Fluorine NMR has recently gained high popularity in drug discovery as it allows efficient and sensitive screening of large numbers of ligands. However, the positive hits found in screening must subsequently be ranked according to their affinity in order to prioritize them for follow-up chemistry. Unfortunately, the primary read-out from the screening experiments, namely the increased relaxation rate upon binding, is not proportional to the affinity of the ligand, as it is polluted by effects such as exchange broadening. Here we present the method CSAR (Chemical Shift-anisotropy-based Affinity Ranking) for reliable ranking of fluorinated ligands by NMR, without the need of isotope labeled protein, titrations or setting up a reporter format. Our strategy is to produce relaxation data that is directly proportional to the binding affinity. This is achieved by removing all other contributions to relaxation as follows: (i) exchange effects are efficiently suppressed by using high power spin lock pulses, (ii) dipolar relaxation effects are approximately subtracted by measuring at two different magnetic fields and (iii) differences in chemical shift anisotropy are normalized using calculated values. A similar ranking can be obtained with the simplified approach FastCSAR that relies on a measurement of a single relaxation experiment at high field (preferably > 600 MHz). An affinity ranking obtained in this simple way will enable prioritizing ligands and thus improve the efficiency of fragment-based drug design.

氟NMR最近在药物研发中获得了很高的知名度，因为它可以高效，灵敏地筛选大量的配体。但是，在筛选中发现的阳性结果必须随后根据其亲和力进行排序，以便优先进行后续化学分析。不幸的是，筛选实验的主要读数，即结合后增加的弛豫速率，与配体的亲和力不成正比，因为它会被诸如交换增宽等作用污染。在这里，我们介绍了CSAR（基于化学位移各向异性的亲和力排名）方法，该方法可通过NMR对氟化配体进行可靠的排名，而无需同位素标记的蛋白质，滴定或建立报告基因形式。我们的策略是产生与结合亲和力成正比的弛豫数据。这可以通过如下消除所有其他对弛豫的贡献来实现：（i）通过使用高功率自旋锁定脉冲有效地抑制交换效应，（ii）通过在两个不同的磁场中进行测量来近似减去偶极弛豫效应，以及（iii）差异使用计算值对化学位移各向异性的值进行归一化。使用简化方法FastCSAR可以获得类似的排名，该方法依赖于在高场（最好> 600 MHz）下对单个弛豫实验的测量。以这种简单方式获得的亲和力排名将使配体优先排序，从而提高基于片段的药物设计的效率。（ii）通过在两个不同的磁场中进行测量来近似减去偶极弛豫效应，并且（iii）使用计算值将化学位移各向异性的差异归一化。使用简化方法FastCSAR可以获得类似的排名，该方法依赖于在高场（最好> 600 MHz）下对单个弛豫实验的测量。以这种简单方式获得的亲和力排名将使配体优先排序，从而提高基于片段的药物设计的效率。（ii）通过在两个不同的磁场中进行测量来近似减去偶极弛豫效应，并且（iii）使用计算值将化学位移各向异性的差异归一化。使用简化方法FastCSAR可以获得类似的排名，该方法依赖于在高场（最好> 600 MHz）下对单个弛豫实验的测量。以这种简单方式获得的亲和力排名将使配体优先排序，从而提高基于片段的药物设计的效率。600 MHz）。以这种简单方式获得的亲和力排名将使配体优先排序，从而提高基于片段的药物设计的效率。600 MHz）。以这种简单方式获得的亲和力排名将使配体优先排序，从而提高基于片段的药物设计的效率。