The CXC chemokine receptor 4 (CXCR4) is involved in chemotaxis and serves as a coreceptor for T-tropic HIV-1 viral entry, thus making this receptor an attractive drug target. Recently, crystal structures of CXCR4 were reported as complexes with the small molecule IT1t and the CVX15 peptide. Follow-up efforts to model different antagonists into the small molecule CXCR4:IT1t crystal structure did not generate poses consistent with either the X-ray crystal structure or site-directed mutagenesis (SDM). Here, we compare the binding pockets of the two CXCR4 crystal structures, revealing differences in helices IV, V, VI, and VII, with major differences for the His203 residue buried in the binding pocket. The small molecule antagonist AMD11070 was docked into both CXCR4 crystal structures. An AMD11070 pose identified from the CXCR4:CVX15 model presented interactions with Asp171, Glu288, Trp94, and Asp97, consistent with published SDM data, thus suggesting it is the bioactive pose. A CXCR4 receptor model was optimized around this pose of AMD11070, and the resulting model correlated HIV-1 inhibition with MM-GBSA docking scores for a congeneric AMD11070-like series. Subsequent NAMFIS NMR results successfully linked the proposed binding pose to an independent experimental structure. These results strongly suggest that not all small molecules will bind to CXCR4 in a similar manner as IT1t. Instead, the CXCR4:CVX15 crystal structure may provide a binding locus for small organic molecules that is more suitable than the secondary IT1t site. This work is expected to provide modeling insights useful for future CXCR4 antagonist and X4-tropic HIV-1 based drug design efforts.

The interaction of CXCR4 with CXCL12 (SDF-1) plays a critical role in cancer metastasis by facilitating the homing of tumor cells to metastatic sites. Based on our previously published work on CXCR4 antagonists, we have synthesized a series of aryl sulfonamides that inhibit the CXCR4/CXCL12 interaction. Analogue bioactivities were assessed with binding affinity and Matrigel invasion assays. Computer modeling was employed to evaluate a selection of the new analogues docked into the CXCR4 X-ray structure and to rationalize discrepancies between the affinity and Matrigel in vitro assays. A lead compound displays nanomolar potency in the binding affinity assay (IC₅₀=8.0 nM) and the Matrigel invasion assay (100 % blockade of invasion at 10 nM). These data demonstrate that benzenesulfonamides are a unique class of CXCR4 inhibitors with high potency.

A novel and efficient synthetic strategy to access unique C-2 substituted steroid analogues 3 and 4 is described. The unusual C-2 aryl ether analogues 3 were shown to act as virtual antagonists of LRH-1 and were prepared as single diastereoisomers, employing a fifteen-step sequence from pregnenolone (9). The key steps include the stereoconvergent nucleophilic displacement of an epimeric mixture of 3-keto 2-bromo steroids, chemoselective carbonylation of an enol triflate and conversion of a thiopyridyl ester into an aryl ketone. The related C-2 benzyl analogues 4 were prepared in a similar manner.

A series of conformationally restrained epothilone analogues with a short bridge between the methyl groups at C6 and C8 was designed to mimic the binding pose assigned to our recently reported EpoA–microtubule binding model. A versatile synthetic route to these bridged epothilone analogues has been successfully devised and implemented. Biological evaluation of the compounds against A2780 human ovarian cancer and PC3 prostate cancer cell lines suggested that the introduction of a bridge between C6–C8 reduced potency by 25–1000 fold in comparison with natural epothilone D. Tubulin assembly measurements indicate these bridged epothilone analogues to be mildly active, but without significant microtubule stabilization capacity. Molecular mechanics and DFT energy evaluations suggest the mild activity of the bridged epo-analogues may be due to internal conformational strain.

Cyclostreptin (CS) is a recently discovered natural product with cytotoxic activity caused by microtubule stabilization. It is the only known microtubule-stabilizing agent (MSA) that covalently binds to tubulin. It also exhibits the fast-binding kinetics seen for other MSAs. Through careful peptide digestion and mass spectrometry analysis, Buey et al. found that two amino acids are labeled by CS: Asn228, near the known taxane-binding site, and Thr220, in the type I microtubule pore. This led Buey et al. to propose Thr220 resides at the site previously predicted to be a way station or low-affinity site. By using molecular dynamics simulations and structural considerations of the microtubule pore and tubulin dimer, we conclude that postulation of a low-affinity site is unnecessary to explain the available experimental data. An alternative explanation views the microtubule pore as a structural entity that presents a substantial kinetic barrier to ligand passage to the known taxane-binding site—an entry point to the microtubule lumen that becomes completely blocked if cyclostreptin is bound at Thr220. Simulations of the free dimer also suggest a common mechanism of microtubule stabilization for taxane site MSAs through their conformational effect on the M-loop. Such an effect explains the low tubulin polymerization caused by cyclostreptin in vitro despite its covalent attachment.