Bimalendu Dasmahapatra

SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1–2 μm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

Structure of Sch 68631: A new hepatitis C virus proteinase inhibitor from Streptomyces sp.

Abstract A novel hepatitis C virus (HCV) proteinase inhibitor, Sch 68631 ( 1 ), was isolated from the fermentation culture broth of Streptomyces sp. The structure of 1 was elucidated by analyses of spectroscopic data and shown to be a new member of the phenanthrenequinone family of compounds.

The synthesis of novel HIV-protease inhibitors

The syntheses, enzyme inhibition and antiviral activity of potent HIV-protease inhibitors containing novel beta-hydroxy ether and thioethers based on the transition state mimetic concept are discussed.

Cell-free expression of the coxsackievirus 3C protease using the translational initiation signal of an insect virus RNA and its characterization.

We have expressed the 3C protease of coxsackievirus B3 (CVB3) in a cell-free system. This expression system employs the translational initiation signal of an insect virus RNA, black beetle virus (BBV) RNA 1, to direct CVB3-specific protein synthesis. Using this expression system, we demonstrate that a biologically active 3C protease is synthesized which possesses both cis and trans processing capabilities. This in vitro-synthesized 3C protease is analogous to the native 3C, which was obtained from cytoplasmic extracts of CVB3-infected HeLa cells, in all biological parameters that were evaluated. In addition, antibody prepared against the 3C protease purified from extracts of CVB3-infected HeLa cells cross-reacts with the 3C protease produced in this cell-free system. Using the translational initiation signal from BBV RNA 1, we also have expressed the CVB3 capsid precursor and part of the P2 region in vitro, and have shown that the capsid precursor is cleaved between 1C (VP3) and 1D (VP1) by the proteolytic activity of in vitro-synthesized 3C in trans. Evidence also is presented to implicate the 2A protein of CVB3 as having proteolytic function.

Expression of functional beta-galactosidase containing the coxsackievirus 3C protease as an internal fusion

Alpha complementation of beta-galactosidase (beta gal) is intracistronic and requires interaction between the alpha donor region (residues 3-41) and alpha acceptor fragment (produced by M15). We have constructed two plasmids which direct the synthesis of hybrid beta gal: coxsackievirus proteins in Escherichia coli. One plasmid, pBD1045, encodes an enzymatically active 3C protease of coxsackievirus B3 fused between the amino-terminal 79 amino acids of beta gal (containing the alpha donor region) and amino acids 80 to 1023 (alpha acceptor region). A second plasmid, pBD1043 encodes an inactive 3C protease and results in a fusion of 260 coxsackievirus amino acids between residues 79 and 80 of the beta gal monomer. Both hybrid proteins expressed by these constructs have beta-galactosidase activity regardless of whether the viral protease (183 amino acids) is autocatalytically cleaved out of the chimeric protein (pBD1045) or remains as part of a fusion protein (pBD1043). The implications of these results for structural flexibility of the complemented beta-galactosidase enzyme are discussed.

Novel HIV- protease inhibitors containing β-hydroxyether and -thioether dipeptide isostere surrogates: modification of the P3 ligand

Studies involving modifications to the P3 position of previously described HIV-protease inhibitors containing beta-hydroxyether and thioether dipeptide isostere replacements led to the discovery of pseudopeptides 8o and 8p with improved antiviral activities.

Inactivation of the human cytomegalovirus protease by diisopropylfluorophosphate

Publisher Summary Cytomegalovirus (CMV) protease is a serine protease, and labeling with diisopropylfluorophosphate (DFP) has identified Ser132 as the active site serine. The structure of the CMV protease containing the diisopropylphosphorylserine at residue 132 (DIP-CMV protease) is likely to resemble that of the tetrahedral transition-state intermediate. As the structure of the DFP-treated serine proteases resembles that of the tetrahedral transition-state intermediate, and the inactivated enzyme would not be susceptible to autoproteolysis, production of DIP-CMV protease would be useful for structure based drug design. The concentrations of DFP sufficient to yield stoichiometric incorporation of inhibitor at the active site of CMV protease, also resulted in substantial incorporation of DIP at a second site or sites. This heterogeneous incorporation would preclude crystallographic studies. For this reason, this chapter has attempted to optimize the conditions for inactivation of CMV protease with DFP, to produce pure DIP-CMV protease with minimum second site incorporation. Initial studies indicated that there was no loss of protease activity in the control samples, even when incubated for 23 hours at 22°C. A 3.8 hour incubation was sufficient to completely inactivate 180 μM protease in the presence of 4.3 mM DFP, while a 2.4 fold excess of the inhibitor inactivated only 50% of the enzyme. This indicates that CMV protease is less reactive with DFP than trypsin or chymotrypsin, and requires an order of magnitude excess of the organophosphate to achieve complete inactivation.