Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX

Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regarding the expression and crystallization of the enzyme. Currently, only one baculovirus-produced CA IX structure in complex with a nonspecific CA inhibitor, acetazolamide, is available in Protein Data Bank. We have developed an efficient system for the production of the catalytic domain of CA IX in methylotrophic yeast Pichia pastoris. The produced protein can be easily crystallized in the presence of inhibitors, as we have demonstrated for several 2-thiophene-sulfonamide compounds. We have also observed significant differences in the binding mode of chemically identical compounds to CA IX and CA II, which can be further exploited in the design of CA IX-specific inhibitor

Targeting Carnitine Biosynthesis: Discovery of New Inhibitors against γ‑Butyrobetaine Hydroxylase

γ-Butyrobetaine hydroxylase (BBOX) catalyzes the conversion of gamma butyrobetaine (GBB) to l-carnitine, which is involved in the generation of metabolic energy from long-chain fatty acids. BBOX inhibitor 3-(1,1,1-trimethylhydrazin-1-ium-2-yl)­propanoate (mildronate), which is an approved, clinically used cardioprotective drug, is a relatively poor BBOX inhibitor and requires high daily doses. In this paper we describe the design, synthesis, and properties of 51 compounds, which include both GBB and mildronate analogues. We have discovered novel BBOX inhibitors with improved IC₅₀ values; the best examples are in the nanomolar range and about 2 orders of magnitude better when compared to mildronate. For six inhibitors, crystal structures in complex with BBOX have been solved to explain their activities and pave the way for further inhibitor design

Plasmepsin Inhibitory Activity and Structure-Guided Optimization of a Potent Hydroxyethylamine-Based Antimalarial Hit

Antimalarial hit <b>1</b><i><b>SR</b></i> (TCMDC-134674) identified in a GlaxoSmithKline cell based screening campaign was evaluated for inhibitory activity against the digestive vacuole plasmepsins (Plm I, II, and IV). It was found to be a potent Plm IV inhibitor with no selectivity over Cathepsin D. A cocrystal structure of <b>1</b><i><b>SR</b></i> bound to Plm II was solved, providing structural insight for the design of more potent and selective analogues. Structure-guided optimization led to the identification of structurally simplified analogues <b>17</b> and <b>18</b> as low nanomolar inhibitors of both, plasmepsin Plm IV activity and <i>P. falciparum</i> growth in erythrocytes

Fragment-Based Discovery of 2‑Aminoquinazolin-4(3<i>H</i>)‑ones As Novel Class Nonpeptidomimetic Inhibitors of the Plasmepsins I, II, and IV

2-Aminoquinazolin-4­(3<i>H</i>)-ones were identified as a novel class of malaria digestive vacuole plasmepsin inhibitors by using NMR-based fragment screening against Plm II. Initial fragment hit optimization led to a submicromolar inhibitor, which was cocrystallized with Plm II to produce an X-ray structure of the complex. The structure showed that 2-aminoquinazolin-4­(3<i>H</i>)-ones bind to the open flap conformation of the enzyme and provided clues to target the flap pocket. Further improvement in potency was achieved via introduction of hydrophobic substituents occupying the flap pocket. Most of the 2-aminoquinazolin-4­(3<i>H</i>)-one based inhibitors show a similar activity against digestive Plms I, II, and IV and >10-fold selectivity versus CatD, although varying the flap pocket substituent led to one Plm IV selective inhibitor. In cell-based assays, the compounds show growth inhibition of Plasmodium falciparum 3D7 with IC₅₀ ∼ 1 μM. Together, these results suggest 2-aminoquinazolin-4­(3<i>H</i>)-ones as perspective leads for future development of an antimalarial agent

<i>N</i>‑Acylbenzenesulfonamide Dihydro-1,3,4-oxadiazole Hybrids: Seeking Selectivity toward Carbonic Anhydrase Isoforms

A series of <i>N</i>-acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids (<b>EMAC8000a–m</b>) was designed and synthesized with the aim to target tumor associated carbonic anhydrase (hCA) isoforms IX and XII. Most of the compounds were selective inhibitors of the tumor associated hCA XII. Moreover, resolution of <b>EMAC8000d</b> racemic mixture led to the isolation of the levorotatory eutomer exhibiting an increase of hCA XII inhibition potency and selectivity with respect to hCA II. Computational studies corroborated these data. Overall our data indicate that both substitution pattern and stereochemistry of dihydro-1,3,4-oxadiazole could be considered as key factors to determine activity and selectivity toward hCA isozymes. These results can provide further indication for the design and optimization of selective hCA inhibitors