Stephen L. Briggs

A Transforming Mutation in the Pleckstrin Homology Domain of Akt1 in Cancer.

Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

Helix-stabilized cyclic peptides as selective inhibitors of steroid receptor–coactivator interactions

The interaction between nuclear receptors and coactivators provides an arena for testing whether protein–protein interactions may be inhibited by small molecule drug candidates. We provide evidence that a short cyclic peptide, containing a copy of the LXXLL nuclear receptor box pentapeptide, binds tightly and selectively to estrogen receptor α. Furthermore, as shown by x-ray analysis, the disulfide-bridged nonapeptide, nonhelical in aqueous solutions, is able to adopt a quasihelical conformer while binding to the groove created by ligand attachment to estrogen receptor α. An i, i+3 linked analog, H-Lys-cyclo(d-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln–NH2 (peptidomimetic estrogen receptor modulator 1), binds with a Ki of 25 nM, significantly better than an i, i+4 bridged cyclic amide, as predicted by molecular modeling design criteria. The induction of helical character, effective binding, and receptor selectivity exhibited by this peptide analog provide strong support for this strategy. The stabilization of minimalist surface motifs may prove useful for the control of other macromolecular assemblies, especially when an amphiphilic helix is crucial for the strong binding interaction between two proteins.

Isolation of three separate anaphylatoxins from complement-activated human serum

Recent methodologies used in preparing anaphylatoxins from complement-activated serum are described. Activation of the alternative pathway generates C3a and C5a; however, activation of the classical pathway is required to generate the anaphylatoxin from C4. This article describes an activation scheme that simultaneously generates all three of the anaphylatoxins (e.g., C3a, C4a and C5a) in human serum and outlines a procedure for isolating each as homogeneous products. Purification of intact anaphylatoxins directly from complement-activated serum takes place only if an exopeptidase in serum, known as carboxypeptidase N (SCPN), is properly inhibited. A new series of mercapto derivatives of arginine analogs are introduced as potent and effective inhibitors of SCPN. These inhibitors permit normal complement activation but prevent degradation of the released activation fragments C3a, C4a or C5a. The SCPN inhibitor previously used was 6-aminohexanoic acid (EACA), but it required a 1 M concentration for effective inhibition, the substituted mercapto-guanido compounds prove to be effective in the mM range.

In vivo efficacy of monoclonal antibody-drug conjugates of three different subisotypes which bind the human tumor-associated antigen defined by the KS1/4 monoclonal antibody.

SummaryA panel of three hybridomas has been isolated each of which secretes a single species of monoclonal antibody (MoAb) directed against the KS1/4 tumor-associated antigen originally described by Varki et al. (Cancer Res 44:681, 1984). These MoAbs were designated L1-(IgG2b), L2-(IgG1), and L4-(IgG2a) KS. Binding specificity, immunoprecipitation, and competitive binding analyses indicated that these MoAbs each recognize the same epitope of the KS1/4 antigen. The immunoprecipitation studies indicated that the MoAbs recognized a major antigenic component of 42 kDa and a minor component of 35 kDa. The L-KS antibodies were evaluated as MoAb-drug conjugates against a variety of human tumor targets grown in vivo as nude mouse xenografts. The MoAb-drug conjugates were constructed using protein-A-purified MoAbs conjugated to 4-desacetyl-vinblastine-3-carboxhydrazide. Efficacy was determined using various dosing protocols on 2–14 day established tumors of lung, pharynx, colon, and skin origin. Control experiments included the use of dual-flank antigen-positive and negative tumors, free MoAbs, free drug, and mixtures of MoAbs and drug. These studies indicated that significant tumor growth supression and actual tumor regression could be achieved by the MoAb — vinca conjugates and that this activity was antigen-mediated. The drug conjugates were more efficacious than free drug or free MoAbs administered either singly or in combination with each other.

Dibasic benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors: 2. Sidechain optimization and demonstration of in vivo efficacy.

Potent, subnanomolar thrombin inhibitors 4, 5, and 6 are developed through side chain optimization of novel, benzo[b]thiophene-based small organic entities 2 and 3 and through SAR additivity studies of the new structural elements identified. X-ray crystallographic studies of 4b-thrombin complex revealed a hydrophobic and an electrostatic interaction of these new elements with thrombin at the S2 and S3 binding sites. In vitro and in vivo pharmacological studies showed that 4, 5, and 6 are potent anticoagulants in human plasma with demonstrated antithrombotic efficacy in a rat model of thrombosis.