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We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in <jats:italic>profilin<\/jats:italic> (N154Y, K124N) and a third one in <jats:italic>actin-1<\/jats:italic> (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant <jats:italic>P<\/jats:italic>. <jats:italic>falciparum<\/jats:italic> proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1\/profilin interaction in <jats:italic>P<\/jats:italic>. <jats:italic>falciparum<\/jats:italic> and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.<\/jats:p>","DOI":"10.1371\/journal.pbio.3002066","type":"journal-article","created":{"date-parts":[[2023,4,13]],"date-time":"2023-04-13T18:29:56Z","timestamp":1681410596000},"page":"e3002066","update-policy":"https:\/\/doi.org\/10.1371\/journal.pbio.corrections_policy","source":"Crossref","is-referenced-by-count":23,"title":["Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1\/profilin dynamics"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9386-0648","authenticated-orcid":true,"given":"Madeline 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