Discovery of MK-4688: an Efficient Inhibitor of the HDM2-p53 Protein-Protein Interaction.

Identification of low-dose, low-molecular-weight, drug-like inhibitors of protein-protein interactions (PPIs) is a challenging area of research. Despite the challenges, the therapeutic potential of PPI inhibition has driven significant efforts toward this goal. Adding to recent success in this area, we describe herein our efforts to optimize a novel purine carboxylic acid-derived inhibitor of the HDM2-p53 PPI into a series of low-projected dose inhibitors with overall favorable pharmacokinetic and physical properties. Ultimately, a strategy focused on leveraging known binding hot spots coupled with biostructural information to guide the design of conformationally constrained analogs and a focus on efficiency metrics led to the discovery of MK-4688 (compound 56), a highly potent, selective, and low-molecular-weight inhibitor suitable for clinical investigation.

Publisher Correction: A mass spectrometry-based proteome map of drug action in lung cancer cell lines.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A mass spectrometry-based proteome map of drug action in lung cancer cell lines.

Mass spectrometry-based discovery proteomics is an essential tool for the proximal readout of cellular drug action. Here, we apply a robust proteomic workflow to rapidly profile the proteomes of five lung cancer cell lines in response to more than 50 drugs. Integration of millions of quantitative protein-drug associations substantially improved the mechanism of action (MoA) deconvolution of single compounds. For example, MoA specificity increased after removal of proteins that frequently responded to drugs and the aggregation of proteome changes across cell lines resolved compound effects on proteostasis. We leveraged these findings to demonstrate efficient target identification of chemical protein degraders. Aggregating drug response across cell lines also revealed that one-quarter of compounds modulated the abundance of one of their known protein targets. Finally, the proteomic data led us to discover that inhibition of mitochondrial function is an off-target mechanism of the MAP2K1/2 inhibitor PD184352 and that the ALK inhibitor ceritinib modulates autophagy.

Identification of G-Quadruplex-Binding Inhibitors of Myc Expression through Affinity Selection-Mass Spectrometry.

The Myc oncogene is overexpressed in many cancers, yet targeting it for cancer therapy has remained elusive. One strategy for inhibition of Myc expression is through stabilization of the G-quadruplex (G4), a G-rich DNA secondary structure found within the Myc promoter; stabilization of G4s has been shown to halt transcription of downstream gene products. Here we used the Automated Ligand Identification System (ALIS), an affinity selection-mass spectrometry method, to identify compounds that bind to the Myc G4 out of a pool of compounds that had previously been shown to inhibit Myc expression in a reporter screen. Using an ALIS-based screen, we identified hits that bound to the Myc G4, a small subset of which bound preferentially relative to G4s from the promoters of five other genes. To determine functionality and specificity of the Myc G4-binding compounds in cell-based assays, we compared inhibition of Myc expression in cells with and without Myc G4 regulation. Several compounds inhibited Myc expression only in the Myc G4-containing line, and one compound was verified to function through Myc G4 binding. Our study demonstrates that ALIS can be used to identify selective nucleic acid-binding compounds from phenotypic screen hits, increasing the pool of drug targets beyond proteins.

Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway.

The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.

Identification of an allosteric binding site for RORγt inhibition.

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3.

The development of resistance to anti-retroviral drugs targeted against HIV is an increasing clinical problem in the treatment of HIV-1-infected individuals. Many patients develop drug-resistant strains of the virus after treatment with inhibitor cocktails (HAART therapy), which include multiple protease inhibitors. Therefore, it is imperative that we understand the mechanisms by which the viral proteins, in particular HIV-1 protease, develop resistance. We have determined the three-dimensional structure of HIV-1 protease NL4-3 in complex with the potent protease inhibitor TL-3 at 2.0 A resolution. We have also obtained the crystal structures of three mutant forms of NL4-3 protease containing one (V82A), three (V82A, M46I, F53L) and six (V82A, M46I, F53L, V77I, L24I, L63P) point mutations in complex with TL-3. The three protease mutants arose sequentially under ex vivo selective pressure in the presence of TL-3, and exhibit fourfold, 11-fold, and 30-fold resistance to TL-3, respectively. This series of protease crystal structures offers insights into the biochemical and structural mechanisms by which the enzyme can overcome inhibition by TL-3 while recovering some of its native catalytic activity.

Is resistance futile?

A global effort has been undertaken to control human immunodeficiency virus (HIV) though the development of vaccines and pharmacologics. Current FDA approved pharmacological inhibitors target two of the three viral enzymes critical to replication and maturation of infectious viral particles: reverse transcriptase (RT) and protease (Pr). Although combination therapies targeting RT and Pr have significantly reduced AIDS related morbidity and mortality, resistance to individual inhibitors is a growing concern. Currently, there are six protease inhibitors in clinical use. These inhibitors target the active site of protease using peptidomimetic transition state analogs based on natural substrates. However, treatment failures arise as a lack of compliance due to HIV-inhibitor pharmacokinetics, toxicity, and tolerance. This allows reduced HIV-inhibitor pressure, increased viral replication, and the emergence of drug resistant mutations. Continued use of protease inhibitors in the face of incomplete viral suppression may result in HIV-1 escape mutants not only being resistant to the protease inhibitor used, but to all clinically available protease inhibitors. Thus, new broad-based protease inhibitors are needed to control the emerging multi-drug, cross-resistant HIV-1. Moreover, given the emergence of cross-resistant HIV-1, there is a need to target novel protease structural sites to reduce the risk of multi-drug cross-resistance. In this review, we discuss the resistance to protease inhibitors and the rationale for new strategies towards drug design for suppressing protease activity. We focus on the structure and function relationship and the influence that drug resistance mutants exert on the evolution of HIV-1 protease.