Replacement of sulfonamide by sulfoximine within a helicase-primase inhibitor with restricted flexibility.

Helicase-primase is an interesting target for the therapy of herpes simplex virus (HSV) infections. Since amenamevir is already approved for varicella-zoster virus (VZV) and HSV in Japan and pritelivir has received breakthrough therapy status for the treatment of acyclovir-resistant HSV infections in immunocompromised patients, the target has sparked interest in me-too approaches. Here, we describe the attempt to improve nervous tissue penetration in Phaeno Therapeutics drug candidate HN0037 to target the latent reservoir of HSV by installing less polar moieties, mainly a difluorophenyl instead of a pyridyl group, and replacing the primary sulfonamide with a methyl sulfoximine moiety. However, all obtained stereoisomers exhibited a weaker inhibitory activity on HSV-1 and HSV-2.

Validation of nuclear receptor RORγ isoform 1 as a novel host-directed antiviral target based on the modulation of cholesterol levels.

Currently, the clinically approved repertoire of antiviral drugs predominantly comprises direct-acting antivirals (DAAs). However, the use of DAAs is frequently limited by adverse effects, restriction to individual virus species, or the induction of viral drug resistance. These issues will likely be resolved by the introduction of host-directed antivirals (HDAs) targeting cellular proteins crucial for viral replication. However, experiences with the development of antiviral HDAs and clinical applications are still in their infancy. With the present study, we explored the human nuclear receptor and transcription factor RORγ isoform 1 (RORγ1), a member of the retinoic acid receptor-related orphan receptor (ROR) family, as a putative target of antiviral HDAs. To this end, cell culture models were used to investigate major viral human pathogens, i.e. the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human cytomegalovirus (HCMV), varicella zoster virus (VZV) and human immunodeficiency virus 1 (HIV-1). Our results demonstrated (i) an antiviral activity of the clinically relevant RORγ modulators cedirogant and others, (ii) that isoform RORγ1 acts as the responsible determinant and drug target in the analyzed cell culture-based models, (iii) a selectivity of the antiviral effect for RORγ1 over related receptors RORα and RORß, (iv) a late-phase inhibition exerted by cedirogant in HCMV replication and (v) a mechanistic link to the cellular cholesterol biosynthesis. Combined, the data highlight this novel RORγ-specific antiviral targeting concept and the developmental potential of RORγ-directed small molecules.

Intermittent therapy with helicase-primase inhibitor IM-250 efficiently controls recurrent herpes disease and reduces reactivation of latent HSV.

Herpes is a contagious life-long infection with persistently high incidence and prevalence, causing significant disease worldwide. Current therapies have efficacy against active HSV infections but no impact on the latent viral reservoir in neurons. Thus, despite treatment, disease recurs from latency and the infectious potential remains unaffected within patients. Here, efficacy of the helicase-primase inhibitor (HPI) IM-250 against chronic neuronal HSV infections utilizing two classic herpes in vivo latency/reactivation animal models (intravaginal guinea pig HSV-2 infection model and ocular mouse HSV-1 infection model) is presented. Intermittent therapy of infected animals with 4-7 cycles of IM-250 during latency silences subsequent recurrences analyzed up to 6 months. In contrast to common experience, our studies show that the latent reservoir is indeed accessible to antiviral therapy altering the latent viral reservoir such that reactivation frequency can be reduced significantly by prior IM-250 treatment. We provide evidence that antiviral treatment during HSV latency can reduce future reactivation from the latent reservoir, supporting a conceptual shift in the antiviral field, and reframing what is achievable with respect to therapy of latent neuronal HSV infections.

Development of a Potent Nurr1 Agonist Tool for In Vivo Applications.

Nuclear receptor related 1 (Nurr1) is a neuroprotective transcription factor and an emerging target in neurodegenerative diseases. Despite strong evidence for a role in Parkinson's and Alzheimer's disease, pharmacological control and validation of Nurr1 are hindered by a lack of suitable ligands. We have discovered considerable Nurr1 activation by the clinically studied dihydroorotate dehydrogenase (DHODH) inhibitor vidofludimus calcium and systematically optimized this scaffold to a Nurr1 agonist with nanomolar potency, strong activation efficacy, and pronounced preference over the highly related receptors Nur77 and NOR1. The optimized compound induced Nurr1-regulated gene expression in astrocytes and exhibited favorable pharmacokinetics in rats, thus emerging as a superior chemical tool to study Nurr1 activation in vitro and in vivo.

Helicase-primase inhibitors from Medshine Discovery Inc. (WO2018/127207 and WO2020/007355) for the treatment of herpes simplex virus infections - structure proposal for Phaeno Therapeutics drug candidate HN0037.

Helicase-primase is an interesting target for small-molecule therapy of herpes simplex virus (HSV) infections. With amenamevir already approved for varicella-zoster virus and with pritelivir's granted breakthrough therapy designation for the treatment of acyclovir-resistant HSV infections in immunocompromised patients, the target has sparked interest for me-too approaches. We describe the opportunities and limitations of the helicase-primase inhibitor patent portfolio from Phaeno Therapeutics and propose the structure of their drug candidate HN0037, which has been in-licensed from Medshine Discovery.

Interplay of Trans- and Cis-Interactions of Glycolipids in Membrane Adhesion.

Glycolipids mediate stable membrane adhesion of potential biological relevance. In this article, we investigate the trans- and cis-interactions of glycolipids in molecular dynamics simulations and relate these interactions to the glycolipid-induced average separations of membranes obtained from neutron scattering experiments. We find that the cis-interactions between glycolipids in the same membrane leaflet tend to strengthen the trans-interactions between glycolipids in apposing leaflets. The trans-interactions of the glycolipids in our simulations require local membrane separations that are significantly smaller than the average membrane separations in the neutron scattering experiments, which indicates an important role of membrane shape fluctuations in glycolipid trans-binding. Simulations at the experimentally measured average membrane separations provide a molecular picture of the interplay between glycolipid attraction and steric repulsion of the fluctuating membranes probed in the experiments.

Helicase primase inhibitors (HPIs) are efficacious for therapy of human herpes simplex virus (HSV) disease in an infection mouse model.

Although the seroprevalence of Herpes simplex virus type 1 (HSV-1) currently amounts to âˆ¼ 67% worldwide, the annual incidence of a severe disease progression, particularly herpes encephalitis, is approximately 2-4 cases per 1,000,000 infections. Nucleoside analogues, such as acyclovir (ACV), valacyclovir (VACV) or famciclovir, are still the therapeutic treatment of choice for HSV infections. However, nucleoside drugs have limited efficacy against severe HSV disease and for treatment of nucleoside-resistant viral strains, alternative therapies such as helicase-primase inhibitors (HPIs) which are highly potent by inhibiting viral replication are under development. In preclinical studies we analyzed the antiviral efficacy of drug candidates of a novel compound class of HPIs for the treatment of HSV to identify the most active eutomer structure in an intranasal infection mouse lethal challenge model. HSV-1 infected BALB/c mice treated with vehicle control developed fatal disease according to humane endpoints after 5-7 days. In contrast, the animals dosed orally once daily with the HPI compounds at 10 or 4 mg/kg/day showed a significantly increased survival (70% and 100% for 10 mg/kg/day; 90% and 100% for 4 mg/kg/day, respectively) compared to the vehicle treatment (0-10%), when therapy was initiated 6 h post HSV-1 inoculation. We observed a significantly improved outcome in clinical parameters and survival over 21 days in the group receiving novel HPI candidates using even the lowest dose of 4 mg/kg/day. With VACV treatment of 75 mg/kg daily survival was also significantly increased (80%-90% for 75 mg/kg/day) but to lesser extent. Initial IM-250 therapy at 10 mg/kg/day could be delayed up to 72 h resulting in significantly increased survival compared to the vehicle control. Furthermore, we detected significantly fewer viral genome copies in the lungs and brains of HPI treated animals compared to vehicle (440-fold reduction for 4 mg/kg/day IM-250 in the brain) or VACV controls by quantitative PCR. In conclusion the preclinical studies of the novel HPI compounds showed superior efficacy in comparison to the current standard HSV treatment represented by VACV with respect to the survival according humane endpoints, the clinical score and virus load in lungs and brains. Thus, candidates of this new drug class are promising antivirals of HSV infections and further translation into clinical trials is warranted.

Retinoic acid-related orphan receptor gamma t (RORγt) inverse agonists/antagonists for the treatment of inflammatory diseases - where are we presently?

Introduction: The transcription factor retinoic acid-related orphan receptor gamma t (RORγt) has been identified as the master regulator of TH17 cell differentiation and IL-17/22 production and is therefore an attractive target for the treatment of inflammatory diseases. Several orally or topically administered small molecule RORγt inverse agonists (RIAs) have progressed up to the end of clinical Phase 2.Areas covered: Based on publications and patent evaluations this review summarizes the evolution of the chemical matter for all 16 pharmaceutical companies, who develop(ed) a clinical-stage RIAs (until March 2021). Structure proposals for some clinical stage RIAs are presented and the outcome of the clinical trials is discussed.Expert opinion: So far, the clinical trials have been plagued with a high attrition rate. Main reasons were lack of efficacy (topical) or safety signals (oral) as well as, amongst other things, thymic lymphomas as seen with BMS-986251 in a preclinical study and liver enzyme elevations in humans with VTP-43742. Possibilities to mitigate these risks could be the use of RIAs with different chemical structures not interfering with thymocytes maturation and no livertox-inducing properties. With new frontrunners (e.g., ABBV-157 (cedirogant), BI 730357 or IMU-935) this is still an exciting time for this treatment approach.

A helicase-primase drug candidate with sufficient target tissue exposure affects latent neural herpes simplex virus infections.

More than 50% of the world population is chronically infected with herpesviruses. Herpes simplex virus (HSV) infections are the cause of herpes labialis (cold sores), genital herpes, and sight-impairing keratitis. Less frequently, life-threatening disseminated disease (encephalitis and generalized viremia) can also occur, mainly in immunocompromised patients and newborns. After primary infection, HSV persists for life in a latent state in trigeminal or sacral ganglia and, triggered by diverse stimuli, disease recurs in more than 30% of patients up to several times a year. Current therapy with nucleoside analogs targeting the viral polymerase is somewhat effective but limited by poor exposure in the nervous system, and latent infections are not affected by therapy. Here, we report on an inhibitor of HSV helicase-primase with potent in vitro anti-herpes activity, a different mechanism of action, a low frequency of HSV resistance, and a favorable pharmacokinetic and safety profile. Improved target tissue exposure results in superior efficacy in preventing and treating HSV infection and disease in animal models as compared to standard of care. Therapy of primary HSV infections with drug candidate IM-250 {(S)-2-(2',5'-difluoro-[1,1'-biphenyl]-4-yl)-N-methyl-N-(4-methyl-5-(S-methylsulfon-imidoyl)thiazol-2-yl)acetamide} not only reduces the duration of disease symptoms or time to healing but also prevents recurrent disease in guinea pigs. Treatment of recurrent infections reduces the frequency of recurrences and viral shedding, and, unlike nucleosidic drugs, IM-250 remains effective for a time after cessation of treatment. Hence, IM-250 has advantages over standard-of-care therapies and represents a promising therapeutic for chronic HSV infection, including nucleoside-resistant HSV.

Finding New Molecular Targets of Familiar Natural Products Using In Silico Target Prediction.

Natural products comprise a rich reservoir for innovative drug leads and are a constant source of bioactive compounds. To find pharmacological targets for new or already known natural products using modern computer-aided methods is a current endeavor in drug discovery. Nature's treasures, however, could be used more effectively. Yet, reliable pipelines for the large-scale target prediction of natural products are still rare. We developed an in silico workflow consisting of four independent, stand-alone target prediction tools and evaluated its performance on dihydrochalcones (DHCs)-a well-known class of natural products. Thereby, we revealed four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1, 17ß-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a thorough strategy on how to perform computational target predictions and guidance on using the respective tools.

Discovery and optimization of new oxadiazole substituted thiazole RORγt inverse agonists through a bioisosteric amide replacement approach.

Starting from previously identified thiazole-2-carboxamides exemplified by compound 1/6, two new series of RORγt inverse agonists with significantly improved aqueous solubility, ADME parameters and oral PK properties were discovered. These scaffolds were identified from a bioisosteric amide replacement approach. Amongst the variety of heterocycles explored, a 1,3,4-oxadiazole led to compounds with the best overall profile for SAR development and in vivo exploration. In an ex vivo mouse PD model, concentration dependent efficacy was demonstrated and compounds 3/5 and 6/3 were profiled in a 5-day rat tolerability study.

Optimization and biological evaluation of thiazole-bis-amide inverse agonists of RORγt.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with small molecule therapeutics. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We recently described the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole scaffold. Herein we describe the successful optimization of this class by incorporation of an additional amide moiety at the 4-position of the thiazole core. In several optimization cycles, we have reduced human PXR activation, improved solubility, and increased potency while maintaining nuclear receptor selectivity. X-ray crystallographic analysis of compound 1g bound in the sterol binding site of the ligand binding domain of RORγt was largely consistent with an earlier structure, guiding further insight into the molecular mechanism for RORγt inhibition with this series. Compound 1g is orally bioavailable, potent in a human whole blood assay and proved to be efficacious in an ex-vivo IL-17A assay, and was selected for preclinical evaluation.

Nonsteroidal FXR Ligands: Current Status and Clinical Applications.

FXR agonists have demonstrated very promising clinical results in the treatment of liver disorders such as primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and nonalcoholic steatohepatitis (NASH). NASH, in particular, is one of the last uncharted white territories in the pharma landscape, and there is a huge medical need and a large potential pharmaceutical market for a NASH pharmacotherapy. Clinical efficacy superior to most other treatment options was shown by FXR agonists such as obeticholic acid (OCA) as they improved various metabolic features including liver steatosis as well as liver inflammation and fibrosis. But OCA's clinical success comes with some major liabilities such as pruritus, high-density lipoprotein cholesterol (HDLc) lowering, low-density lipoprotein cholesterol (LDLc) increase, and a potential for drug-induced liver toxicity. Some of these effects can be attributed to on-target effects exerted by FXR, but with others it is not clear whether it is FXR- or OCA-related. Therefore a quest for novel, proprietary FXR agonists is ongoing with the aim to increase FXR potency and selectivity over other proteins and to overcome at least some of the OCA-associated clinical side effects through an improved pharmacology. In this chapter we will discuss the historical and ongoing efforts in the identification and development of nonsteroidal, which largely means non-bile acid-type, FXR agonists for clinical use.

Identification and biological evaluation of thiazole-based inverse agonists of RORγt.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with a small molecule. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We describe the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole core. Acid analog 1j demonstrated oral bioavailability in rats and was potent in a human whole blood assay, suggesting potential utility in treating autoimmune and inflammatory diseases such as psoriasis. X-ray crystallographic data helped to elucidate the molecular mechanism for RORγt inhibition with this series.

RORγt inhibitors as potential back-ups for the phase II candidate VTP-43742 from Vitae Pharmaceuticals: patent evaluation of WO2016061160 and US20160122345.

Retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt or RORc2) is a key transcription factor for the differentiation of naïve proinflammatory CD4+ T cells and the production of T helper-17 (TH17) cells. Inhibiting RORγt activity is thought to be beneficial in targeting a variety of inflammatory and autoimmune disorders. Recently Vitae Pharmaceuticals (to be acquired by Allergan) reported positive top-line results from a Phase 2a clinical trial of RORγt inhibitor VTP-43742 in psoriatic patients. The compound was reported to demonstrate a clear signal of efficacy over a short four-week period and no drug-related cardiac abnormalities were observed; however, in the 700 mg dose group reversible transaminase elevations were observed in four patients, which prompted the company to cancel testing VTP-43742 at a initially planned third, higher dose. In Vitae Pharmaceuticals latest patent applications, WO2016061160 and US20160122345, potential dihydropyrrolopyridine back-up compounds of clinical candidate VTP-43742 (covered in WO2015116904) are disclosed. In light of the recently announced RORγt back-up molecule VTP-45489, the improvements of the new compounds are discussed and their potential impact is elucidated.

Novel substituted isoxazole FXR agonists with cyclopropyl, hydroxycyclobutyl and hydroxyazetidinyl linkers: Understanding and improving key determinants of pharmacological properties.

Several isoxazole-containing series of FXR agonists have been published over the last 15years, subsequent to the prototypical amphiphilic 'hammerhead'-type structure that was originally laid out by GW4064, the first potent synthetic FXR agonist. A set of novel compounds where the hammerhead is connected to the terminal carboxylic acid-bearing aryl or heteroaryl moiety by either a cyclopropyl, a hydroxycyclobutyl or a hydroxyazetidinyl linker was synthesized in order to improve upon the ADME properties of such isoxazoles. The resulting compounds all demonstrated high potencies at the target receptor FXR but with considerable differences in their physicochemical and in vivo profiles. The structure-activity relationships for key chemical features that have a major impact on the in vivo pharmacology of this series are discussed.

Retinoid-related orphan receptor gamma t (RORγt) inhibitors from Vitae Pharmaceuticals (WO2015116904) and structure proposal for their Phase I candidate VTP-43742.

Retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt or RORC2) is a key transcription factor for the differentiation of naïve proinflammatory CD4(+) T cells and the production of T helper-17 (TH17) cells. Inhibiting RORγt activity is thought to be beneficial in targeting a variety of inflammatory and autoimmune disorders, however current candidates remain to be validated in the clinic. Recently Vitae Pharmaceuticals successfully finished its Phase 1 single ascending dose clinical study with their proprietary RORγt inverse agonist VTP-43742. On the basis of the reported promising results, Vitae Pharmaceuticals could currently be considered as having the leading clinical candidate in the RORγt inverse agonist category. This prompts the interest on the exact chemical structure of their clinical candidate. The first relevant patent application (WO2014179564) from Vitae Pharmaceuticals describes RORγt inverse agonists with a 5,6-dihydro-4H-pyrrolo[3,4-d]thiazole core, while in the second and latest patent application (WO2015116904) this element has changed towards a 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine core. By combining information from Vitae's patent applications and trustworthy online information, the potential elucidation of the chemical structure of clinical candidate VTP-43742 is described.

Retinoid-related orphan receptor γ t modulators: comparison of Glenmark's me-too patent application (WO2015008234) with the originator application from Merck Sharp and Dohme (WO2012106995).

Retinoic acid receptor-related orphan nuclear receptor γ t (RORγt) is a key transcription factor for the development of TH17 cells. Inhibiting RORγt activity is thought to be beneficial in targeting a variety of inflammatory and autoimmune disorders. In their patent application WO2015008234, Glenmark applied a scaffold-hop approach regarding to the originator application from Merck Sharp & Dohme (WO2012106995): a 6-membered annelated aromatic moiety is replaced by an annelated 5-membered heteroaryl (exemplified and claimed is however only thiophene), resulting in potent RORγt inverse agonists with a thieno[3,2-b]pyrrole or thieno[3,2-c]pyrazole core. Based on the patent disclosure, the novelty and utility of these me-too compounds is discussed.

Knocking on FXR's door: the "hammerhead"-structure series of FXR agonists - amphiphilic isoxazoles with potent in vitro and in vivo activities.

The Farnesoid X Receptor (FXR) was recently validated in clinical studies using the bile acid analogue Obeticholic Acid (OCA) as an attractive drug target for liver diseases such as Primary Biliary Cirrhosis (PBC) or Non-alcoholic Steatohepatitis (NASH). OCA, however, turned out to induce cholesterol- related side effects upon prolonged treatment and it shows bile acid like pharmacokinetics. The quest for synthetic non-steroidal FXR agonists with general drug likeliness and improved pharmacokinetic and - dynamic properties has started more than a decade ago: The first non-steroidal and selective FXR agonist with decent submicromolar potency, GW4064, was patented in 1998 and published in 2000. Since then, many pharmaceutical companies have taken GW4064 as a structural template for their efforts in identifying novel patentable FXR agonists with the GW-derived trisubstituted isoxazole general structure. However, so far only one compound out of these different series has made it into the early stages of clinical development: The Px-102/Px-104 from Phenex is currently tested in a phase IIa study in patients with Non-Alcoholic Fatty Liver Disease (NAFLD). In this review we try to summarize from the patent and scientific literature the attempts to improve the GW4064 structure into different directions. Furthermore, we suggest directions for further improvements of this special class of synthetic FXR agonists which all display the typical "hammerhead"-conformation in the FXR ligand binding pocket that provides the basis for their impressive in vitro and in vivo potencies.

Identification of the first inverse agonist of retinoid-related orphan receptor (ROR) with dual selectivity for RORß and RORγt.

Retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt) is a key transcription factor for the development of Th17 cells. Inhibiting RORγt activity is thought to be beneficial in targeting a variety of inflammatory and autoimmune disorders. Recently N-(5-(arylcarbonyl)thiazol-2-yl)amides were described as RORγt antagonists with in vivo efficacy in experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA) via oral administration. So far no selective small molecule ligands have been revealed for RORß. We show, that one compound of this class, namely N-[5-(2-chloro-benzoyl)-4-(3-chlorophenyl)-thiazol-2-yl]-2-(4-ethanesulfonyl-phenyl)-acetamide (4) is a potent dual inverse agonist towards RORγt and RORß devoid of activity to 18 other human nuclear receptors and thus can serve as chemical probe to deepen our understanding about RORß and its biology.