靶向GPCR的藥物研發(fā)
一、小分子藥物和抗體藥物的特點(diǎn)和優(yōu)勢
G蛋白偶聯(lián)受體（GPCR）是膜蛋白，在信號轉導和細胞通訊中發(fā)揮著(zhù)重要作用。因此，GPCR通常成為藥物治療靶點(diǎn)，包括小分子藥物和抗體藥物。靶向GPCR的小分子藥物和抗體藥物具有各自的特性和優(yōu)勢[1]。
小分子藥物通常用于通過(guò)與GPCR的正構或變構結合位點(diǎn)結合來(lái)調節其活性。小分子藥物的優(yōu)點(diǎn)是可以口服，并且容易穿透細胞膜到達目標。小分子藥物的制造成本相對較低，可以大批量生產(chǎn)。 然而，小分子藥物通常具有脫靶效應，可能對目標受體的特異性較低，而導致副作用[2]。
相比之下，抗體藥物是大型蛋白質(zhì)，可以結合于GPCRs的特定表位。它們通常高度特異于靶標受體，并且可以通過(guò)改造在血液中有更長(cháng)的半衰期。，抗體藥物可以激活或阻斷下游信號通路，并且與小分子藥物相比，不容易發(fā)生非特異性作用。然而抗體藥物需要通過(guò)靜脈注射進(jìn)入體內，這可能會(huì )限制其方便性[3]。
總體而言，小分子藥物和抗體藥物對GPCRs的靶向有互補的優(yōu)點(diǎn)和局限性，如何選擇取決于特定的治療應用和目標受體的特性。
二、 GPCR藥物發(fā)現過(guò)程中潛在激動(dòng)劑或拮抗劑的驗證方法
為了驗證潛在的GPCR激動(dòng)劑或拮抗劑的功效，研究人員使用各種測定方法，包括細胞實(shí)驗，放射性配基結合實(shí)驗和功能驗證。
（由G蛋白介導的GPCR信號通路）
細胞實(shí)驗（Cell-based assays）
細胞實(shí)驗通常用于驗證潛在的GPCR激動(dòng)劑或拮抗劑。這些實(shí)驗涉及測量細胞內信號轉導途徑在配體結合后的變化?？梢詼y量的信號轉導途徑包括腺苷酸?；福╝denylyl cyclase）的激活、細胞內鈣離子釋放以及MAP激酶途徑的激活[4]。
一些商業(yè)試劑盒可用于驗證GPCR活性的細胞實(shí)驗。例如，Promega提供GloSensor cAMP測定系統，允許實(shí)時(shí)監測活細胞中的cAMP水平[5]。同樣，Molecular Devices的FLIPR鈣濃度測定系統（FLIPR Calcium Assay）通過(guò)測量鈣動(dòng)員實(shí)現GPCR活性的高通量篩選[6]。
放射性配基結合實(shí)驗 （Radioligand binding assays）
放射性配基結合實(shí)驗是驗證潛在的GPCR激動(dòng)劑或拮抗劑的另一種常用方法。這種方法使用放射標記的配體測量化合物對受體的結合親和力。放射性配基結合實(shí)驗非常敏感，可以檢測到即使很小的結合親和力變化。
可用于GPCR的放射性配基結合實(shí)驗的商業(yè)試劑盒有PerkinElmer的膜靶系統。該試劑盒提供了多種GPCR的預驗證膜和配體，簡(jiǎn)化了測定設置并最小化了變異性。
功能驗證（Functional assays）
功能驗證實(shí)驗用于測量 GPCR 激活的下游效應，例如離子通道的激活或細胞內第二信使的釋放。 這種方法不僅可用于驗證 GPCR 的潛在激動(dòng)劑或拮抗劑，并且通常用于確定這些化合物的作用機制[7]。
有幾種商業(yè)試劑盒可用于GPCR活性的功能測定。 例如，DiscoverX的PathHunter分析系統使用基因編碼的報告系統測量下游信號通路的激活。 同樣，Promega的Beta-Glo檢測系統可測量 β-arrestin 的激活，β-arrestin是 GPCR 脫敏和內化的關(guān)鍵介質(zhì)[8]。
總之，驗證 GPCR 的潛在激動(dòng)劑或拮抗劑是藥物開(kāi)發(fā)的重要一步。通過(guò)使用這些檢測方法，研究人員可以鑒定出對 GPCR 具有高親和力和特異性的化合物，從而開(kāi)發(fā)出有效且安全的療法。
G protein-coupled receptors (GPCRs) are membrane proteins that play important roles in signal transduction and cellular communication. As such, GPCRs are often targeted by therapeutics, including small molecule drugs and antibody drugs. Small molecule drugs and antibody drugs targeting G protein-coupled receptors (GPCRs) have different characteristics and advantages [1].
Small molecule drugs are often used to modulate the activity of GPCRs by binding to their orthosteric or allosteric binding sites. They have the advantage of being orally available and can easily penetrate cell membranes to reach their target. Small molecule drugs have a relatively low manufacturing cost and can be produced in large quantities. However, they can have off-target effects and may be less specific to their target receptor, which can result in side effects [2].
In contrast, antibody drugs are large proteins that bind to specific epitopes on GPCRs. They can be highly specific to their target receptor and can be engineered to have a longer half-life in the bloodstream. Antibody drugs are less likely to have off-target effects compared to small molecule drugs, and they can also activate or block downstream signaling pathways. However, they are not orally available and need to be administered through injection or infusion, which can limit their convenience and accessibility [3].
Overall, small molecule drugs and antibody drugs targeting GPCRs have complementary strengths and limitations, and the choice between them depends on the specific therapeutic application and the properties of the targeted receptor.
To validate the efficacy of potential GPCR agonists or antagonists, researchers use various assays, including cell-based assays, radioligand binding assays, and functional assays.
Cell-based assays
Cell-based assays are commonly used to validate potential GPCR agonists or antagonists. These assays involve measuring changes in intracellular signaling pathways in response to ligand binding. Examples of signaling pathways that can be measured include the activation of adenylyl cyclase, the release of intracellular calcium stores, and the activation of MAP kinase pathways [4].
There are several commercial kits available for cell-based assays of GPCR activity. For example, Promega offers the GloSensor cAMP Assay System, which allows for real-time monitoring of cAMP levels in live cells [5]. Similarly, the FLIPR Calcium Assay from Molecular Devices enables high-throughput screening of GPCR activity through the measurement of calcium mobilization [6].
