Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

Activity of Tricyclic Pyrrolopyrimidine Gyrase B Inhibitor against Mycobacterium abscessus.

Tricyclic pyrrolopyrimidines (TPPs) are a new class of antibacterials inhibiting the ATPase of DNA gyrase. TPP8, a representative of this class, is active against Mycobacterium abscessus in vitro. Spontaneous TPP8 resistance mutations mapped to the ATPase domain of M. abscessus DNA gyrase, and the compound inhibited DNA supercoiling activity of recombinant M. abscessus enzyme. Further profiling of TPP8 in macrophage and mouse infection studies demonstrated proof-of-concept activity against M. abscessus ex vivo and in vivo.

Structure-Guided Drug Design of 6-Substituted Adenosine Analogues as Potent Inhibitors of Mycobacterium tuberculosis Adenosine Kinase.

Mycobacterium tuberculosis adenosine kinase (MtbAdoK) is an essential enzyme of Mtb and forms part of the purine salvage pathway within mycobacteria. Evidence suggests that the purine salvage pathway might play a crucial role in Mtb survival and persistence during its latent phase of infection. In these studies, we adopted a structural approach to the discovery, structure-guided design, and synthesis of a series of adenosine analogues that displayed inhibition constants ranging from 5 to 120 nM against the enzyme. Two of these compounds exhibited low micromolar activity against Mtb with half maximal effective inhibitory concentrations of 1.7 and 4.0 µM. Our selectivity and preliminary pharmacokinetic studies showed that the compounds possess a higher degree of specificity against MtbAdoK when compared with the human counterpart and are well tolerated in rodents, respectively. Finally, crystallographic studies showed the molecular basis of inhibition, potency, and selectivity and revealed the presence of a potentially therapeutically relevant cavity unique to the MtbAdoK homodimer.

Thiazomycin, nocathiacin and analogs show strong activity against clinical strains of drug-resistant Mycobacterium tuberculosis.

Thiazolyl peptides are a class of natural products with potent Gram-positive antibacterial activities. Lack of aqueous solubility precluded this class of compounds from advancing to clinical evaluations. Nocathiacins and thiazomycins are sub-classes of thiazolyl peptides that are endowed with structural features amenable for chemical modifications. Semi-synthetic modifications of nocathiacin led to a series of analogs with improved water solubility, while retaining potency and antibacterial spectrum. We studied the activities of a selection of two natural products (nocathiacin and thiazomycin) as well as seven polar semi-synthetic analogs against twenty clinical strains of Mycobacterium tuberculosis with MDR phenotypes. Two compounds show useful activity against H37Rv strain with MIC values ⩽1 µM, two (⩽0.5 µm) and three (⩽10 µm). These two derivatives showed MIC values ⩽2.5 µm against most of the 20 MDR strains regardless their resistance profile. Specifically, these lack cross-resistance to rifampicin, isoniazid and moxifloxacin.

Structure activity relationship of pyridoxazinone substituted RHS analogs of oxabicyclooctane-linked 1,5-naphthyridinyl novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-6).

Oxabicyclooctane linked 1,5-naphthyridinyl-pyridoxazinones are novel broad-spectrum bacterial topoisomerase inhibitors (NBTIs) targeting bacterial DNA gyrase and topoisomerase IV at a site different than quinolones. Due to lack of cross-resistance to known antibiotics they present excellent opportunity to combat drug-resistant bacteria. A structure activity relationship of the pyridoxazinone moiety is described in this Letter. Chemical synthesis and activities of NBTIs with substitutions at C-3, C-4 and C-7 of the pyridoxazinone moiety with halogens, alkyl groups and methoxy group has been described. In addition, substitutions of the linker NH proton and its transformation into amide analogs of AM-8085 and AM-8191 have been reported. Fluoro, chloro, and methyl groups at C-3 of the pyridoxazinone moiety retained the potency and spectrum. In addition, a C-3 fluoro analog showed 4-fold better oral efficacy (ED50 3.9 mg/kg) as compared to the parent AM-8085 in a murine bacteremia model of infection of Staphylococcus aureus. Even modest polarity (e.g., methoxy) is not tolerated at C-3 of the pyridoxazinone unit. The basicity and NH group of the linker is important for the activity when CH2 is at the linker position-8. However, amides (with linker position-8 ketone) with a position-7 NH or N-methyl group retained potency and spectrum suggesting that neither basicity nor hydrogen-donor properties of the linker amide NH is essential for the activity. This would suggest likely an altered binding mode of the linker position-7,8 amide containing compounds. The amides showed highly improved hERG (functional IC50 >30 µM) profile.

Structure activity relationship of C-2 ether substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-5).

Oxabicyclooctane linked novel bacterial topoisomerase inhibitors (NBTIs) are new class of recently reported broad-spectrum antibacterial agents. They target bacterial DNA gyrase and topoisomerase IV and bind to a site different than quinolones. They show no cross-resistance to known antibiotics and provide opportunity to combat drug-resistant bacteria. A structure activity relationship of the C-2 substituted ether analogs of 1,5-naphthyridine oxabicyclooctane-linked NBTIs are described. Synthesis and antibacterial activities of a total of 63 analogs have been summarized representing alkyl, cyclo alkyl, fluoro alkyl, hydroxy alkyl, amino alkyl, and carboxyl alkyl ethers. All compounds were tested against three key strains each of Gram-positive and Gram-negative bacteria as well as for hERG binding activities. Many key compounds were also tested for the functional hERG activity. Six compounds were evaluated for efficacy in a murine bacteremia model of Staphylococcus aureus infection. Significant tolerance for the ether substitution (including polar groups such as amino and carboxyl) at C-2 was observed for S. aureus activity however the same was not true for Enterococcus faecium and Gram-negative strains. Reduced clogD generally showed reduced hERG activity and improved in vivo efficacy but was generally associated with decreased overall potency. One of the best compounds was hydroxy propyl ether (16), which mainly retained the potency, spectrum and in vivo efficacy of AM8085 associated with the decreased hERG activity and improved physical property.

Human monoclonal antibody HCV1 effectively prevents and treats HCV infection in chimpanzees.

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5-1.0 log(10) reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.

Sustained viral response in a hepatitis C virus-infected chimpanzee via a combination of direct-acting antiviral agents.

Efforts to develop novel, interferon-sparing therapies for treatment of chronic hepatitis C (HCV) infection are contingent on the ability of combination therapies consisting of direct antiviral inhibitors to achieve a sustained virologic response. This work demonstrates a proof of concept that coadministration of the nucleoside analogue MK-0608 with the protease inhibitor MK-7009, both of which produced robust viral load declines as monotherapy, to an HCV-infected chimpanzee can achieve a cure of infection.

Identification and biological evaluation of a series of 1H-benzo[de]isoquinoline-1,3(2H)-diones as hepatitis C virus NS5B polymerase inhibitors.

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for inhibition. Herein, we present 1H-benzo[de]isoquinoline-1,3(2H)-diones as a new series of selective inhibitors of HCV NS5B polymerase. The HTS hit 1 shows submicromolar potency in two different HCV replicons (1b and 2b) and displays no activity on other polymerases (HIV-RT, Polio-pol, GBV-b-pol). These inhibitors act during the pre-elongation phase by binding to NS5B non-nucleoside binding site Thumb Site II as demonstrated by crystal structure of compound 1 with the DeltaC55-1b and DeltaC21-2b enzymes and by mutagenesis studies. SAR in this new series reveals inhibitors, such as 20, with low micromolar activity in the HCV replicon and with good activity/toxicity window in cells.

Phosphoramidate prodrugs of 2'-C-methylcytidine for therapy of hepatitis C virus infection.

The application of a phosphoramidate prodrug approach to 2'-C-methylcytidine (NM107), the first nucleoside inhibitor of the hepatitis C virus (HCV) NS5B polymerase, is reported. 2'-C-Methylcytidine, as its valyl ester prodrug (NM283), was efficacious in reducing the viral load in patients infected with HCV. Several of the phosphoramidates prepared demonstrated a 10- to 200-fold superior potency with respect to the parent nucleoside in the cell-based replicon assay. This is due to higher levels of 2'-C-methylcytidine triphosphate in the cells. These prodrugs are efficiently activated and converted to the triphosphate in hepatocytes of several species. Our SAR studies ultimately led to compounds that gave high levels of NTP in hamster and rat liver after subcutaneous dosing and that were devoid of the toxic phenol moiety usually found in ProTides.

PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination.

Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.

Robust antiviral efficacy upon administration of a nucleoside analog to hepatitis C virus-infected chimpanzees.

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide and is associated with an increased incidence of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Currently approved therapies to treat HCV infection consist of combinations of pegylated alpha interferon and ribavirin which result in a sustained viral response in 40 to 60% of patients. Efforts to develop improved therapies include the development of direct inhibitors of virally encoded enzymes such as the viral RNA-dependent RNA polymerase. A nucleoside analog, 2'-C-methyl-7-deaza-adenosine (MK-0608), has been shown to inhibit viral RNA replication in the subgenomic HCV genotype 1b replicon, with a 50% effective concentration (EC(50)) of 0.3 microM (EC(90) = 1.3 microM). To determine efficacy in vivo, MK-0608 was administered to HCV-infected chimpanzees, resulting in dose- and time-dependent decreases in plasma viral loads. In separate experiments, chimpanzees dosed for 7 days with MK-0608 at 0.2 and 2 mg per kg of body weight per day by intravenous administration experienced average reductions in viral load of 1.0 and >5 log(10) IU/ml, respectively. Two other HCV-infected chimpanzees received daily doses of 1 mg MK-0608 per kg via oral administration. After 37 days of oral dosing, one chimpanzee with a high starting viral load experienced a reduction in viral load of 4.6 log(10), and the viral load in the other chimpanzee fell below the limit of quantification (LOQ) of the HCV TaqMan assay (20 IU/ml). Importantly, viral load remained below the LOQ throughout the duration of dosing and for at least 12 days after dosing ended. The results demonstrate a robust antiviral effect on the administration of MK-0608 to HCV-infected chimpanzees.

Liver-targeted prodrugs of 2'-C-methyladenosine for therapy of hepatitis C virus infection.

2'-C-Methyladenosine exhibits impressive inhibitory activity in the cell-based hepatitis C virus (HCV) subgenomic replicon assay, by virtue of intracellular conversion to the corresponding nucleoside triphosphate (NTP) and inhibition of NS5B RNA-dependent RNA polymerase (RdRp). However, rapid degradation by adenosine deaminase (ADA) limits its overall therapeutic potential. To reduce ADA-mediated deamination, we prepared cyclic 1-aryl-1,3-propanyl prodrugs of the corresponding nucleoside monophosphate (NMP), anticipating cytochrome P450 3A-mediated oxidative cleavage to the NMP in hepatocytes. Lead compounds identified in a primary rat hepatocyte screen were shown to result in liver levels of NTP predictive of efficacy after intravenous dosing to rats. The oral bioavailability of the initial lead was below 5%; therefore, additional analogues were synthesized and screened for liver NTP levels after oral administration to rats. Addition of a 2',3'-carbonate prodrug moiety proved to be a successful strategy, and the 1-(4-pyridyl)-1,3-propanyl prodrug containing a 2',3'-carbonate moiety displayed oral bioavailability of 39%.

Synthesis and HCV inhibitory properties of 9-deaza- and 7,9-dideaza-7-oxa-2'-C-methyladenosine.

As a part of an ongoing medicinal chemistry effort to identify inhibitors of the Hepatitis C Virus RNA replication, we report here the synthesis and biological evaluation of 9-deaza- and 7,9-dideaza-7-oxa-2'-C-methyladenosine. The parent 2'-C-methyladenosine shows excellent intracellular inhibitory activity but poor pharmacokinetic profile. Replacement of the nucleoside-defining 9-N of 2'-C-methyladenosine with a carbon atom was designed to yield metabolically more stable C-nucleosides. Modifications at position 7 were designed to exploit the importance of the hydrogen bond accepting properties of this heteroatom in modulating the adenosine deaminase (ADA) mediated 6-N deamination. 7-Oxa-7,9-dideaza-2'-C-methyladenosine was found to be a moderately active inhibitor of intracellular HCV RNA replication, whereas 9-deaza- 2'-C-methyladenosine showed only weak activity despite excellent overlap of both of the synthesized target compounds with 2'-C-methyladenosine's three dimensional structure. Position 7 of the nucleobase proved to be an effective handle for modulating ADA-mediated degradation, with the rate of degradation correlating with the hydrogen-bonding properties at this position.

Muscle phenotype and mutation load in 51 persons with the 3243A>G mitochondrial DNA mutation.

BACKGROUND: Mitochondrial disorders are generally not associated with a clear phenotype-genotype relationship, which complicates the understanding of the disease and genetic counseling. OBJECTIVE: To investigate the relationship between the muscle and blood mitochondrial DNA mutation load and phenotype. DESIGN: Survey. SETTING: The Neuromuscular Research Unit, Rigshospitalet, Copenhagen, Denmark. PARTICIPANTS: Fifty-one persons with the 3243A>G point mutation of mitochondrial DNA, and 20 healthy control subjects. METHODS: We recorded the maximal oxygen uptake (Vo(2)max), maximal workload, resting and peak-exercise plasma lactate levels, muscle and blood mutation load, muscle morphology, and presence of diabetes mellitus and hearing impairment in all subjects. RESULTS: Muscle mutation load (mean +/- SE, 50% +/- 5%; range, 2%-95%) correlated with Vo(2)max and resting plasma lactate level (P<.001; R>/=0.64). All persons except 5 with a muscle mutation load above 50% had abnormal Vo(2)max and morphology on muscle biopsy findings. Persons with hearing impairment and diabetes mellitus had a muscle mutation load above 65%. The mutation load in blood (mean +/- SE, 18% +/- 3%; range, 0%-61%) did not correlate with Vo(2)max, resting plasma lactate levels, or presence of hearing impairment or diabetes mellitus. CONCLUSIONS: This study demonstrates a close relationship between the muscle mutation load and phenotype in persons carrying the 3243A>G mutation. The lack of correlation between the mutation load in blood and symptoms from other tissues emphasizes the importance of assessing phenotype-genotype correlations in the same tissue in mitochondrial disease. The results indicate that the threshold of muscle mutation load at which oxidative impairment occurs can be as low as 50%, which is as much as 40% lower than that suggested by in vitro studies.

Aerobic training is safe and improves exercise capacity in patients with mitochondrial myopathy.

Exercise intolerance is a prominent symptom in patients with mitochondrial myopathy (MM), but it is still unsettled whether exercise training is safe and beneficial for patients with MM. To address this, we studied the effect of 12 weeks cycle training on exercise capacity, quality of life and underlying molecular and cellular events in five patients with single large-scale deletions, one with a microdeletion and 14 with point mutations of mitochondrial DNA (mtDNA), and 13 healthy subjects. Each training session lasted 30 min, and was performed at an intensity of 70% of VO2max (maximal oxygen uptake). Each subject performed 50 training sessions in 12 weeks. All subjects were evaluated before and after training, and 13 MM patients were studied after 8 weeks of deconditioning. Evaluation included VO2max and mutation load and mtDNA quantity, mitochondrial enzymatic activity, and number of centrally nucleated, apoptotic, ragged red and cytochrome oxidase (COX)-negative fibres in muscle biopsies from the quadriceps muscle. After 12 weeks of training, VO2max and muscle citrate synthase increased in MM (26 and 67%) and healthy (17 and 65%) subjects, while mtDNA quantity in muscle only increased in the MM patients (81%). In the MM patients, training did not change mtDNA mutation load in muscle, mitochondrial enzyme complex activities, muscle morphology and plasma creatine kinase. After deconditioning, VO2max and citrate synthase activity returned to values before training, while muscle mtDNA mutation load decreased. These findings show that aerobic training efficiently improves oxidative capacity in MM patients. Based on unchanged levels of mutant load in muscle, morphological findings on muscle biopsy and plasma creatine kinase levels during training, the treatment appears to be safe. Regular, supervised aerobic exercise is therefore recommended in MM patients with the studied mutations.

A genotype 2b NS5B polymerase with novel substitutions supports replication of a chimeric HCV 1b:2b replicon containing a genotype 1b NS3-5A background.

HCV diversity suggests that evaluation of HCV inhibitors for broad genotypic efficacy is warranted. The replicon system enables cell-culture compound efficacy evaluation against an active replication complex, and a functional replicon dependent upon a genotype 2b polymerase would augment existing cell-culture efficacy studies that are presently limited to genotype 1a, 1b, and 2a replicons. We made a chimeric Neo(r) 1b:2b replicon where genotype 2b NS5B was inserted into a genotype 1b NS3-5A background and transfected replicon RNA to generate Neo(r) cell lines. All cell lines contained novel substitutions within NS5B which were subsequently engineered into the parental 1b:2b replicon and shown to enhance replication to various degrees. A single NS5B M31I substitution enhanced replication to levels sufficiently robust to quantify sensitivity to HCV inhibitors in a transient replication assay. The M31I 1b:2b replicon was similarly sensitive to an active-site nucleoside inhibitor of NS5B as genotype 1b replicons, but was insensitive to two non-nucleoside inhibitors which were otherwise efficacious against the genotype 1b replicons. This work describes a novel HCV replicon sustained by a genotype 2b polymerase that is sufficiently robust for quantifiable analysis in a transient replication assay, and demonstrates its utility in characterizing anti-HCV compounds for cross-genotypic efficacy.

Orally bioavailable highly potent HIV protease inhibitors against PI-resistant virus.

Efforts directed to identifying potent HIV protease inhibitors (PI) have yielded a class of compounds that are not only very active against wild-type (NL4-3) HIV virus but also very potent against a panel of PI-resistant viral isolates. Chemistry and biology are described.

Inhibitory effect of 2'-substituted nucleosides on hepatitis C virus replication correlates with metabolic properties in replicon cells.

Nucleosides have been widely used in the treatment of viral diseases, but relatively few have been identified as inhibitors of hepatitis C virus (HCV). The modified ribonucleosides, 2'-C-methyl-adenosine and 2'-O-methyl-cytidine, are potent inhibitors of HCV replication which specifically target the NS5B polymerase. Herein, a more extensive characterization of the effect of these compounds upon HCV replication in subgenomic replicons is reported. A highly selective antireplicative effect induced by the nucleosides in replicon-containing cell lines was maintained during an exponential growth period with potencies which paralleled the reduction of both positive- and negative-strand RNA replication. Moreover, the inhibitory effect closely correlated with the intrinsic metabolic properties of differing replicon clonal lines. Interestingly, while 2'-C-methyl-adenosine elicited similar inhibitory potencies in different cell lines, 2'-O-methyl-cytidine was found to be inactive in one replicon cell line tested, although the corresponding triphosphates comparably inhibited the in vitro activity of replication complexes isolated from these cells and the activity of NS5B polymerase using synthetic templates. The lack of antireplicative effect, attributed to poor intracellular conversion of the 2'-O-methyl-cytidine nucleoside to the active 5'-triphosphate, was reversed using a monophosphate prodrug. Thus, although replicon cells are useful for evaluating the effect of inhibitors upon HCV replication, these findings have important implications for their use in the identification and characterization of nucleosides and other chemotherapeutic agents requiring cellular metabolism.

Synthesis and evaluation of S-acyl-2-thioethyl esters of modified nucleoside 5'-monophosphates as inhibitors of hepatitis C virus RNA replication.

Several triphosphates of modified nucleosides (1-6) were identified as inhibitors (IC(50) = 0.08-3.8 microM) of hepatitis C virus RNA-dependent RNA polymerase (RdRp). Although the initial SAR developed by determining the ability of the triphosphates to inhibit the in vitro activity of the HCV RdRp identified several potent inhibitors, none of the corresponding nucleosides exhibited significant inhibitory potency in a cell-based replicon assay. To improve upon the activity, bis(tBu-S-acyl-2-thioethyl) nucleoside 5'-monophosphate esters (7-12) were synthesized, and these derivatives exhibited improved potency compared to the corresponding nucleosides in the cell-based assay. Analysis of the intracellular metabolism demonstrated that the S-acyl-2-thioethyl (SATE) prodrug is metabolized to the 5'-triphosphate 40- to 155-fold more efficiently compared to the corresponding nucleoside. The prodrug approach involving bis(tBuSATE)cytidine 5'-monophosphate ester significantly reduced the deamination of cytidine derivatives by cellular deaminases. Additionally, chromosomal aberration studies with the SATE prodrug in cells showed no statistically relevant increase in aberrations compared to the concurrent controls.