts; however, the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF‐β1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF‐β1. Additionally, inhibiting Src activation prevented Erk activation, Smads 2 and 3 activation and nuclear translocation by TGF‐β1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway directly by mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co‐activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF‐β1‐induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. These data demonstrate that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF‐β1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. J. Cell. Physiol. 224: 691–701, 2010.

Targeting protein palmitoylation: selective inhibitors and implications in disease

Introduction: Palmitoylation describes the enzymatic attachment of the 16-carbon fatty acid, palmitate, to specific cysteines of proteins via a labile thioester bond. This post-translational modification increases the lipophilicity of the modified protein, thus regulating its subcellular distribution and function. The transfer of palmitate to a substrate is mediated by palmitoyl acyltransferases (PATs), while depalmitoylation is catalyzed by acyl protein thioesterases (APTs). Nearly one-third of the 23 genes that encode PATs are linked to human diseases, representing important targets for drug development. Areas covered: In this review, the authors summarize the recent technical advances in the field of palmitoylation and how they will affect our ability to understand palmitoylation and its relevance to human disease. They also review the current literature describing existing palmitoylation inhibitors. The aim of this article is to increase the awareness of the importance of palmitoylation in disease by reviewing the recent progress made in identifying pharmacological modulators of PATs/APTs. It also aims to provide suggestions for general considerations in the development of selective and potent PAT inhibitors. Expert opinion: Developing therapeutically useful pharmacological modulators of palmitoylation will require that they be developed within the context of well-characterized PAT/APT-related signaling systems. The successful development of potent, specific drugs in similarly complex systems suggests that development of useful drugs targeting PATs is feasible.

Ets-1 Is Essential for Connective Tissue Growth Factor (CTGF/CCN2) Induction by TGF-β1 in Osteoblasts

Background Ets-1 controls osteoblast differentiation and bone development; however, its downstream mechanism of action in osteoblasts remains largely undetermined. CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of Ets-1 for CCN2 induction by TGF-β1 in primary osteoblasts. Results We demonstrated that Ets-1 is expressed and induced by TGF-β1 treatment in osteoblasts, and that Ets-1 over-expression induces CCN2 protein expression and promoter activity at a level similar to TGF-β1 treatment alone. Additionally, we found that simultaneous Ets-1 over-expression and TGF-β1 treatment synergize to enhance CCN2 induction, and that CCN2 induction by TGF-β1 treatment was impaired using Ets-1 siRNA, demonstrating the requirement of Ets-1 for CCN2 induction by TGF-β1. Site-directed mutagenesis of eight putative Ets-1 motifs (EBE) in the CCN2 promoter demonstrated that specific EBE sites are required for CCN2 induction, and that mutation of EBE sites in closer proximity to TRE or SBE (two sites previously shown to regulate CCN2 induction by TGF-β1) had a greater effect on CCN2 induction, suggesting potential synergetic interaction among these sites for CCN2 induction. In addition, mutation of EBE sites prevented protein complex binding, and this protein complex formation was also inhibited by addition of Ets-1 antibody or Smad 3 antibody, demonstrating that protein binding to EBE motifs as a result of TGF-β1 treatment require synergy between Ets-1 and Smad 3. Conclusions This study demonstrates that Ets-1 is an essential downstream signaling component for CCN2 induction by TGF-β1 in osteoblasts, and that specific EBE sites in the CCN2 promoter are required for CCN2 promoter transactivation in osteoblasts.

Estrogen receptors (ERα versus ERβ): friends or foes in human biology?

Abstract Most of the biological effects of estrogens are mediated via the estrogen receptors (ERs) at the level of gene regulation. Recently, new information regarding the role of ERs in physiology, pathology and the mechanisms through which estrogens bring about these functions has emerged. The physiological effects of estrogen are manifested through two ER isoforms – ERα and ERβ – which display distinct regions of sequence homology. The crystal structures of these receptors bound to their specific ligands (e.g. agonists or antagonists) have revealed much about how ligand binding alters receptor structure/conformation and the interaction with coactivators or corepressors as well as how it determines the cellular response to a ligand. ERs are involved in the variety of physiological and pathological activities and different cells and tissues have shown divergent responses to these two receptor isoforms. The discovery of sub-isoforms of ER alpha and beta has further complicated our understanding of how the interaction between ERs and its ligands contribute to the development of disease. Nevertheless, continuing efforts in the study of ERs have helped us to more clearly define their role in disease and to develop novel, ER-targeted therapeutics.

Naturally occurring osmolyte, trehalose induces functional conformation in an intrinsically disordered activation domain of glucocorticoid receptor.

Intrinsically disordered (ID) regions are frequently found in the activation domains of many transcription factors including nuclear hormone receptors. It is believed that these ID regions promote molecular recognition by creating large surfaces suitable for interactions with their specific protein binding partners, which is a critical component of gene regulation by transcription factors. It has been hypothesized that conditional folding of these activation domains may be a prerequisite for their efficient interaction with specific coregulatory proteins, and subsequent transcriptional activity leading to the regulation of target gene(s). In this study, we tested whether a naturally occurring osmolyte, trehalose can promote functionally ordered conformation in glucocorticoid receptors major activation function domain, AF1, which is found to exist as an ID protein, and requires an efficient interaction with coregulatory proteins for optimal activity. Our data show that trehalose induces an ordered conformation in AF1 such that its interaction with steroid receptor coactivator-1 (SRC-1), a critical coregulator of glucocorticoid receptors activity, is greatly enhanced.

Antiproliferative factor regulates connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells

Connective tissue growth factor (CTGF/CNN2) is a novel APF target gene. A novel mechanism is described by which the APF cellular receptor, cytoskeleton-associated protein 4 (CKAP4), mediates APF-induced CTGF transcription.