Novel series of tunable µOR modulators with enhanced brain penetration for the treatment of opioid use disorder, pain and neuropsychiatric indications.

Structural optimization of a previously reported agonist of µOR, PZM21 is described resulting in the discovery of a novel series of amides with at least 4-folds enhanced CNS penetration in rat. Furthermore, these efforts yielded compounds with varying levels of efficacy on the receptor ranging from high efficacy agonists such as compound 20 to antagonists, such as 24. The correlation between in vitro activation of µOR and relative activity in models of analgesia for these compounds is discussed. The compelling results obtained in these studies demonstrate the potential utility of these newly discovered compounds in the treatment of pain and opioid use disorder.

Structure-guided discovery of a novel, potent, and orally bioavailable 3,5-dimethylisoxazole aryl-benzimidazole BET bromodomain inhibitor.

The bromodomain and extra-terminal (BET) family of proteins, consisting of the bromodomains containing protein 2 (BRD2), BRD3, BRD4, and the testis-specific BRDT, are key epigenetic regulators of gene transcription and has emerged as an attractive target for anticancer therapy. Herein, we describe the discovery of a novel potent BET bromodomain inhibitor, using a systematic structure-based approach focused on improving potency, metabolic stability, and permeability. The optimized dimethylisoxazole aryl-benzimidazole inhibitor exhibited high potency towards BRD4 and related BET proteins in biochemical and cell-based assays and inhibited tumor growth in two proof-of-concept preclinical animal models.

Loss of TRIM33 causes resistance to BET bromodomain inhibitors through MYC- and TGF-ß-dependent mechanisms.

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-ß receptor expression and signaling, and blocking TGF-ß receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-ß signaling with BET bromodomain inhibition may offer new therapeutic benefits.

GS-5806 inhibits pre- to postfusion conformational changes of the respiratory syncytial virus fusion protein.

GS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproduced in vitro using a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity.

Phenotypic screening with deep learning identifies HDAC6 inhibitors as cardioprotective in a BAG3 mouse model of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by reduced cardiac output, as well as thinning and enlargement of left ventricular chambers. These characteristics eventually lead to heart failure. Current standards of care do not target the underlying molecular mechanisms associated with genetic forms of heart failure, driving a need to develop novel therapeutics for DCM. To identify candidate therapeutics, we developed an in vitro DCM model using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) deficient in B-cell lymphoma 2 (BCL2)-associated athanogene 3 (BAG3). With these BAG3-deficient iPSC-CMs, we identified cardioprotective drugs using a phenotypic screen and deep learning. From a library of 5500 bioactive compounds and siRNA validation, we found that inhibiting histone deacetylase 6 (HDAC6) was cardioprotective at the sarcomere level. We translated this finding to a BAG3 cardiomyocyte-knockout (BAG3cKO) mouse model of DCM, showing that inhibiting HDAC6 with two isoform-selective inhibitors (tubastatin A and a novel inhibitor TYA-018) protected heart function. In BAG3cKO and BAG3E455K mice, HDAC6 inhibitors improved left ventricular ejection fraction and reduced left ventricular diameter at diastole and systole. In BAG3cKO mice, TYA-018 protected against sarcomere damage and reduced Nppb expression. Based on integrated transcriptomics and proteomics and mitochondrial function analysis, TYA-018 also enhanced energetics in these mice by increasing expression of targets associated with fatty acid metabolism, protein metabolism, and oxidative phosphorylation. Our results demonstrate the power of combining iPSC-CMs with phenotypic screening and deep learning to accelerate drug discovery, and they support developing novel therapies that address underlying mechanisms associated with heart disease.

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.

Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor Derived from the Sanglifehrin Macrocycle.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

The structure of the extracellular region of human hepsin reveals a serine protease domain and a novel scavenger receptor cysteine-rich (SRCR) domain.

Hepsin is an integral membrane protein that may participate in cell growth and in maintaining proper cell morphology and is overexpressed in a number of primary tumors. We have determined the 1.75 A resolution structure of the extracellular component of human hepsin. This structure includes a 255-residue trypsin-like serine protease domain and a 109-residue region that forms a novel, poorly conserved, scavenger receptor cysteine-rich (SRCR) domain. The two domains are associated with each other through a single disulfide bond and an extensive network of noncovalent interactions. The structure suggests how the extracellular region of hepsin may be positioned with respect to the plasma membrane.

Discovery of an oral respiratory syncytial virus (RSV) fusion inhibitor (GS-5806) and clinical proof of concept in a human RSV challenge study.

GS-5806 is a novel, orally bioavailable RSV fusion inhibitor discovered following a lead optimization campaign on a screening hit. The oral absorption properties were optimized by converting to the pyrazolo[1,5-a]-pyrimidine heterocycle, while potency, metabolic, and physicochemical properties were optimized by introducing the para-chloro and aminopyrrolidine groups. A mean EC50 = 0.43 nM was found toward a panel of 75 RSV A and B clinical isolates and dose-dependent antiviral efficacy in the cotton rat model of RSV infection. Oral bioavailability in preclinical species ranged from 46 to 100%, with evidence of efficient penetration into lung tissue. In healthy human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a mean 4.2 log10 reduction in peak viral load and a significant reduction in disease severity compared to placebo. In conclusion, a potent, once daily, oral RSV fusion inhibitor with the potential to treat RSV infection in infants and adults is reported.

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay.

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.

Design of novel, potent, and selective human beta-tryptase inhibitors based on alpha-keto-[1,2,4]-oxadiazoles.

A series of novel alpha-keto-[1,2,4]-oxadiazoles has been synthesized as human tryptase inhibitors for evaluation as a new class of anti-asthmatic agent. The inhibitor design is focused on using a prime-side hydrophobic pocket and the S2 pocket of beta-tryptase to achieve inhibition potency and selectivity over other serine proteases.

Design and synthesis of selective keto-1,2,4-oxadiazole-based tryptase inhibitors.

Using a scaleable, directed library approach based on orthogonally protected advanced intermediates, we have prepared a series of potent keto-1,2,4-oxadiazoles designed to explore the P(2) binding pocket of human mast cell tryptase, while building in a high degree of selectivity over human trypsin and other serine proteases.

Identification of metabolites of the tryptase inhibitor CRA-9249: observation of a metabolite derived from an unexpected hydroxylation pathway.

The metabolites of the tryptase inhibitor CRA-9249 were identified after exposure to liver microsomes. CRA-9249 was found to be degraded rapidly in liver microsomes from rabbit, dog, cynomolgus monkey, and human, and less rapidly in microsomes from rat. The key metabolites included cleavage of an aryl ether, in addition to an unexpected hydroxylation of the amide side chain adjacent to the amide nitrogen. The chemical structures of both metabolites were confirmed by synthesis and comparison to material isolated from the liver microsomes. Several suspected hydroxylated metabolites were also synthesized and analyzed as part of the structure identification process.

Novel, potent, selective, and orally bioavailable human betaII-tryptase inhibitors.

The synthesis of novel [1,2,4]oxadiazoles and their structure-activity relationship (SAR) for the inhibition of tryptase and related serine proteases is presented. Elaboration of the P'-side afforded potent, selective, and orally bioavailable tryptase inhibitors.

Structure-guided design of peptide-based tryptase inhibitors.

Improved peptide-based inhibitors of human beta tryptase were discovered using information gleaned from tripeptide library screening and structure-guided design methods, including fragment screening. Our efforts sought to improve this class of inhibitors by replacing the traditional Lys or Arg P1 element. The optimized compounds display low nanomolar potency against the mast cell target and several hundred-fold selectivity with respect to serine protease off targets. Thus, replacement of Lys/Arg at P1 in a peptide-like scaffold does not need to be accompanied by a loss in target affinity.

Discovery and structure-activity relationship of 2-phenyl-oxazole-4-carboxamide derivatives as potent apoptosis inducers.

As a continuation of our efforts to discover novel apoptosis inducers as anticancer agents using a cell-based caspase HTS assay, 2-phenyl-oxazole-4-carboxamide derivatives were identified. The structure-activity relationships of this class of molecules were explored. Compound 1k, with EC(50) of 270 nM and GI(50) of 229 nM in human colorectal DLD-1 cells, was selected and demonstrated the ability to cleave PARP and displayed DNA laddering, the hallmarks of apoptosis. Compound 1k showed 63% tumor growth inhibition in human colorectal DLD-1 xenograft mouse model at 50 mpk, bid.

Highly potent non-peptidic inhibitors of the HCV NS3/NS4A serine protease.

Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A led to the identification of a potent Zn(2+)-dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn(2+) conditions (K(i)=27nM). This compound (46) binds also to NS3/NS4A in a Zn(2+) independent fashion (K(i)=1microM). The SAR of this class of compounds under Zn(2+) conditions is highly divergent compared to the SAR in the absence of Zn(2+), suggesting two distinct binding modes.