Hazem Abdelkarim

Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2.

The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs.

Novel peptide nanoparticle–biased antagonist of CCR3 blocks eosinophil recruitment and airway hyperresponsiveness

Background: Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason might be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. Objective: We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G protein signaling but enable or promote receptor internalization. Methods: Self‐assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by means of dynamic light scattering and nuclear magnetic resonance. Inhibitory activity on CCR3 signaling was assessed in vitro by using flow cytometry, confocal microscopy, and Western blot analysis in a CCR3+ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed by using a triple‐allergen mouse asthma model. Results: R321 self‐assembles into nanoparticles and binds directly to CCR3, altering receptor function. Half‐maximal inhibitory concentration values for eotaxin‐induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of extracellular signal‐regulated kinase 1/2 activation and not the late phase generally associated with &bgr;‐arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo R321 effectively blocks eosinophil recruitment into the blood, lungs, and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model. Conclusions: R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism might hold significant therapeutic promise by eluding the formation of drug tolerance. Graphical abstract Figure. No caption available.

α-Synuclein interacts directly but reversibly with psychosine: implications for α-synucleinopathies

Aggregation of α-synuclein, the hallmark of α-synucleinopathies such as Parkinson’s disease, occurs in various glycosphingolipidoses. Although α-synuclein aggregation correlates with deficiencies in the lysosomal degradation of glycosphingolipids (GSL), the mechanism(s) involved in this aggregation remains unclear. We previously described the aggregation of α-synuclein in Krabbe’s disease (KD), a neurodegenerative glycosphingolipidosis caused by lysosomal deficiency of galactosyl-ceramidase (GALC) and the accumulation of the GSL psychosine. Here, we used a multi-pronged approach including genetic, biophysical and biochemical techniques to determine the pathogenic contribution, reversibility, and molecular mechanism of aggregation of α-synuclein in KD. While genetic knock-out of α-synuclein reduces, but does not completely prevent, neurological signs in a mouse model of KD, genetic correction of GALC deficiency completely prevents α-synuclein aggregation. We show that psychosine forms hydrophilic clusters and binds the C-terminus of α-synuclein through its amino group and sugar moiety, suggesting that psychosine promotes an open/aggregation-prone conformation of α-synuclein. Dopamine and carbidopa reverse the structural changes of psychosine by mediating a closed/aggregation-resistant conformation of α-synuclein. Our results underscore the therapeutic potential of lysosomal correction and small molecules to reduce neuronal burden in α-synucleinopathies, and provide a mechanistic understanding of α-synuclein aggregation in glycosphingolipidoses.

Biased antagonism of CXCR4 avoids antagonist tolerance

Selectively blocking G protein signaling but not GPCR internalization may provide therapeutic benefit. The right kind of bias AMD3100, an antagonist of the chemokine receptor CXCR4, prevents the accumulation of leukemic cells in the bone marrow, which promotes the efficacy of chemotherapeutic agents. However, tolerance to AMD3100 can develop, which leads to receptor accumulation on the cell surface and retention of cells in the bone marrow. Hitchinson et al. showed that a peptide derived from CXCR4 avoided the development of tolerance by acting as a biased antagonist of G protein signaling but not β-arrestin–mediated internalization of CXCR4. Similar properties were shared by a nonpeptide, small-molecule inhibitor that also did not stimulate tolerance. These results suggest that the use of biased GPCR antagonists could be of therapeutic benefit in patients who have developed tolerance to nonbiased antagonists. Repeated dosing of drugs targeting G protein–coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and β-arrestin1/2–dependent receptor endocytosis. We demonstrated that biased antagonists of G protein–dependent chemotaxis but not β-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit β-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.

Partial agonist activity of α1-adrenergic receptor antagonists for chemokine (C-X-C motif) receptor 4 and atypical chemokine receptor 3

We observed in PRESTO-Tango β-arrestin recruitment assays that the α1-adrenergic receptor (AR) antagonist prazosin activates chemokine (C-X-C motif) receptor (CXCR)4. This prompted us to further examine this unexpected pharmacological behavior. We screened a panel of 14 α1/2- and β1/2/3-AR antagonists for CXCR4 and atypical chemokine receptor (ACKR)3 agonist activity in PRESTO-Tango assays against the cognate agonist CXCL12. We observed that multiple α1-AR antagonists activate CXCR4 (CXCL12 = prazosin = cyclazosin > doxazosin) and ACKR3 (CXCL12 = prazosin = cyclazosin > alfuzosin = doxazosin = phentolamine > terazosin = silodosin = tamsulosin). The two strongest CXCR4/ACKR3 activators, prazosin and cyclazosin, were selected for a more detailed evaluation. We found that the drugs dose-dependently activate both receptors in β-arrestin recruitment assays, stimulate ERK1/2 phosphorylation in HEK293 cells overexpressing each receptor, and that their effects on CXCR4 could be inhibited with AMD3100. Both α1-AR antagonists induced significant chemical shift changes in the 1H-13C-heteronuclear single quantum correlation spectrum of CXCR4 and ACKR3 in membranes, suggesting receptor binding. Furthermore, prazosin and cyclazosin induced internalization of endogenous CXCR4/ACKR3 in human vascular smooth muscle cells (hVSMC). While these drugs did not in induce chemotaxis in hVSMC, they inhibited CXCL12-induced chemotaxis with high efficacy and potency (IC50: prazosin—4.5 nM, cyclazosin 11.6 pM). Our findings reveal unexpected pharmacological properties of prazosin, cyclazosin, and likely other α1-AR antagonists. The results of the present study imply that prazosin and cyclazosin are biased or partial CXCR4/ACKR3 agonists, which function as potent CXCL12 antagonists. Our findings could provide a mechanistic basis for previously observed anti-cancer properties of α1-AR antagonists and support the concept that prazosin could be re-purposed for the treatment of disease processes in which CXCR4 and ACKR3 are thought to play significant pathophysiological roles, such as cancer metastases or various autoimmune pathologies.

Design, Synthesis, Molecular Modeling, and Biological Evaluation of Novel Amine-based Histone Deacetylase Inhibitors

Histone deacetylases (HDACs) are promising drug targets for a variety of therapeutic applications. Herein we describe the design, synthesis, biological evaluation in cellular models of cancer, and preliminary drug metabolism and pharmacokinetic studies (DMPK) of a series of secondary and tertiary N‐substituted 7‐aminoheptanohydroxamic acid‐based HDAC inhibitors. Introduction of an amino group with one or two surface binding groups (SBGs) yielded a successful strategy to develop novel and potent HDAC inhibitors. The secondary amines were found to be generally more potent than the corresponding tertiary amines. Docking studies suggested that the SBGs of tertiary amines cannot be favorably accommodated at the gorge region of the binding site. The secondary amines with naphthalen‐2‐ylmethyl, 5‐phenylthiophen‐2‐ylmethyl, and 1H‐indol‐2‐ylmethyl (2 j) substituents exhibited the highest potency against class I HDACs: HDAC1 IC50 39–61 nm, HDAC2 IC50 260–690 nm, HDAC3 IC50 25–68 nm, and HDAC8 IC50 320–620 nm. The cytotoxicity of a representative set of secondary and tertiary N‐substituted 7‐aminoheptanoic acid hydroxyamide‐based inhibitors against HT‐29, SH‐SY5Y, and MCF‐7 cancer cells correlated with their inhibition of HDAC1, 2, and 3 and was found to be similar to or better than that of suberoylanilide hydroxamic acid (SAHA). Compounds in this series increased the acetylation of histones H3 and H4 in a time‐dependent manner. DMPK studies indicated that secondary amine 2 j is metabolically stable and has plasma and brain concentrations >23‐ and >1.6‐fold higher than the IC50 value for class I HDACs, respectively. Overall, the secondary and tertiary N‐substituted 7‐aminoheptanoic acid hydroxyamide‐based inhibitors exhibit excellent lead‐ and drug‐like properties and therapeutic capacity for cancer applications.