There is a significant emphasis in chemical biology and medicinal chemistry on targeting enzymes. An enzymatic target has the advantage of being poised for small molecule recognition, and such recognition events are likely to modulate enzymatic activity. Small molecules like acetylsalicylic acid (Aspirin), amethopterin (Methotrexate), pregabalin (Lyrica), and sildenafil citrate (Viagra) target physiologically important enzymes to elicit therapeutic effects. Academic chemists have therefore focused attention on such targets in their scholarly efforts. While this has yielded interesting and important results, an alternative strategy would be to explore non-enzymatic targets of physiologic importance. With this in mind, the work herein defines the protein encoded by the Sonic Hedgehog (Shh) gene as a medically relevant target that has no known enzymatic activity. With a small-molecule microarray (SMM) screening approach, a synthetic, macrocyclic small molecule was discovered that binds to the Shh protein. Robotnikinin was synthesized and characterized as a more potent Shh-binding molecule that elicited inhibitory effects on the Shh signaling pathway in vitro and in in vivo mouse model studies.