James M. Chen

Design, synthesis, and anti-HIV activity of 4′-modified carbocyclic nucleoside phosphonate reverse transcriptase inhibitors

A diphosphate of a novel cyclopentyl based nucleoside phosphonate with potent inhibition of HIV reverse transcriptase (RT) (20, IC(50)=0.13 microM) has been discovered. In cell culture the parent phosphonate diacid 9 demonstrated antiviral activity EC(50)=16 microM, within two-fold of GS-9148, a prodrug of which is currently under clinical investigation, and within 5-fold of tenofovir (PMPA). In vitro cellular metabolism studies using 9 confirmed that the active diphosphate metabolite is produced albeit at a lower efficiency relative to GS-9148.

Benzodiazepine inhibitors of the MMPs and TACE.

A series of benzodiazepine inhibitors of the MMPs and TACE has been developed. These compounds display an interesting selectivity profile and should be useful tools for exploring the biological relevance of such selectivity.

Conformational effects in the p53 protein of mutations induced during chemical carcinogenesis: Molecular dynamic and immunologic analyses

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen that induces the rare sentinel neoplasm angiosarcoma of the liver, has been associated with specific A → T transversions at the first base of codons 249 and 255 of the p53 gene. These mutations result in an Arg→Trp amino acid substitution at residue 249 and an Ile→Phe amino acid substitution at residue 255 in a highly conserved region in the DNA-binding core domain of the p53 protein. To determine the effects of these substitutions on the three-dimensional structure of the p53 protein, we have performed molecular dynamics calculations on this core domain of the wild-type and the Trp-249 and Phe-255 mutants to compute the average structures of each of the three forms. Comparisons of the computed average structures show that both mutants differ substantially from the wild-type structure in certain common, discrete regions. One of these regions (residues 204–217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure but accessible in both mutant structures. In order to confirm this conformational shift, tumor tissue and serum from vinyl chloride-exposed individuals with angiosarcomas of the liver were examined by immunohistochemistry and enzyme-linked immunosorbent assay. Individuals with tumors that contained the p53 mutations were found to have detectable mutant p53 protein in their tumor tissue and serum, whereas individuals with tumors without mutations and normal controls did not.

N1-Heterocyclic pyrimidinediones as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A series of N1-heterocyclic pyrimidinediones were extensively evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Inhibitor 1 is active against NNRTI-resistant viruses including RT mutant K103N. The co-crystal structure of inhibitor 1 with HIV-1 RT revealed that H-bonds are formed with K101 and K103. Efforts to improve the suboptimal pharmacokinetic profile of 1 resulted in the discovery of compound 13, which represents the lead compound in this series with improved pharmacokinetics and similar potency as inhibitor 1.

Synthesis And Anti-Hiv Activity Of Cyclic Pyrimidine Phosphonomethoxy Nucleosides And Their Prodrugs: A Comparison Of Phosphonates And Corresponding Nucleosides

Cyclic phosphonomethoxy pyrimidine nucleosides that are bioisosteres of the monophosphate metabolites of HIV reverse transcriptase (RT) inhibitors AZT, d4T, and ddC have been synthesized. The RT inhibitory activities of the phosphonates were reduced for both dideoxy (dd) and dideoxydidehydro (d4) analogs compared to the nucleosides. Bis-isopropyloxymethylcarbonyl (BisPOC) prodrugs were prepared on selected compounds and provided > 150-fold improvements in antiviral activity.

Inhibition of Oncogenic and Activated Wild-Type ras-p21 Protein-Induced Oocyte Maturation by Peptides from the ras-Binding Domain of the raf-p74 Protein, Identified from Molecular Dynamics Calculations

In the preceding paper we found from molecular dynamics calculations that the structure of the ras-binding domain (RBD) of raf changes predominantly in three regions depending upon whether it binds to ras-p21 protein or to its inhibitor protein, rap-1A. These three regions of the RBD involve residues from the protein–protein interaction interface, e.g., between residues 60 and 72, residues 97–110, and 111–121. Since the rap-1A–RBD complex is inactive, these three regions are implicated in ras-p21-induced activation of raf. We have therefore co-microinjected peptides corresponding to these three regions, 62–76, 97–110, and 111–121, into oocytes with oncogenic p21 and microinjected them into oocytes incubated in in insulin, which activates normal p2l. All three peptides, but not a control peptide, strongly inhibit both oncogenic p21- and insulin-induced oocyte maturation. These findings corroborate our conclusions from the theoretical results that these three regions constitute raf effector domains. Since the 97–110 peptide is the strongest inhibitor of oncogenic p21, while the 111–121 peptide is the strongest inhibitor of insulin-induced oocyte maturation, the possibility exists that oncogenic and activated normal p21 proteins interact differently with the RBD of raf.

Comparison of the Computed Three-Dimensional Structures of Oncogenic Forms (Bound to GDP) of the ras-Gene-Encoded p21 Protein with the Structure of the Normal (Non-Transforming) Wild-Type Protein

Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.

Structural effects of the binding of GTP to the wild-type and oncogenic forms of the ras-gene-encoded p21 proteins.

Molecular dynamics calculations have been performed to determine the average structures ofras-gene-encoded p21 proteins bound to GTP, i.e., the normal (wild-type) protein and two oncogenic forms of this protein, the Val 12- and Leu 61-p21 proteins. We find that the average structures for all of these proteins exhibit low coordinate fluctuations (which are highest for the normal protein), indicating convergence to specific structures. From previous dynamics calculations of the average structures of these proteins bound to GDP, major regional differences were found among these proteins (Monacoet al. (1995),J. Protein Chem., in press). We now find that the average structures of the oncogenic proteins are more similar to one another when the proteins are bound to GTP than when they are bound to GDP (Monacoet al. (1995),J. Protein Chem., in press). However, they still differ in structureat specific amino acid residues rather than in whole regions, in contradistinction to the results found for the p21-GDP complexes. Two exceptions are the regions 25–32, in anα-helical region, and 97–110. The two oncogenic (Val 12- and Leu 61-) proteins have similar structures which differ significantly in the region of residues 97–110. This region has recently been identified as being critical in the interaction of p21 with kinase target proteins. The differences in structure between the oncogenic proteins suggest the existence of more than one oncogenic form of the p21 protein that can activate different signaling pathways.

N1-Alkyl pyrimidinediones as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A series of N1-alkyl pyrimidinediones were designed, synthesized and evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Our efforts identified compound 10b, which represents the lead compound in this series with pharmacokinetics and antiviral potency that may support once-daily dosing.

Mechanism of Resistance to GS-9148 Conferred by the Q151L Mutation in HIV-1 Reverse Transcriptase

ABSTRACT GS-9148 is an investigational phosphonate nucleotide analogue inhibitor of reverse transcriptase (RT) (NtRTI) of human immunodeficiency virus type 1 (HIV-1). This compound is an adenosine derivative with a 2′,3′-dihydrofuran ring structure that contains a 2′-fluoro group. The resistance profile of GS-9148 is unique in that the inhibitor can select for the very rare Q151L mutation in HIV-1 RT as a pathway to resistance. Q151L is not stably selected by any of the approved nucleoside or nucleotide analogues; however, it may be a transient intermediate that leads to the related Q151M mutation, which confers resistance to multiple compounds that belong to this class of RT inhibitors. Here, we employed pre-steady-state kinetics to study the impact of Q151L on substrate and inhibitor binding and the catalytic rate of incorporation. Most importantly, we found that the Q151L mutant is unable to incorporate GS-9148 under single-turnover conditions. Interference experiments showed that the presence of GS-9148–diphosphate, i.e., the active form of the inhibitor, does not reduce the efficiency of incorporation for the natural counterpart. We therefore conclude that Q151L severely compromises binding of GS-9148–diphosphate to RT. This effect is highly specific, since we also demonstrate that another NtRTI, tenofovir, is incorporated with selectivity similar to that seen with wild-type RT. Incorporation assays with other related compounds and models based on the RT/DNA/GS-9148–diphosphate crystal structure suggest that the 2′-fluoro group of GS-9148 may cause steric hindrance with the side chain of the Q151L mutant.