Marilyn C. Darkes

Pharmacology of AMD3465: A small molecule antagonist of the chemokine receptor CXCR4

CXCR4 is widely expressed in multiple cell types, and is involved in neonatal development, hematopoiesis, and lymphocyte trafficking and homing. Disruption of the CXCL12/CXCR4 interaction has been implicated in stem cell mobilization. Additionally CXCR4 is a co-receptor for HIV. Selective small molecule antagonists of CXCR4 therefore have therapeutic potential. AMD3465 is an N-pyridinylmethylene monocyclam CXCR4 antagonist which can block infection of T-tropic, CXCR4-using HIV. Using the CCRF-CEM T-cell line which expresses CXCR4 we have demonstrated that AMD3465 is an antagonist of SDF-1 ligand binding (K(i) of 41.7+/-1.2nM), and inhibits SDF-1 mediated signaling as shown by inhibition of GTP binding, calcium flux, and inhibition of chemotaxis. AMD3465 is selective for CXCR4 and does not inhibit chemokine-stimulated calcium flux in cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7, nor does it inhibit binding of LTB(4) to its receptor, BLT1. The pharmacokinetics of AMD3465 was investigated in mice and dogs. Absorption was rapid following subcutaneous administration. AMD3465 was cleared from dog plasma in a biphasic manner with a terminal half-life of 1.56-4.63h. Comparison of exposure to the intravenous and subcutaneous doses indicated 100% bioavailability following subcutaneous administration. AMD3465 caused leukocytosis when administered subcutaneously in mice and dogs, with peak mobilization occurring between 0.5 and 1.5h following subcutaneous dosing in mice and with maximum peak plasma concentration of compound preceding peak mobilization in dogs, indicating that AMD3465 has the potential to mobilize hematopoietic stem cells. These data demonstrate the therapeutic potential for the CXCR4 antagonist AMD3465.

A ruthenium (III) polyaminocarboxylate complex, a novel nitric oxide scavenger, enhances graft survival and decreases nitrosylated heme protein in models of acute and delayed cardiac transplant rejection.

Nitric oxide (NO) derived from the up-regulation of inducible NO synthase (iNOS) is believed to play an important role in organ rejection. In experimental models of acute cardiac transplant rejection (i.e., without immunosuppression), treatment using NOS inhibitors to prevent acute rejection have yielded conflicting results. This is most likely due to potential inhibition of constitutive NOS. Accordingly, agents that trap NO directly may have some advantage. In the current study, we evaluated the actions of a ruthenium-based NO scavenger, AMD6221, to inhibit the nitrosylation of myocardial protein and to prolong cardiac allograft survival in a model of acute cardiac transplant rejection (without immunosuppression). In addition, we evaluated the efficacy of AMD6221 used in combination with low-dose cyclosporine (CsA) (i.e., a model of delayed graft rejection). Heterotopic abdominal cardiac transplantation was performed using rat strains with disparities at major and minor histocompatibility loci. Grafts were harvested on postoperative day 6 for histologic examination or analysis of myocardial protein nitrosylation using electron paramagnetic resonance (EPR) spectroscopy. Other animals were monitored twice daily to determine rejection times. Plasma was also taken at postoperative day 6 for determining the concentration of NO by-products (nitrate plus nitrite). Treatment with AMD6221 either prolonged graft survival and/or caused a marked decrease in myocardial nitrosylprotein formation as determined by EPR spectroscopy. In vivo scavenging of NO by AMD6221 was verified by high-performance liquid chromatography analysis of nitrosylated-drug in plasma samples. Low-dose CsA given alone or in combination with AMD6221 completely blocked formation of myocardial nitrosylprotein complexes. Whereas low-dose CsA alone prolonged graft survival, combination therapy with CsA plus AMD6221 produced a synergistic effect on graft survival. These studies indicate that treatment with a ruthenium-based NO scavenger, such as AMD6221, may be an effective regimen used alone or in combination with CsA to protect myocardial proteins from posttranscriptional modification and to prolong cardiac graft survival.

HIV-1 entry inhibition by small-molecule CCR5 antagonists: A combined molecular modeling and mutant study using a high-throughput assay

Based on the attrition rate of CCR5 small molecule antagonists in the clinic the discovery and development of next generation antagonists with an improved pharmacology and safety profile is necessary. Herein, we describe a combined molecular modeling, CCR5-mediated cell fusion, and receptor site-directed mutagenesis approach to study the molecular interactions of six structurally diverse compounds (aplaviroc, maraviroc, vicriviroc, TAK-779, SCH-C and a benzyloxycarbonyl-aminopiperidin-1-yl-butane derivative) with CCR5, a coreceptor for CCR5-tropic HIV-1 strains. This is the first study using an antifusogenic assay, a model of the interaction of the gp120 envelope protein with CCR5. This assay avoids the use of radioactivity and HIV infection assays, and can be used in a high throughput mode. The assay was validated by comparison with other established CCR5 assays. Given the hydrophobic nature of the binding pocket several binding models are suggested which could prove useful in the rational drug design of new lead compounds.

The molecular pharmacology of AMD11070: An orally bioavailable CXCR4 HIV entry inhibitor

In order to enter and infect human cells HIV must bind to CD4 in addition to either the CXCR4 or the CCR5 chemokine receptor. AMD11070 was the first orally available small molecule antagonist of CXCR4 to enter the clinic. Herein we report the molecular pharmacology of AMD11070 which is a potent inhibitor of X4 HIV-1 replication and the gp120/CXCR4 interaction. Using the CCRF-CEM T cell line that endogenously expresses CXCR4 we have demonstrated that AMD11070 is an antagonist of SDF-1α ligand binding (IC50 = 12.5 ± 1.3 nM), inhibits SDF-1 mediated calcium flux (IC50 = 9.0 ± 2.0 nM) and SDF-1α mediated activation of the CXCR4 receptor as measured by a Eu-GTP binding assay (IC50 =39.8 ± 2.5 nM) or a [(35)S]-GTPγS binding assay (IC50 =19.0 ± 4.1 nM), and inhibits SDF-1α stimulated chemotaxis (IC50 =19.0 ± 4.0 nM). AMD11070 does not inhibit calcium flux of cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7, or ligand binding to CXCR7 and BLT1, demonstrating selectivity for CXCR4. In addition AMD11070 is able to inhibit the SDF-1β isoform interactions with CXCR4; and N-terminal truncated variants of CXCR4 with equal potency to wild type receptor. Further mechanistic studies indicate that AMD11070 is an allosteric inhibitor of CXCR4.

Prospective CCR5 Small Molecule Antagonist Compound Design Using a Combined Mutagenesis/Modeling Approach

The viral resistance of marketed antiviral drugs including the emergence of new viral resistance of the only marketed CCR5 entry inhibitor, maraviroc, makes it necessary to develop new CCR5 allosteric inhibitors. A mutagenesis/modeling approach was used (a) to remove the potential hERG liability in an otherwise very promising series of compounds and (b) to design a new class of compounds with an unique mutant fingerprint profile depending on residues in the N-terminus and the extracellular loop 2. On the basis of residues, which were identified by mutagenesis as key interaction sites, binding modes of compounds were derived and utilized for compound design in a prospective manner. The compounds were then synthesized, and in vitro evaluation not only showed that they had good antiviral potency but also fulfilled the requirement of low hERG inhibition, a criterion necessary because a potential approved drug would be administered chronically. This work utilized an interdisciplinary approach including medicinal chemistry, molecular biology, and computational chemistry merging the structural requirements for potency with the requirements of an acceptable in vitro profile for allosteric CCR5 inhibitors. The obtained mutant fingerprint profiles of CCR5 inhibitors were used to translate the CCR5 allosteric binding site into a general pharmacophore, which can be used for discovering new inhibitors.