针对趋化因子受体CCR5的SPA-WGA药物筛选方法

目的建立针对趋化因子受体CCR5的药物筛选SPA-WGA法[35S]GTPγS结合实验方法。方法通过降低SPA-WGA小球用量和对其他各种因素进行优化,如细胞膜用量、GDP浓度、体系孵育时间和样品连续测量时间,建立了SPA-WGA法[35S]GTPγS结合实验方法。结果最后优化得到实验条件为:100μl反应体系,0.1 mg SPA-WGA小球,10μgCHO-CCR5细胞膜,10μmol.L-1GDP和0.3 nmo.lL-1[35S]GTPγS,室温孵育1.5 h并且在1 200 m in之内完成所有样品测量。该法与传统抽滤法比较,测量得到RANTES的EC50分别为:1.40 nmol.L-1和2.90 nmol.L-1,测量得到SCH-C的IC50分别为:2.95 nmo.lL-1和2.73 nmo.lL-1,测量结果相似,均呈现出较好的剂量效应关系。利用该法筛选54个化合物,筛到3个IC50在1~10 nmol.L-1的化合物。这3个化合物是否具有H IV-1进入抑制剂的潜在开发价值,还有待体外抗H IV-1实验的进一步验证和筛选。结论此方法操作简便,灵敏性和重现性好,且可高通量和自动化,该法适用于能与Gi家族蛋白偶联的GPCR的高通量药物筛选。     

The influence of G protein subtype on agonist action at D2 dopamine receptors

In previous studies, we have shown that agonists influence the ability of D2 dopamine receptors to couple to G proteins and here we extend this work. The human D2Short dopamine receptor and a natural polymorphism of this D(2Short)(Ser311Cys), have been studied by co-expressing the receptors in insect cells with Gbeta1gamma2 and either Galpha(o), Galpha(i1), Galpha(i2) or Galpha(i3) G protein subunits. These preparations have been used to study the G protein coupling profiles of the two receptors and the influence of agonists. Receptor/G protein coupling was analysed in dopamine/[3H]spiperone competition binding experiments and through stimulation of [35S]GTPgammaS binding. Although the Ser311Cys polymorphism itself had no appreciable effect on the G protein coupling specificity of the D2 receptor, agonist stimulation of [35S]GTPgammaS binding, revealed that both dopamine and (+)-3PPP showed a clear preference for Galpha(o) compared to the Galpha(i) subtypes, but quinpirole did not. These results indicate that agonists are able to stabilise different receptor conformations with different abilities to couple to G proteins.     

Differential regulation of serotonin-1A receptor-stimulated [35S]GTP gamma S binding in the dorsal raphe nucleus by citalopram and escitalopram

The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10 microM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram.     

Activation of G proteins and extracellular signal-regulated kinase 1/2 phosphorylation via human dopamine D4.4 receptors: differential pathway-dependent potencies of receptor agonists

Agonist activity at recombinant human dopamine D_4.4 receptors was compared in stably transfected CHO cells using two functional readouts: G protein activation by [³⁵S]GTPγS binding and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2). Results with a large series of agonists reveal markedly higher relative agonist efficacy in the pERK1/2 assay compared with [³⁵S]GTPγS binding, while potencies were generally higher in the latter readout. Whereas efficacies were highly correlated when comparing both tests, potencies determined using the pERK1/2 assay were neither correlated with those for G protein activation nor with binding affinities. In order to examine if these differences may be attributable to distinct assay conditions (5 min incubation for pERK1/2 compared with binding equilibrium conditions for [³⁵S]GTPγS), selected compounds were tested in a modified short-duration [³⁵S]GTPγS binding assay. In these experiments, potencies were generally reduced; however, compounds exhibiting comparably high potency in the pERK1/2 assay were not affected by this duration-dependent potency shift. We conclude that assay parameters such as signal amplification and incubation time have to be considered with respect to the appropriate choice of experimental approaches that best reflect agonist activity at dopamine D₄ receptors in vivo. This paper is especially dedicated to the memory of our appreciated colleague and co-author, Dr. Liesbeth Bruins Slot, who left us much too soon the 4th of April, 2008.     

Comparison between the interaction of steroids with [35S]TBPS binding to cerebral cortical and to pituitary membranes: correlation with inhibition of prolactin release

Summary It has been shown previously that 5-pregnan-3-ol-20-one (53P) can inhibit prolactin release from anterior pituitary gland cells in culture through an interaction with a specific modulatory site on the GABAA receptor complex in anterior pituitary gland membranes. In the present work, this receptor site has been labelled with [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) to enable a study of the relative binding affinities (RBA) of different steroids for the GABAA receptor complex to be made.We have found a high correlation (r = + 0.88) between the inhibition of [³⁵S]TBPS binding to anterior pituitary membranes and the inhibition of [³⁵S]TBPS binding to cerebral cortical membranes by nine different steroids. There was also a high correlation between the inhibition of prolactin release from anterior pituitary gland cells in culture by these steroids and the inhibition of [³⁵S]TBPS binding to anterior pituitary membranes (r = + 0.99) or to cortical membranes (r = + 0.81).These observations suggest that the measurement of prolactin release from anterior pituitary gland cells in culture is a good indicator of the functional activity of drugs that bind to the allosteric modulatory TBPS-binding site on the GABA_A-receptor complex. Correspondence to M. Vincens at the above address     

Effects of chronic treatment with citalopram on cannabinoid and opioid receptor-mediated G-protein coupling in discrete rat brain regions

Rationale There is growing interest in investigating the mechanisms of action of selective serotonin reuptake inhibitors (SSRIs), beyond their association with the serotonergic system, due to their wide therapeutic potential for disorders including depression, pain and addiction. Objective The aim of this study was to investigate whether chronic treatment with the SSRI, citalopram, alters the functional coupling of G_i/o-associated cannabinoid type 1 (CB₁) and μ-opioid receptors in selected areas of rat brain implicated in psychiatric disorders and pain. Methods Using an autoradiographic approach, the effects of the cannabinoid receptor agonist, HU210 (in the presence or absence of the CB₁ receptor antagonist AM251), or the μ-opioid receptor agonist, [d-Ala²,N–Me–Phe4,Gly⁵-ol]-enkephalin (DAMGO; in the presence or absence of the μ-opioid receptor antagonist d-Phe–Cys–Tyr-d-Trp–Orn–Thr–Pen–Thr–NH₂), on [³⁵S]GTPγS binding in discrete brain regions of citalopram-treated (10 mg kg⁻¹ day⁻¹ for 14 days by subcutaneous minipump) and control rats were investigated. Results The HU210-induced increase in [³⁵S]GTPγS binding observed in the hypothalamic paraventricular nucleus of control rats was abolished after chronic treatment with citalopram. Reduced response to HU210 in rats receiving chronic treatment with citalopram was also observed in the hippocampus and medial geniculate nucleus. Citalopram had no significant effect on DAMGO-induced [³⁵S]GTPγS binding in the brain regions investigated, with the exception of the medial geniculate nucleus where a modest impairment was observed. Conclusions These data provide evidence for reduced cannabinoid receptor-mediated G-protein coupling in the hypothalamus, hippocampus and medial geniculate nucleus of rats chronically treated with citalopram, effects which may, in part, underlie the mechanism of action of SSRIs. Hesketh and Brennan have contributed equally to this work.     

5-HT-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5-HT1B receptors in rat striatal membranes

Receptor-mediated guanine nucleotide-binding regulatory protein (G protein) activation or functional coupling between receptors and G proteins has been investigated by means of agonist-induced [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding, especially for the receptor subtypes negatively coupled to adenylyl cyclase through G_i type G proteins. In the present study, 5-HT-stimulated [³⁵S]GTPγS binding to rat stritatal membranes was pharmacologically characterized in detail with the help of an extensive series of 5-HT receptor ligands. The optimum experimental conditions for the concentrations of GDP, MgCl₂ and NaCl in the assay buffer were initially determined, and the standard assay was performed with 20 μM GDP, 5 mM MgCl₂ and 100 mM NaCl. The specific [³⁵S]GTPγS binding was stimulated by several compounds that had been shown to be agonists at 5-HT_1B/1D receptors. The negative logarithmic values of the concentration eliciting half-maximal effect (pEC₅₀) for these agonists were significantly correlated with their pK _i’s reported in the previous study of 5-HT_1B receptor binding in rat frontal cortical membranes. The increase in specific [³⁵S]GTPγS binding in response to 1 μM 5-HT was potently inhibited by several 5-HT_1B/1D receptor antagonists as well as β-adrenoceptor antagonists such as S(−)-cyanopindolol. On the other hand, 3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol HCl (BRL15572), a selective antagonist against human 5-HT_1D receptors, was inactive as an antagonist at least up to 1 μM. Additionally, the concentration-response curve for 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine (L694247) was shifted rightward in parallel by the addition of S(−)-cyanopindolol at concentrations of 10 and 100 nM, indicative of the competitive inhibitory manner. The specific [³⁵S]GTPγS binding was reduced by 1′-methyl-5-([2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl)-2,3,6,7-tetrahydrospirospiro(furo[2,3-f]indole-3,4′-piperidine) (SB224289) and methiothepin in a concentration-dependent manner. The inhibitory curve by either compound was shifted to the right by 10 and 100 nM S(−)-cyanopindolol, suggesting that these two drugs behaved as inverse agonists at 5-HT_1B receptors in the present functional assay system. 5-HT-stimulated [³⁵S]GTPγS binding to rat striatal membranes serves as a simple but useful method of investigating the functional interaction between the native 5-HT_1B receptors and their coupled G proteins in this brain region.     

Aging alters in a region-specific manner serotonin transporter sites and 5-HT(1A) receptor-G protein interactions in hamster brain

Key proteins regulating serotonergic activity, specifically the serotonin transporter and 5-HT(1A) receptor, were examined in the midbrain raphe nuclei of young (3-4 months) and old (17-19 months) hamsters (N=7-10/group). An age-related decrease in the maximal density of serotonin transporter sites labelled with [(3)H]paroxetine (fmol/mg protein, Old: 396+/-13; Young: 487+/-27) was observed in the dorsal raphe nucleus (DRN) but not the median raphe nucleus (MRN), without affecting the affinity of [(3)H]paroxetine. In the DRN and MRN, the stimulation of [(35)S]GTP gamma S binding by the 5-HT(1A) receptor agonist 8-OH-DPAT, or the number of 5-HT(1A) receptor sites labeled with [(3)H] MPPF, was not different in old versus young animals. Thus in the DRN, aging decreased serotonin transporter sites without changing 5-HT(1A) receptor activation of G proteins or 5-HT(1A) receptor density. In the CA(1) region of hippocampus, 8-OH-DPAT-stimulated [(35)S]GTP gamma S binding was increased in the older animals (% above basal, Old: 141+/-21; Young: 81+/-17) without changing specific [(3)H] MPPF binding sites, suggesting that the capacity of 5-HT(1A) receptors to activate G proteins is enhanced. Aging also appears to enhance this capacity in the dentate gyrus, because this region exhibited a constant level of 8-OH-DPAT-stimulated [(35)S]GTP gamma S binding in spite of an age-related decrease in the number of [(3)H] MPPF binding sites (fmol/mg protein, Old: 203+/-21; Young: 429+/-51).     

Quantitative stoichiometry of G-proteins activated by mu-opioid receptors in postmortem human brain

Paradoxically, the potencies (EC(50)) of agonists stimulating [35S]GTPgammaS binding are several orders of magnitude lower than their affinities in receptor binding assays. We have investigated the quantitative stoichiometry of mu-opioid receptor-G-protein coupling in postmortem human brain. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) displaced [3H]naloxone binding in a biphasic pattern. The ratio between K(i-low) and EC(50) of DAMGO stimulating [35S]GTPgammaS binding was lower than one. The K(A) of DAMGO was calculated following mu-opioid receptor alkylation by beta-funaltrexamine from [35S]GTPgammaS binding data using the "nested hyperbolic method", yielding K(A)/EC(50)>1. Thus, only 1.2 +/- 0.2% of mu-opioid receptors was needed to be occupied to achieve the half-maximal effect of DAMGO. The estimated ratio between the G-proteins activated by 10 microM DAMGO (determined by isotopic dilution curves) and the occupied-mu-opioid receptors was 1304. In conclusion, we have determined the stoichiometric and the kinetic parameters in the mu-opioid receptor-G-protein system.     

Importance of a N-terminal aspartate in the internalization of the neuropeptide Y Y2 receptor

With human neuropeptide Y Y(2) receptor expressed in the Chinese hamster ovary (CHO) cells, the Asp(35)Ala mutation, and especially the change of Pro(34)Asp(35) to Ala(34)Ala(35), decrease the compartmentalization and strongly accelerate internalization of the receptor. These changes are not associated with alterations in agonist affinity, G-protein interaction, dimerization, or level of expression of the mutated receptors relative to the wildtype receptor. The proline-flanked aspartate in the N-terminal extracellular segment of the neuropeptide Y Y(2) receptor thus apparently has a large role in anchoring and compartmentalization of the receptor. However, the Pro(34)Ala mutation does not significantly affect the embedding and cycling of the receptor.     

Histamine H3 and dopamine D2 receptor-mediated [35S]GTPgamma[S] binding in rat striatum: evidence for additive effects but lack of interactions

The interactions in the rat striatum between H(3) receptors (H(3)Rs) and D(2) receptors (D(2)Rs) were investigated with the [(35)S]GTPgamma[S] binding assay. The H(3)R agonist (R)alpha-methylhistamine increased [(35)S]GTPgamma[S] binding to striatal membranes with an EC(50)=14+/-5 nM and a maximal effect of +19+/-1%. This effect was inhibited by the H(3)R antagonist ciproxifan with a K(i)=1.0+/-0.3 nM. The D(2)R agonist quinpirole increased [(35)S]GTPgamma[S] binding to the same membranes with an EC(50)=1.5+/-0.5 microM and a maximal effect of +28+/-2%. Its effect was blocked by haloperidol with a K(i)=0.3+/-0.1 nM. The maximal effects of the H(3)R and D(2)R agonists were additive (+46+/-3%). However, D(2)R ligands did not modify the effects of H(3)R ligands and vice versa. Ciproxifan behaved as an H(3)R inverse agonist and decreased [(35)S]GTPgamma[S] binding. Haloperidol had no effect and did not change the inverse agonist effect of ciproxifan. Administrations for 10 days of ciproxifan (1.5mg/kg/day) or haloperidol (0.5mg/kg/day) did not change the effects of quinpirole and (R)alpha-methylhistamine, respectively. These data suggest that striatal H(3)Rs and D(2)Rs do not interact through their coupling to G-proteins. However, a hyperactivity of histaminergic and dopaminergic neurons being observed in schizophrenia, the additive activations of H(3)Rs and D(2)Rs suggest that they cooperate to generate some schizophrenic symptoms. Such a postsynaptic mechanism may underlie the antipsychotic-like effects of H(3)R inverse agonists and supports their therapeutic interest, alone or as adjunctive treatment with neuroleptics.     

(35)S]GTPγS binding and opioid tolerance and efficacy in mouse spinal cord

The present study examined efficacy of a series of opioid agonists and then using chronic in vivo treatment protocols, determined tolerance to opioid agonist stimulated [³⁵S]GTPγS (guanosine 5′-O-(3-[³⁵S] thio)triphosphate) binding in mouse spinal cord membranes and compared it directly to spinal analgesic tolerance. The [³⁵S]GTPγS binding assay was used to estimate efficacy (E_max and τ; Operational Model of Agonism) of a series of opioid agonists for G-protein activation in mouse spinal cord. The rank order of opioid agonist efficacy determined in the [³⁵S]GTPγS assay using the Operational Model and E_max was similar. These efficacy estimates correlated with historical analgesic efficacy estimates. For tolerance studies, mice were continuously treated s.c. for 7 days with morphine, oxycodone, hydromorphone, etorphine or fentanyl and [³⁵S]GTPγS studies were conducted in spinal cord membranes. Other mice were tested in i.t. analgesia dose response studies (tailflick). Tolerance to DAMGO ([D-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin) or morphine stimulated [³⁵S]GTPγS binding (decrease in E_max) was observed following etorphine and fentanyl treatment only. These treatment protocols downregulate μ-opioid receptor density whereas morphine, oxycodone and hydromorphone do not. Spinal analgesic tolerance was observed following all treatment protocols examined (morphine, oxycodone and etorphine). Opioid antagonist treatment that specifically upregulates (chronic naltrexone) or downregulates (clocinnamox) μ-opioid receptor density produced a corresponding change in opioid agonist stimulated [³⁵S]GTPγS binding. Although receptor downregulation and G-protein uncoupling are among potential mechanisms of opioid tolerance, the present results suggest that uncoupling in mouse spinal cord plays a minor role and that the [³⁵S]GTPγS assay is particularly responsive to changes in μ-opioid receptor density.     

Failure ofγ-hydroxybutyrate to alter the function of the GABAA receptor complex in the rat cerebral cortex

The present study was designed to evaluate the possible interaction of-hydroxybutyrate (GHB) with the GABA_A receptor complex in the rat cerebral cortex. To this purpose we studied the effect of in vitro addition and in vivo administration of GHB on the biochemical parameters currently used to evaluate the function of the GABAergic system. In vitro addition of increasing concentrations of GHB failed to modify [³H]flunitrazepam (³H]FNZ) binding and the modulatory action of GABA on this binding. Moreover, unlike diazepam, GHB did not modify in vitro both muscimol-stimulated³⁶Cl^– uptake and t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to rat cerebral cortex. In vivo administration of sedative and hypnotic doses of GHB (300–750 mg/kg IP) failed to induce in 60 min any significant change in the [³⁵S]TBPS binding to unwashed cortical membranes. Moreover, GHB also failed to antagonize the increase in [³⁵S]TBPS binding (+55%) induced by isoniazid (350 mg/kg SC). In contrast, at the highest doses used, this drug completely antagonized the seizure activity induced by isoniazid. In conclusion, our data show that GHB fails to alter the function of the GABA_A/benzodiazepine/ionophore receptor complex in the rat cerebral cortex.     

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor CCR5

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca(2+) mobilisation in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1alpha (D26A) (MIP-1alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5), MIP-1beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin-stimulated cAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCL5, CCL8 and CCL13 were able to stimulate Ca(2+) mobilisation through CCR5, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca(2+) responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca(2+) mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca(2+) mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCR5.     

Terbutaline increases the cervical resistance of the pregnant rat in vitro

Cervical ripening is a crucial process leading to delivery. Early dilation of the pregnant cervix can contribute to premature labour. The maturity of the cervix can be characterized by its resistance to mechanical stretching. Although a number of compounds are considered to increase cervical resistance (e.g., progesterone, nitric oxide synthase inhibitors and nonsteroidal anti-inflammatory drugs), none of them seem to be safe for clinical application. Other compounds, such as ₂-adrenergic receptor (₂-AR) agonists, have been used for several decades to stop premature myometrium contractions, but their cervical action has never been investigated. The aim of this study was to detect the effects of the ₂-AR agonist terbutaline on nonpregnant and late-pregnant (day 18, 20, 21 or 22) cervices isolated from Sprague–Dawley rats. Cervical resistance was measured by means of a mechanical stretching test in vitro, the ₂-AR density was determined by Western blot analysis, the ₂-AR mRNA was determined by RT-PCR, while the G-protein activation following cervical ₂-AR stimulation with terbutaline was evaluated via a [³⁵S]GTPS binding assay.Terbutaline at 10^–6 M increased the cervical resistance of the late-pregnant samples in vitro from day18 to day 22, but did not alter the resistance of the nonpregnant samples. This cervical resistance-increasing effect was concentration dependent and antagonized with propranolol on day 21. Terbutaline was ineffective on cervical samples when gradual stretching was omitted. RT-PCR and Western blot studies revealed increased ₂-AR mRNA and ₂-AR levels respectively on day 18 of pregnancy compared with the nonpregnant cervix, but no further changes were detected up to the end of pregnancy. The [³⁵S]GTPS binding assay demonstrated a decreased G-protein activation on the days of pregnancy investigated, but no activation was found in the nonpregnant samples. The degree of decrease in G-protein activation by terbutaline was in harmony with its cervical resistance-increasing action. On day 21, the G-protein activation-decreasing effect of terbutaline was antagonized with propranolol.We presume that the cervical resistance-increasing effect of terbutaline is a consequence of its G-protein activation-decreasing property via ₂-ARs, which finally leads to an increased muscle resistance against mechanical stretching. This action of terbutaline seems unique among the smooth muscles, and may open up a new perspective in the prevention of premature labour. Clinical experience indicates that ₂-AR agonists will not be sufficient to stop the overall process, but their combination with more potent inhibitors of uterine contractions may be of clinical benefit.     

DSM-RX78, a new phosphodiesterase inhibitor, suppresses superoxide anion production in activated human neutrophils and attenuates hemorrhagic shock-induced lung injury in rats

Neutrophils are activated following hemorrhagic shock and the accumulation of neutrophils in the lung is associated with lung injury. This research investigated the effects of a semisynthetic 2-benzoylaminobenzoic acid derivative, methyl 2-(2-fluorobenzamido)benzoate (DSM-RX78), on superoxide anion (O₂⁻) production in formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-activated human neutrophils, and on lung injury in Sprague-Dawley rats subjected to trauma-hemorrhage. DSM-RX78 concentration-dependently inhibited O₂⁻ production, but not elastase release, in FMLP-activated human neutrophils. DSM-RX78 displayed no superoxide-scavenging ability, and it failed to alter the subcellular NADPH oxidase activity. Significantly, DSM-RX78 increased cAMP formation and protein kinase (PK)A activity in FMLP-activated neutrophils, which occurred through the selective inhibition of cAMP-specific phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function or cGMP-specific PDE activity. These results show that DSM-RX78 is a new inhibitor of cAMP-specific PDE. Moreover, DSM-RX78 reduced FMLP-induced phosphorylation of protein kinase B (Akt), but not calcium mobilization. The inhibitory effects of DSM-RX78 on O₂⁻ production and Akt phosphorylation were reversed by PKA inhibitors, suggesting that DSM-RX78 regulates O₂⁻ production of human neutrophils by promoting cAMP/PKA-dependent inhibition of Akt activation. On the other hand, administration of DSM-RX78 significantly attenuated the increase in myeloperoxidase activity and edema in the lung, as well as protein concentrations in bronchoalveolar lavage fluid in rats after trauma-hemorrhagic shock. In summary, these results strongly suggest that DSM-RX78 exerts anti-inflammatory effects, which result from the elevation of cAMP levels and PKA activity through its inhibition of cAMP-specific PDE. Also, our findings show that DSM-RX78 attenuates hemorrhagic shock-induced lung injury in rats.     

Small molecules targeting the innate immune cGAS‒STING‒TBK1 signaling pathway

Multiple cancer immunotherapies including chimeric antigen receptor T cell and immune checkpoint inhibitors (ICIs) have been successfully developed to treat various cancers by motivating the adaptive anti-tumor immunity. Particularly, the checkpoint blockade approach has achieved great clinic success as evidenced by several U.S. Food and Drug Administration (FDA)-approved anti-programmed death receptor 1/ligand 1 or anti-cytotoxic T lymphocyte associated protein 4 antibodies. However, the majority of cancers have low clinical response rates to these ICIs due to poor tumor immunogenicity. Indeed, the cyclic guanosine monophosphate-adenosine monophosphate synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS‒STING‒TBK1) axis is now appreciated as the major signaling pathway in innate immune response across different species. Aberrant signaling of this pathway has been closely linked to multiple diseases, including auto-inflammation, virus infection and cancers. In this perspective, we provide an updated review on the latest progress on the development of small molecule modulators targeting the cGAS‒STING‒TBK1 signaling pathway and their preclinical and clinical use as a new immune stimulatory therapy. Meanwhile, highlights on the clinical candidates, limitations and challenges, as well as future directions in this field are also discussed. Further, small molecule inhibitors targeting this signaling axis and their potential therapeutic use for various indications are discussed as well.