PROTAC-mediated degradation of HIV-1 Nef efficiently restores cell-surface CD4 and MHC-I expression and blocks HIV-1 replication.

The HIV-1 Nef accessory factor enhances the viral life cycle in vivo, promotes immune escape of HIV-infected cells, and represents an attractive antiretroviral drug target. However, Nef lacks enzymatic activity and an active site, complicating traditional occupancy-based drug development. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on an existing hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the cereblon E3 ubiquitin ligase thalidomide-binding domain in vitro and triggered Nef degradation in a T cell expression system. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo.

PROTAC-mediated Degradation of HIV-1 Nef Efficiently Restores Cell-surface CD4 and MHC-I Expression and Blocks HIV-1 Replication.

The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo where it promotes immune escape of HIV-infected cells and viral persistence. While these features identify Nef as an attractive antiretroviral drug target, Nef lacks enzymatic activity and an active site, complicating development of occupancy-based drugs. Here we describe the development of proteolysis targeting chimeras (PROTACs) for the targeted degradation of Nef. Nef-binding compounds, based on a previously reported hydroxypyrazole core, were coupled to ligands for ubiquitin E3 ligases via flexible linkers. The resulting bivalent PROTACs induced formation of a ternary complex between Nef and the Cereblon E3 ubiquitin ligase, resulting in ubiquitylation of Nef and proteolytic degradation. Nef-directed PROTACs efficiently rescued Nef-mediated MHC-I and CD4 downregulation in T cells and suppressed HIV-1 replication in donor PBMCs. Targeted degradation of Nef is anticipated to reverse all HIV-1 Nef functions and may help restore adaptive immune responses against HIV-1 reservoir cells in vivo .

Antiretroviral Drug Discovery Targeting the HIV-1 Nef Virulence Factor.

While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs.

Tight-Binding Hydroxypyrazole HIV-1 Nef Inhibitors Suppress Viral Replication in Donor Mononuclear Cells and Reverse Nef-Mediated MHC-I Downregulation.

The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo and promotes immune escape of infected cells via downregulation of cell-surface MHC-I. Previously, we discovered small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in T cell lines. These compounds also restore cell-surface MHC-I expression in HIV-infected CD4 T cells from AIDS patients, enabling recognition and killing by autologous cytotoxic T lymphocytes (CTLs). In this study, we describe the synthesis and evaluation of a diverse set of analogs based on the original hydroxypyrazole Nef inhibitor core. All analogs were screened for the interaction with recombinant HIV-1 Nef by surface plasmon resonance (SPR) and for antiretroviral activity in TZM-bl reporter cells infected with HIV-1. Active analogs were ranked on the basis of an activity score that integrates three aspects of the SPR data (affinity, residence time, and extent of binding) with antiretroviral activity. The top scoring compounds bound tightly to Nef by SPR, with KD values in the low nM to pM range, and displayed very slow dissociation from their Nef target. These analogs also suppressed HIV-1 replication in donor peripheral blood mononuclear cells (PBMCs) with IC50 values in the 1-10 nM range without cytotoxicity, inhibited Nef-mediated IL-2-inducible tyrosine kinase (Itk) and hematopoietic cell kinase (Hck) activation, and rescued MHC-I downregulation in a Nef-transfected T cell line. The development of Nef inhibitors based on the structure-activity relationships defined here has promise as a new approach to antiretroviral therapy that includes a path to eradication of HIV-infected cells via the adaptive immune response.

Discovery of Potent Inhibitors of 11ß-Hydroxysteroid Dehydrogenase Type 1 Using a Novel Growth-Based Protocol of in Silico Screening and Optimization in CONTOUR.

With the continuous progress in ultralarge virtual libraries which are readily accessible, it is of great interest to explore this large chemical space for hit identification and lead optimization using reliable structure-based approaches. In this work, a novel growth-based screening protocol has been designed and implemented in the structure-based design platform CONTOUR. The protocol was used to screen the ZINC database in silico and optimize hits to discover 11ß-HSD1 inhibitors. In contrast to molecular docking, the virtual screening process makes significant improvements in computational efficiency without losing chemical equities through partitioning 1.8 million ZINC compounds into fragments, docking fragments to form key hydrogen bonds with anchor residues, reorganizing molecules into molecular fragment trees using matched fragments and common substructures, and then regrowing molecules with the help of developed intelligent growth features inside the protein binding site to find hits. The growth-base screening approach is validated by the high hit rate. A total of 50 compounds have been selected for testing; of these, 15 hits having diverse scaffolds are found to inhibit 11ß-HSD1 with IC50 values of less than 1 µM in a biochemical enzyme assay. The best hit which exhibits an enzyme IC50 of 33 nM is further developed to a novel series of bicyclic 11ß-HSD1 inhibitors with the best inhibition of enzyme IC50 of 3.1 nM. The final lead candidate exhibits IC50 values of 7.2 and 21 nM in enzyme and adipocyte assays, respectively, displayed greater than 1000-fold of selectivity over 11ß-HSD2 and two other related hydroxysteroid dehydrogenases, and can serve as good starting points for further optimization to develop clinical candidates.

Conformational control in structure-based drug design.

In structure-based drug design, the basic goal is to design molecules that fit complementarily to a given binding pocket. Since such computationally modeled molecules may not adopt the intended bound conformation outside the binding pocket, one challenge is to ensure that the designed ligands adopt similar low energy conformations both inside and outside of the binding pocket. Computational chemistry methods and conformational preferences of small molecules from PDB and Cambridge Structural Database (CSD) can be used to predict the bound structures of the designed molecules. Herein, we review applications of conformational control in structure-based drug design using selected examples from the recent medicinal chemistry literature. The main purpose is to highlight some intriguing conformational features that can be applied to other drug discovery programs.

Discovery of BI 135585, an in vivo efficacious oxazinanone-based 11ß hydroxysteroid dehydrogenase type 1 inhibitor.

A potent, in vivo efficacious 11ß hydroxysteroid dehydrogenase type 1 (11ß HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11ß HSD1 activity in human adipocytes with an IC50 of 4.3nM and in primary human adipose tissue with an IC80 of 53nM. Oral administration of 11j to cynomolgus monkey inhibited 11ß HSD1 activity in adipose tissue. Compound 11j exhibited >1000× selectivity over other hydroxysteroid dehydrogenases, displays desirable pharmacodynamic properties and entered human clinical trials in 2011.

Brain penetrant liver X receptor (LXR) modulators based on a 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole core.

Liver X receptor (LXR) agonists have been reported to lower brain amyloid beta (Aß) and thus to have potential for the treatment of Alzheimer's disease. Structure and property based design led to the discovery of a series of orally bioavailable, brain penetrant LXR agonists. Oral administration of compound 18 to rats resulted in significant upregulation of the expression of the LXR target gene ABCA1 in brain tissue, but no significant effect on Aß levels was detected.

Non-canonical modulators of nuclear receptors.

Like G protein-coupled receptors (GPCRs) and protein kinases, nuclear receptors (NRs) are a rich source of pharmaceutical targets. Over 80 NR-targeting drugs have been approved for 18 NRs. The focus of drug discovery in NRs has hitherto been on identifying ligands that bind to the canonical ligand binding pockets of the C-terminal ligand binding domains (LBDs). Due to the development of drug resistance and selectivity concerns, there has been considerable interest in exploring other, non-canonical ligand binding sites. Unfortunately, the potencies of compounds binding at other sites have generally not been sufficient for clinical development. However, the situation has changed dramatically over the last 3years, as compounds with sufficient potency have been reported for several NR targets. Here we review recent developments in this area from a medicinal chemistry point of view in the hope of stimulating further interest in this area of research.

Discovery of a Novel, Orally Efficacious Liver X Receptor (LXR) ß Agonist.

This article describes the application of Contour to the design and discovery of a novel, potent, orally efficacious liver X receptor ß (LXRß) agonist (17). Contour technology is a structure-based drug design platform that generates molecules using a context perceptive growth algorithm guided by a contact sensitive scoring function. The growth engine uses binding site perception and programmable growth capability to create drug-like molecules by assembling fragments that naturally complement hydrophilic and hydrophobic features of the protein binding site. Starting with a crystal structure of LXRß and a docked 2-(methylsulfonyl)benzyl alcohol fragment (6), Contour was used to design agonists containing a piperazine core. Compound 17 binds to LXRß with high affinity and to LXRα to a lesser extent, and induces the expression of LXR target genes in vitro and in vivo. This molecule served as a starting point for further optimization and generation of a candidate which is currently in human clinical trials for treating atopic dermatitis.

The use of spirocyclic scaffolds in drug discovery.

Owing to their inherent three-dimensionality and structural novelty, spiro scaffolds have been increasingly utilized in drug discovery. In this brief review, we highlight selected examples from the primary medicinal chemistry literature during the last three years to demonstrate the versatility of spiro scaffolds. With recent progress in synthetic methods providing access to spiro building blocks, spiro scaffolds are likely to be used more frequently in drug discovery.

The medicinal chemistry of liver X receptor (LXR) modulators.

LXRs have been of interest as targets for the treatment of atherosclerosis for over a decade. In recent years, LXR modulators have also garnered interest for potential use in the treatment of inflammation, Alzheimer's disease (AD), dermatological conditions, hepatic steatosis, and oncology. To date, no LXR modulator has successfully progressed beyond phase I clinical trials. In this Perspective, we summarize published medicinal chemistry efforts in the context of the available crystallographic data, druglikeness, and isoform selectivity. In addition, we discuss the challenges that need to be overcome before an LXR modulator can reach clinical use.

Biphenyl/diphenyl ether renin inhibitors: filling the S1 pocket of renin via the S3 pocket.

Structure-based design led to the discovery of a novel class of renin inhibitors in which an unprecedented phenyl ring filling the S1 site is attached to the phenyl ring filling the S3 pocket. Optimization for several parameters including potency in the presence of human plasma, selectivity against CYP3A4 inhibition and improved rat oral bioavailability led to the identification of 8d which demonstrated antihypertensive efficacy in a transgenic rat model of human hypertension.

Structure-based design and synthesis of 1,3-oxazinan-2-one inhibitors of 11ß-hydroxysteroid dehydrogenase type 1.

Structure based design led directly to 1,3-oxazinan-2-one 9a with an IC(50) of 42 nM against 11ß-HSD1 in vitro. Optimization of 9a for improved in vitro enzymatic and cellular potency afforded 25f with IC(50) values of 0.8 nM for the enzyme and 2.5 nM in adipocytes. In addition, 25f has 94% oral bioavailability in rat and >1000× selectivity over 11ß-HSD2. In mice, 25f was distributed to the target tissues, liver, and adipose, and in cynomolgus monkeys a 10 mg/kg oral dose reduced cortisol production by 85% following a cortisone challenge.

Discovery of VTP-27999, an Alkyl Amine Renin Inhibitor with Potential for Clinical Utility.

Structure guided optimization of a series of nonpeptidic alkyl amine renin inhibitors allowed the rational incorporation of additional polar functionality. Replacement of the cyclohexylmethyl group occupying the S1 pocket with a (R)-(tetrahydropyran-3-yl)methyl group and utilization of a different attachment point led to the identification of clinical candidate 9. This compound demonstrated excellent selectivity over related and unrelated off-targets, >15% oral bioavailability in three species, oral efficacy in a double transgenic rat model of hypertension, and good exposure in humans.

Structure based design of 11ß-HSD1 inhibitors.

Controlling elevated tissue-specific levels of cortisol may provide a novel therapeutic approach for treating metabolic syndrome. This concept has spurred large scale medicinal chemistry efforts in the pharmaceutical industry for the design of 11ß-HSD1 inhibitors. High resolution X-ray crystal structures of inhibitors in complex with the enzyme have facilitated the structure-based design of diverse classes of molecules. A summary of binding modes, trends in structure-activity relationships, and the pharmacodynamic data of inhibitors from each class is presented.

Discovery and optimization of adamantyl carbamate inhibitors of 11ß-HSD1.

Synthesis of 2-adamantyl carbamate derivatives of piperidines and pyrrolidines led to the discovery of 9a with an IC(50) of 15.2 nM against human 11ß-HSD1 in adipocytes. Optimization for increased adipocyte potency, metabolic stability and selectivity afforded 11k and 11l, both of which were >25% orally bioavailable in rat.

Spirocyclic ureas: orally bioavailable 11 beta-HSD1 inhibitors identified by computer-aided drug design.

Structure-guided drug design led to the identification of a class of spirocyclic ureas which potently inhibit human 11beta-HSD1 in vitro. Lead compound 10j was shown to be orally bioavailable in three species, distributed into adipose tissue in the mouse, and its (R) isomer 10j2 was efficacious in a primate pharmacodynamic model.

Optimization of orally bioavailable alkyl amine renin inhibitors.

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC(50) of 0.83nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10mg/kg resulted in >20h reduction of blood pressure in a double transgenic rat model of hypertension.