Para-Halogenation of Amphetamine and Methcathinone Increases the Mitochondrial Toxicity in Undifferentiated and Differentiated SH-SY5Y Cells.

Halogenation of amphetamines and methcathinones has become a common method to obtain novel psychoactive substances (NPS) also called "legal highs". The para-halogenated derivatives of amphetamine and methcathinone are available over the internet and have entered the illicit drug market but studies on their potential neurotoxic effects are rare. The primary aim of this study was to explore the neurotoxicity of amphetamine, methcathinone and their para-halogenated derivatives 4-fluoroamphetamine (4-FA), 4-chloroamphetamine (PCA), 4-fluoromethcathinone (4-FMC), and 4-chloromethcathinone (4-CMC) in undifferentiated and differentiated SH-SY5Y cells. We found that 4-FA, PCA, and 4-CMC were cytotoxic (decrease in cellular ATP and plasma membrane damage) for both cell types, whereby differentiated cells were less sensitive. IC50 values for cellular ATP depletion were in the range of 1.4 mM for 4-FA, 0.4 mM for PCA and 1.4 mM for 4-CMC. The rank of cytotoxicity observed for the para-substituents was chloride > fluoride > hydrogen for both amphetamines and cathinones. Each of 4-FA, PCA and 4-CMC decreased the mitochondrial membrane potential in both cell types, and PCA and 4-CMC impaired the function of the electron transport chain of mitochondria in SH-SY5Y cells. 4-FA, PCA, and 4-CMC increased the ROS level and PCA and 4-CMC induced apoptosis by the endogenous pathway. In conclusion, para-halogenation of amphetamine and methcathinone increases their neurotoxic properties due to the impairment of mitochondrial function and induction of apoptosis. Although the cytotoxic concentrations were higher than those needed for pharmacological activity, the current findings may be important regarding the uncontrolled recreational use of these compounds.

Allosteric targeting resolves limitations of earlier LFA-1 directed modalities.

Integrins are a family of cell surface receptors well-recognized for their therapeutic potential in a wide range of diseases. However, the development of integrin targeting medications has been impacted by unexpected downstream effects, reflecting originally unforeseen interference with the bidirectional signalling and cross-communication of integrins. We here selected one of the most severely affected target integrins, the integrin lymphocyte function-associated antigen-1 (LFA-1, αLß2, CD11a/CD18), as a prototypic integrin to systematically assess and overcome these known shortcomings. We employed a two-tiered ligand-based virtual screening approach to identify a novel class of allosteric small molecule inhibitors targeting this integrin's αI domain. The newly discovered chemical scaffold was derivatized, yielding potent bis-and tris-aryl-bicyclic-succinimides which inhibit LFA-1 in vitro at low nanomolar concentrations. The characterisation of these compounds in comparison to earlier LFA-1 targeting modalities established that the allosteric LFA-1 inhibitors (i) are devoid of partial agonism, (ii) selectively bind LFA-1 versus other integrins, (iii) do not trigger internalization of LFA-1 itself or other integrins and (iv) display oral availability. This profile differentiates the new generation of allosteric LFA-1 inhibitors from previous ligand mimetic-based LFA-1 inhibitors and anti-LFA-1 antibodies, and is projected to support novel immune regulatory regimens selectively targeting the integrin LFA-1. The rigorous computational and experimental assessment schedule described here is designed to be adaptable to the preclinical discovery and development of novel allosterically acting compounds targeting integrins other than LFA-1, providing an exemplary approach for the early characterisation of next generation integrin inhibitors.

Hyperthermia Increases Neurotoxicity Associated with Novel Methcathinones.

Hyperthermia is one of the severe acute adverse effects that can be caused by the ingestion of recreational drugs, such as methcathinones. The effect of hyperthermia on neurotoxicity is currently not known. The primary aim of our study was therefore to investigate the effects of hyperthermia (40.5 °C) on the neurotoxicity of methcathinone (MC), 4-chloromethcathinone (4-CMC), and 4-methylmethcathinone (4-MMC) in SH-SY5Y cells. We found that 4-CMC and 4-MMC were cytotoxic (decrease in cellular ATP and plasma membrane damage) under both hyper- (40.5 °C) and normothermic conditions (37 °C), whereby cells were more sensitive to the toxicants at 40.5 °C. 4-CMC and 4-MMC impaired the function of the mitochondrial electron transport chain and increased mitochondrial formation of reactive oxygen species (ROS) in SH-SY5Y cells, which were accentuated under hyperthermic conditions. Hyperthermia was associated with a rapid expression of the 70 kilodalton heat shock protein (Hsp70), which partially prevented cell death after 6 h of exposure to the toxicants. After 24 h of exposure, autophagy was stimulated by the toxicants and by hyperthermia but could only partially prevent cell death. In conclusion, hyperthermic conditions increased the neurotoxic properties of methcathinones despite the stimulation of protective mechanisms. These findings may be important for the understanding of the mechanisms and clinical consequences of the neurotoxicity associated with these compounds.

Anti-αLß2 antibodies reveal novel endocytotic cross-modulatory functionality.

BACKGROUND AND PURPOSE: Antibodies targeting cell surface receptors are considered to enable highly selective therapeutic interventions for immune disorders and cancer. Their biological profiles are found, generally, to represent the net effects of antibody-target interactions. The former therapeutic anti-integrin αLß2 antibody efalizumab seems to defeat this paradigm by eliciting, via mechanisms currently unknown, much broader effects than would be predicted based on its target specificity. EXPERIMENTAL APPROACH: To elucidate the mechanisms behind these broad effects, we investigated in primary human lymphocytes in vitro the effects of anti-αLß2 antibodies on the expression of αLß2 as well as unrelated α4 integrins, in comparison to Fab fragments and small-molecule inhibitors. KEY RESULTS: We demonstrate that anti-αLß2 mAbs directly induce the internalization of α4 integrins. The endocytotic phenomenon is a direct consequence of their antibody nature. It is inhibited when monovalent Fab fragments or small-molecule inhibitors are used. It is independent of crosslinking via anti-Fc mAbs and of αLß2 activation. The cross-modulatory effect is unidirectional and not observed in a similar fashion with the α4 integrin antibody natalizumab. CONCLUSION AND IMPLICATIONS: The present study identifies endocytotic cross-modulation as a hitherto unknown non-canonical functionality of anti-αLß2 antibodies. This cross-modulation has the potential to fundamentally alter an antibody's benefit risk profile, as evident with efalizumab. The newly described phenomenon may be of relevance to other therapeutic antibodies targeting cluster-forming receptors. Thus, pharmacologists should be cognizant of this action when investigating such antibodies.

Downstream effect profiles discern different mechanisms of integrin αLß2 inhibition.

The integrin leucocyte function-associated antigen-1 (αLß2, LFA-1) plays crucial roles in T cell adhesion, migration and immunological synapse (IS) formation. Consequently, αLß2 is an important therapeutic target in autoimmunity. Three major classes of αLß2 inhibitors with distinct modes of action have been described to date: Monoclonal antibodies (mAbs), small molecule α/ß I allosteric and small molecule α I allosteric inhibitors. The objective of this study was to systematically compare these three modes of αLß2 inhibition for their αLß2 inhibitory as well as their potential agonist-like effects. All inhibitors assessed were found to potently block αLß2-mediated leucocyte adhesion. None of the inhibitors induced ZAP70 phosphorylation, indicating absence of agonistic outside-in signalling. Paradoxically, however, the α/ß I allosteric inhibitor XVA143 induced conformational changes within αLß2 characteristic for an intermediate affinity state. This effect was not observed with the α I allosteric inhibitor LFA878 or the anti-αLß2 mAb efalizumab. On the other hand, efalizumab triggered the unscheduled internalization of αLß2 in CD4+ and CD8+ T cells while LFA878 and XVA143 did not affect or only mildly reduced αLß2 surface expression, respectively. Moreover, efalizumab, in contrast to the small molecule inhibitors, disturbed the fine-tuned internalization/recycling of engaged TCR/CD3, concomitantly decreasing ZAP70 expression levels. In conclusion, different modes of αLß2 inhibition are associated with fundamentally different biologic effect profiles. The differential established here is expected to provide important translational guidance as novel αLß2 inhibitors will be advanced from bench to bedside.

A novel multi-parameter assay to dissect the pharmacological effects of different modes of integrin αLß2 inhibition in whole blood.

BACKGROUND AND PURPOSE: The integrin αLß2 plays central roles in leukocyte adhesion and T cell activation, rendering αLß2 an attractive therapeutic target. Compounds with different modes of αLß2 inhibition are in development, currently. Consequently, there is a foreseeable need for bedside assays, which allow assessment of the different effects of diverse types of αLß2 inhibitors in the peripheral blood of treated patients. EXPERIMENTAL APPROACH: Here, we describe a flow cytometry-based technology that simultaneously quantitates αLß2 conformational change upon inhibitor binding, αLß2 expression and T cell activation at the single-cell level in human blood. Two classes of allosteric low MW inhibitors, designated α I and α/ß I allosteric αLß2 inhibitors, were investigated. The first application revealed intriguing inhibitor class-specific profiles. KEY RESULTS: Half-maximal inhibition of T cell activation was associated with 80% epitope loss induced by α I allosteric inhibitors and with 40% epitope gain induced by α/ß I allosteric inhibitors. This differential establishes that inhibitor-induced αLß2 epitope changes do not directly predict the effect on T cell activation. Moreover, we show here for the first time that α/ß I allosteric inhibitors, in contrast to α I allosteric inhibitors, provoked partial downmodulation of αLß2, revealing a novel property of this inhibitor class. CONCLUSIONS AND IMPLICATIONS: The multi-parameter whole blood αLß2 assay described here may enable therapeutic monitoring of αLß2 inhibitors in patients' blood. The assay dissects differential effect profiles of different classes of αLß2 inhibitors.