Joseph Markowitz

Crystal structure of phosphodiesterase 4D and inhibitor complex1

Cyclic nucleotide phosphodiesterases (PDEs) regulate physiological processes by degrading intracellular second messengers, adenosine‐3′,5′‐cyclic phosphate or guanosine‐3′,5′‐cyclic phosphate. The first crystal structure of PDE4D catalytic domain and a bound inhibitor, zardaverine, was determined. Zardaverine binds to a highly conserved pocket that includes the catalytic metal binding site. Zardaverine fills only a portion of the active site pocket. More selective PDE4 inhibitors including rolipram, cilomilast and roflumilast have additional functional groups that can utilize the remaining empty space for increased binding energy and selectivity. In the crystal structure, the catalytic domain of PDE4D possesses an extensive dimerization interface containing residues that are highly conserved in PDE1, 3, 4, 8 and 9. Mutations of R358D or D322R among these interface residues prohibit dimerization of the PDE4D catalytic domain in solution.

Design of Inhibitors for S100B

S100B interacts with the p53 protein in a calcium-dependent manner and down-regulates its function as a tumor suppressor. Therefore, inhibiting the S100B-p53 interaction represents a new approach for restoring functional wild-type p53 in cancers with elevated S100B such as found in malignant melanoma. A discussion of the biological rational for targeting S100B and a description of methodologies relevant to the discovery of compounds that inhibit S100B-p53 binding, including computational techniques, structural biology techniques, and cellular assays, is presented.

A Search for Inhibitors of S100B, a Member of the S100 Family of Calcium-Binding Proteins

Typically, malignant melanoma has wild-type p53, and yet this cancer proliferates. S100B, which binds p53 and is up-regulated in melanoma, down-regulates wild-type p53 tumor suppressor function. Inhibitors of the S100B-p53 interaction were identified using computer aided drug design (CADD) combined with NMR methodologies and represent potentially new chemotherapeutics for melanoma.

Simultaneous Inhibition of Key Growth Pathways in Melanoma Cells and Tumor Regression by a Designed Bidentate Constrained Helical Peptide

Protein–protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium‐regulated protein, plays a crucial role in the proliferation of melanoma cells through protein–protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight‐binding peptide, TRTK‐12. The helical conformation of the peptide was constrained by the substitution of α‐amino isobutyric acid—an amino acid having high helical propensity—in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell‐penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild‐type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein–protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein–protein interactions for de novo drug development.