Specificity for a CCR5 Inhibitor Is Conferred by a Single Amino Acid Residue: ROLE OF ILE198.

The chemokine receptors CCR5 and CCR2b share 89% amino acid homology. CCR5 is a co-receptor for HIV and CCR5 antagonists have been investigated as inhibitors of HIV infection. We describe the use of two CCR5 antagonists, Schering-C (SCH-C), which is specific for CCR5, and TAK-779, a dual inhibitor of CCR5 and CCR2b, to probe the CCR5 inhibitor binding site using CCR5/CCR2b chimeric receptors. Compound inhibition in the different chimeras was assessed by inhibition of chemokine-induced calcium flux. SCH-C inhibited RANTES (regulated on activation, normal T cell expressed and secreted) (CCL5)-mediated calcium flux on CCR5 with an IC50 of 22.8 nM but was inactive against monocyte chemoattractant protein-1 (CCL2)-mediated calcium flux on CCR2b. However, SCH-C inhibited CCL2-induced calcium flux against a CCR5/CCR2b chimera consisting of transmembrane domains IV-VI of CCR5 with an IC50 of 55 nM. A sequence comparison of CCR5 and CCR2b identified a divergent amino acid sequence located at the junction of transmembrane domain V and second extracellular loop. Transfer of the CCR5 sequence KNFQTLKIV into CCR2b conferred SCH-C inhibition (IC50 of 122 nM) into the predominantly CCR2b chimera. Furthermore, a single substitution, R206I, conferred partial but significant inhibition (IC50 of 1023 nM) by SCH-C. These results show that a limited amino acid sequence is responsible for SCH-C specificity to CCR5, and we propose a model showing the interaction with CCR5 Ile(198).

RARα-PLZF oncogene inhibits C/EBPα function in myeloid cells.

In acute promyelocytic leukemia, granulocytic differentiation is arrested at the promyelocyte stage. The variant t(11;17) translocation produces two fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα) and RARα-PLZF, both of which participate in leukemia development. Here we provide evidence that the activity of CCAAT/enhancer binding protein α (C/EBPα), a master regulator of granulocytic differentiation, is severely impaired in leukemic promyelocytes with the t(11;17) translocation compared with those associated with the t(15;17) translocation. We show that RARα-PLZF inhibits myeloid cell differentiation through interactions with C/EBPα tethered to DNA, using ChIP and DNA capture assays. Furthermore, RARα-PLZF recruits HDAC1 and causes histone H3 deacetylation at C/EBPα target loci, thereby decreasing the expression of C/EBPα target genes. In line with these results, HDAC inhibitors restore in part C/EBPα target gene expression. These findings provide molecular evidence for a mechanism through which RARα-PLZF acts as a modifier oncogene that subverts differentiation in the granulocytic lineage by associating with C/EBPα and inhibiting its activity.

Discovery of a series of aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)benzamides as TRPA1 antagonists.

We describe the discovery and advancement of a novel series of TRPA1 antagonist having an aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)benzamide scaffold. The physical and in vitro DMPK profiles are discussed.

Discovery of a 4-aryloxy-1H-pyrrolo[3,2-c]pyridine and a 1-aryloxyisoquinoline series of TRPA1 antagonists.

A series of TRPA1 antagonists is described having a 4-aryloxy-1H-pyrrolo[3,2-c]pyridine or a 1-aryloxyisoquinoline scaffold. These compounds have high ligand efficiency and favorable physical properties and may thus serve as scaffolds for further optimization.

Discovery of P2X3 selective antagonists for the treatment of chronic pain.

Purinergic receptor P2X3 has been linked to analgesia in a number of pre-clinical models of pain, and is expressed in the human pain perception pathway. Only few P2X3-selective antagonists have been reported to date. This Letter describes the SAR and in vivo analgesic profile of a novel scaffold of selective P2X3 antagonists.

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.

Design and synthesis of pyridin-2-yloxymethylpiperidin-1-ylbutyl amide CCR5 antagonists that are potent inhibitors of M-tropic (R5) HIV-1 replication.

A novel series of CCR5 antagonists were identified based on the redesign of Schering C. An SAR was established based on inhibition of CCR5 (RANTES) binding and these compounds exhibited potent inhibition of R5 HIV-1 replication in peripheral blood mononuclear cells.

A time-resolved fluorescent lanthanide (Eu)-GTP binding assay for chemokine receptors as targets in drug discovery.

Chemokines are a family of chemoattractant cytokines involved in leukocyte trafficking, activation, development, and hematopoeisis. Chemokines and their receptors have been implicated in several disease processes, particularly inflammatory and autoimmune disorders and cancer, and are therefore attractive targets for drug development. Chemokine receptors are members of the seven-transmembrane, G protein-coupled receptor (GPCR) family. As such they can be studied using GPCR assays such as ligand binding, G protein activation, and downstream signaling processes such as intracellular calcium flux. In this respect assessing GPCR activation by GTP binding is an important tool to study the early stage of signal transduction. Previously this has been done using the radiolabeled non-hydrolyzable GTP analogue [(35)S]GTPgammaS. In order to avoid the problems involved in working with radioactivity, a new non-radioactive version of the assay has been developed using a europium-labeled GTP analogue in which europium-GTP binding can be assayed using time-resolved fluorescence. We have adapted this assay for chemokine receptors. In this chapter, using the chemokine receptor CXCR4 as an example, we describe the steps for assay optimization. In addition we describe adaptation of this assay for the high-throughput screening of chemokine antagonists.

Comparison of the potential multiple binding modes of bicyclam, monocylam, and noncyclam small-molecule CXC chemokine receptor 4 inhibitors.

CXC chemokine receptor (CXCR)4 is an HIV coreceptor and a chemokine receptor that plays an important role in several physiological and pathological processes, including hematopoiesis, leukocyte homing and trafficking, metastasis, and angiogenesis. This receptor belongs to the class A family of G protein-coupled receptors and is a validated target for the development of a new class of antiretroviral therapeutics. This study compares the interactions of three structurally diverse small-molecule CXCR4 inhibitors with the receptor and is the first report of the molecular interactions of the nonmacrocyclic CXCR4 inhibitor (S)-N'-(1H-benzimidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydroquinolin-8-yl)butene-1,4-diamine (AMD11070). Fourteen CXCR4 single-site mutants representing amino acid residues that span the entire putative ligand binding pocket were used in this study. These mutants were used in binding studies to examine how each single-site mutation affected the ability of the inhibitors to compete with (125)I-stromal-derived factor-1alpha binding. Our data suggest that these CXCR4 inhibitors bind to overlapping but not identical amino acid residues in the transmembrane regions of the receptor. In addition, our results identified amino acid residues that are involved in unique interactions with two of the CXCR4 inhibitors studied. These data suggest an extended binding pocket in the transmembrane regions close to the second extracellular loop of the receptor. Based on site-directed mutagenesis and molecular modeling, several potential binding modes were proposed for each inhibitor. These mechanistic studies might prove to be useful for the development of future generations of CXCR4 inhibitors with improved clinical pharmacology and safety profiles.

Characterization of the molecular pharmacology of AMD3100: a specific antagonist of the G-protein coupled chemokine receptor, CXCR4.

The chemokine receptor CXCR4 is widely expressed on different cell types, is involved in leukocyte chemotaxis, and is a co-receptor for HIV. AMD3100 has been shown to be a CXCR4 receptor antagonist, and to block HIV infection of T-tropic, X4-using, virus in vitro and in vivo. AMD3100 is an effective mobilizer of hematopoietic stem cells and is being investigated in clinical trials in multiple myeloma and non-Hodgkins lymphoma patients. Using the CCRF-CEM T-cell line that constitutively expresses CXCR4 we confirmed that AMD3100 was an antagonist of SDF-1/CXCL12 ligand binding (IC50=651+/-37 nM). We have also shown that AMD3100 inhibits SDF-1 mediated GTP-binding (IC50=27+/-2.2 nM), SDF-1 mediated calcium flux (IC50=572+/-190 nM), and SDF-1 stimulated chemotaxis (IC50=51+/-17 nM). AMD3100 did not inhibit calcium flux against cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7 when stimulated with their cognate ligands, nor did it inhibit receptor binding of LTB4. AMD3100 did not, on its own, induce a calcium flux in the CCRF-CEM cells, which express multiple GPCRs including CXCR4, CCR4 and CCR7. Furthermore, AMD3100 neither stimulated GTP-binding, an assay for GPCR activation, in CEM cell membranes; nor chemotaxis of CCRF-CEM cells. These data therefore demonstrate that AMD3100 is a specific antagonist of CXCR4, is not cross-reactive with other chemokine receptors, and is not an agonist of CXCR4.

The development of an europium-GTP assay to quantitate chemokine antagonist interactions for CXCR4 and CCR5.

Chemokine receptors have been implicated in several disease processes such as acute and chronic inflammation, cancer, and allograft rejection and are therefore targets for drug development. The chemokine receptors CCR5 and CXCR4 are of particular interest as they serve as entry cofactors for human immunodeficiency virus. These receptors are members of the G protein-coupled receptor (GPCR) family. In this respect, assessing GPCR activation by GTP binding is an important tool to study the early stage of signal transduction. The assay normally utilizes the non-hydrolysable GTP analogue guanosine 5'-gamma-[35S]thiotriphosphate. In order to avoid the problems involved in working with radioactivity, a new non-radioactive version of the assay was developed using a europium-labeled GTP analogue in which europium-GTP binding can be assayed using time-resolved fluorescence. The assay was optimized for CXCR4 and CCR5 and validated for screening of chemokine antagonists using the small molecule CXCR4 antagonist AMD3100 and CCR5 antagonists.

Novel methodology to identify TRPV1 antagonists independent of capsaicin activation.

TRPV1 was originally characterized as an integrator of various noxious stimuli such as capsaicin, heat, and protons. TRPV1-null mice exhibit a deficiency in sensing noxious heat stimuli, suggesting that TRPV1 is one of the main heat sensors on nociceptive primary afferent neurons and a candidate target for heat hypersensitivity in chronic pain. Several different potent and selective TRPV1 antagonists have been developed by more than 50 companies since the characterization of the receptor in 1997. A consequence of this competitive interest is the crowding of patentable chemical space, because very similar in vitro screening assays are used. To circumvent this issue and to expand our understanding of TRPV1 biology, we sought to take advantage of recent advancements in automated patch-clamp technology to design a novel screening cascade. This SAR-driving assay identified novel modulators that blocked the depolarization-induced activation of outwardly-rectifying TRPV1 currents independent of agonist stimulation, and we correlated the pharmacology to three other innovative assays for higher-throughput screening. Ultimately, we have identified a screening paradigm that would have good predictive value for future TRPV1 drug discovery projects and novel chemical space with a higher probability of gaining intellectual property coverage.

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-1ß 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.

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.

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.

Durable engraftment of AMD3100-mobilized autologous and allogeneic peripheral-blood mononuclear cells in a canine transplantation model.

Peripheral-blood mononuclear cells (PBMCs) mobilized with AMD3100, a CXCR4 antagonist, combined with granulocyte colony-stimulating factor (G-CSF) have reconstituted autologous hematopoiesis in cancer patients following myeloablative conditioning. The engraftment potential of PBMCs mobilized with AMD3100 alone, however, has remained unproven. We therefore studied AMD3100-mobilized PBMCs in a canine model. Four dogs received 920 cGy total body irradiation (TBI) before infusion of autologous AMD3100-mobilized PBMCs (median CD34 cell count, 3.9 x 10(6)/kg). Neutrophil (> 0.5 x 10(9)/L [500/microL]) and platelet (> 20 x 10(9)/L [> 20 000/microL]) recoveries occurred at medians of 9 (range, 7-10) days and 25 (range, 23-38) days, respectively, after TBI, and all dogs had normal marrow function at 1 year after transplantation. To evaluate the long-term engraftment potential of AMD3100-mobilized PBMCs, 5 dogs were given 920 cGy TBI followed by infusion of AMD3100-mobilized PBMCs (median CD34 cell dose, 4.7 x 10(6)/kg) from their dog leukocyte antigen (DLA)-identical littermates. Neutrophil and platelet recoveries occurred at medians of 8 (range, 8-10) days and 26 (range, 26-37) days, respectively, after TBI. With a median follow-up of 53 (range, 33-61) weeks, recipients' marrow function was normal, and blood-donor chimerism levels were 97% to 100%. In summary, both autologous and allogeneic AMD3100-mobilized PBMCs led to prompt and durable engraftment in dogs after 920 cGy TBI.

Natriuretic peptide receptor A activation stabilizes a membrane-distal dimer interface.

We have shown previously (Rondeau, J.-J., McNicoll, N., Gagnon, J., Bouchard, N., Ong, H., and De Léan, A. (1995) Biochemistry 34, 2130-2136) that atrial natriuretic peptide (ANP) stabilizes a dimeric form of the natriuretic peptide receptor A (NPRA) by simultaneously interacting with both receptor subunits. However, the first crystallographic study of unliganded NPRA extracellular domain documented a V-shaped dimer involving a membrane-proximal dimer interface and separate binding sites for ANP on each monomer. We explored the possibility of an alternative A-shaped dimer involving a membrane-distal dimer interface by substituting an unpaired solvent-exposed cysteine for Trp(74) in the amino-terminal lobe of full-length NPRA. The predicted spacing between Trp(74) from both subunits drastically differs, depending on whether the V-shaped (84 A) or the A-shaped (8 A) dimer model is considered. In contrast with the expected results for the reported V-shaped dimer, the NPRA(W74C) mutant was constitutively covalently dimeric. Also, the subunits spontaneously reassociated following transient disulfide reduction by dithiothreitol and reoxidation. However, ANP could neither bind to nor activate NPRA(W74C). Permanent disulfide opening by reduction with dithiothreitol and alkylation with N-ethylmaleimide rescued ANP binding to NPRA(W74C). The NPRA mutant could be maintained as a covalent dimer while preserving its function by crosslinking with the bifunctional alkylating agent phenylenedimaleimides (PDM), the ortho-substituted oPDM being more efficient than mPDM or pPDM. These results indicate that the membrane-distal lobe of the NPRAM extracellular domains are dynamically interfacing in the unliganded state and that ANP binding stabilizes the receptor dimer with more stringent spacing at the dimer interface.

Allotopic antagonism of the non-peptide atrial natriuretic peptide (ANP) antagonist HS-142-1 on natriuretic peptide receptor NPR-A.

The microbial polysaccharide HS-142-1 has been documented as an antagonist of natriuretic peptides. It inhibits activation and peptide binding to both guanylate receptors natriuretic peptide receptor (NPR)-A and NPR-B, but has no effect on the non-cyclase receptor NPR-C. At first sight the effect of HS-142-1 on peptide binding appears to be surmountable, suggesting that it might be competitive despite its chemically divergent nature. We explored its mode of action on wild-type NPR-A (WT), on a disulphide-bridged constitutively active mutant (C423S) and on truncated mutants lacking either their cytoplasmic domain (DeltaKC) or both the cytoplasmic and the transmembrane domains (ECD). On the WT, HS-142-1 inhibited atrial natriuretic peptide (ANP) binding with a pK value of 6.51 +/- 0.07 (K(d)=0.31 microM). It displayed a similar effect on the C423S mutant (pK=6.31 +/- 0.11), indicating that its action might not be due to interference with receptor dimerization. HS-142-1 also inhibited ANP binding to DeltaKC with a pK of 7.05 +/- 0.05 (K(d)=0.089 microM), but it was inactive on ANP binding to ECD at a concentration of 10(-4) M, suggesting that the antagonism was not competitive at the peptide-binding site located on the ECD and that the transmembrane domain might be required. HS-142-1 also enhanced dissociation of NPR-A-bound (125)I-ANP in the presence of excess unlabelled ANP, implying an allotopic (allosteric) mode of action for the antagonist.

Investigation of the volatile organic substances that cause the characteristic odor of pentachlorophenol treated wood utility poles.

The nature of the volatile organic compounds that could be at the origin of the characteristic odor of treated wood utility poles was investigated by the study of compositional changes in the chromatographic profiles of fresh-pentachlorophenol (PCP) solvent samples and weathered samples collected from an in-service red pine pole. Over 99 peaks were identified in the chromatogram of the fresh solvent from which a large portion of the C3-, C4-, C5-, C6-alkylbenzene isomers and early eluting n-alkanes was missing from the analysis of weathered samples. Three domains in the chromatographic profile (volatile, semivolatile, and nonvolatile components) were confirmed by assessing the headspace of fresh-PCP solvent samples using direct syringe sampling and solid-phase microextraction. A first level of field validation was achieved using an emission cell for measuring substances emanating from sapwood specimens at different temperatures. The average latent heat of vaporization (deltaHvap) of the PCP-solvent components was estimated at 99.9 kJ/mol from these results. Finally, the analysis of airborne substances at a treating plant and a utility pole storage site confirmed that the C4-, C5-, and C6-alkylbenzene isomers could contribute to the characteristic odor perceived by humans.