Identification and Optimization of Thienopyridine Carboxamides as Inhibitors of HIV Regulatory Complexes.

Viral regulatory complexes perform critical functions during virus replication and are important targets for therapeutic intervention. In HIV, the Tat and Rev proteins form complexes with multiple viral and cellular factors to direct transcription and export of the viral RNA. These complexes are composed of many proteins and are dynamic, making them difficult to fully recapitulate Therefore, we developed a cell-based reporter assay to monitor the assembly of viral complexes for inhibitor screening.

Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation.

Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis.

Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis.

Unlabelled: Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic.

Simple one-pot synthesis of disulfide fragments for use in disulfide-exchange screening.

Disulfide exchange screening is a method for evaluating the binding of small molecule fragments to proteins that have at least one accessible cysteine. While operationally simple, it does require a large library of small fragment molecules bearing disulfide-containing side chains. These specialized fragments are not available commercially and this has limited the adoption of the method.

Turning a protein kinase on or off from a single allosteric site via disulfide trapping.

There is significant interest in identifying and characterizing allosteric sites in enzymes such as protein kinases both for understanding allosteric mechanisms as well as for drug discovery. Here, we apply a site-directed technology, disulfide trapping, to interrogate structurally and functionally how an allosteric site on the Ser/Thr kinase, 3-phosphoinositide-dependent kinase 1 (PDK1)--the PDK1-interacting-fragment (PIF) pocket--is engaged by an activating peptide motif on downstream substrate kinases (PIFtides) and by small molecule fragments. By monitoring pairwise disulfide conjugation between PIFtide and PDK1 cysteine mutants, we defined the PIFtide binding orientation in the PIF pocket of PDK1 and assessed subtle relationships between PIFtide positioning and kinase activation.

Structure-activity relationships of 9-substituted-9-dihydroerythromycin-based motilin agonists: optimizing for potency and safety.

A series of 9-dihydro-9-acetamido-N-desmethyl-N-isopropyl erythromycin A analogues and related derivatives was generated as motilin agonists. The compounds were optimized for potency while showing both minimal antibacterial activity and hERG inhibition. As the substituent on the amide was increased in lipophilicity the potency and hERG inhibition increased, while polar groups lowered potency, without significantly impacting hERG inhibition.

A series of 23,24-dihydrodiscodermolide analogues with simplified lactone regions.

A collection of seven new 23,24-dihydrodiscodermolide analogues have been synthesized with modifications to the lactone ring, some of which show antiproliferative activities similar to discodermolide.

Toward understanding how the lactone moiety of discodermolide affects activity.

A series of simplified discodermolide analogues have been designed and synthesized in an attempt to understand the role of the lactone ring. These synthetic efforts have led to an unsubstituted butyrolactone 9 being generated, which shows improved activity over the natural product.

Design, synthesis, and evaluation of analogues of (+)-14-normethyldiscodermolide.

[Structure: see text] The design, syntheses, and biological evaluation of nine totally synthetic analogues of the microtubule-stabilizing agent (+)-14-normethyldiscodermolide (2) are reported. Simplification at the C(21)-C(24) terminal diene and at the C(1)-C(5) lactone moieties reveals significant structure-activity relationships.

Design, synthesis, and evaluation of carbamate-substituted analogues of (+)-discodermolide.

[Structure: see text] The design, syntheses, and biological evaluation of 22 totally synthetic analogues of the potent microtubule-stabilizing agent (+)-discodermolide (1) have been achieved. Structure-activity relationships of the C(19) carbamate were defined, exploiting two synthetically simplified scaffolds, as well as the parent (+)-discodermolide framework.

Design, synthesis and cytotoxicity of 7-deoxy aryl discodermolide analogues.

A series of 7-deoxy discodermolide analogues in which the lactone fragment 'C' was replaced by aryl substituents were designed, synthesized, and evaluated for cytotoxicity.