Decision making in the pharmaceutical industry - A tale of three antibiotics.

There is a mounting crisis in treatment of bacterial diseases. The appearance of nosocomial infections produced by multi-drug resistant bacteria is rapidly increasing and at the same time the pharmaceutical industry has been abandoning new antibiotic discovery. To help understand why, we investigated the decision-making processes behind three novel antibiotics that were initially discovered in the late 1980's and early 1990's: daptomycin, linezolid, and lysobactin. Each antibiotic was investigated by two highly qualified scientific organizations that came to opposing opinions regarding the clinical utility and commercial potential of the drug. After reviewing the literature and interviewing key scientific staff members working on each of these molecules, we have identified factors needed to generate positive development decisions. Organizational factors included decision timing, therapeutic area focus, organizational support for risk taking and the presence of a project champion. Technical factors included investment in the optimization of dosing for improved drug exposure, toxicological evaluation of the purified eutomer from a diastereomer and the failure to develop an effective research formulation.

Correction to "Chiral Cyclohexane 1,3-Diones as Inhibitors of Mutant SOD1-Dependent Protein Aggregation for the Treatment of ALS".

[This corrects the article DOI: 10.1021/ml3000963.].

Tertiary Amine Pyrazolones and Their Salts as Inhibitors of Mutant Superoxide Dismutase 1-Dependent Protein Aggregation for the Treatment of Amyotrophic Lateral Sclerosis.

Pyrazolone derivatives have previously been found to be inhibitors of Cu/Zn superoxide dismutase 1 (SOD1)-dependent protein aggregation, which extended survival of an amyotrophic lateral sclerosis (ALS) mouse model. On the basis of ADME analysis, we describe herein a new series of tertiary amine-containing pyrazolones and their structure-activity relationships. Further conversion to the conjugate salts greatly improved their solubility. Phosphate compound 17 exhibited numerous benefits both to cellular activity and to CNS-related drug-like properties in vitro and in vivo, including microsomal stability, tolerated toxicity, and blood-brain barrier permeation. These results indicate that tertiary amine pyrazolones comprise a valuable class of ALS drug candidates.

Deuteration and fluorination of 1,3-bis(2-phenylethyl)pyrimidine-2,4,6(1H,3H,5H)-trione to improve its pharmacokinetic properties.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons, leading to muscle weakness, paralysis, and death, most often from respiratory failure. Over 200 pyrimidine-2,4,6-trione (PYT) small molecules, which prevent aggregation and reduce the associated toxicity of mutant superoxide dismutase 1 (SOD1) found in patients with familial ALS, have been synthesized and tested. One of the compounds (1,3-bis(2-phenylethyl)pyrimidine-2,4,6(1H,3H,5H)-trione, (1) was previously found to have an excellent combination of potency efficacy, and some desirable pharmacokinetic properties. To improve the solubility and metabolic stability properties of this compound, deuterium and fluorine were introduced into 1. New analogs with better solubility, plasma stability, and human microsome stability were identified.

Proteasome activation is a mechanism for pyrazolone small molecules displaying therapeutic potential in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1(G93A) cells. PC12-SOD1(G93A) cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1(G93A) cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS.

Arylazanylpyrazolone derivatives as inhibitors of mutant superoxide dismutase 1 dependent protein aggregation for the treatment of amyotrophic lateral sclerosis.

The arylsulfanylpyrazolone and aryloxanylpyrazolone scaffolds previously were reported to inhibit Cu/Zn superoxide dismutase 1 dependent protein aggregation and to extend survival in the ALS mouse model. However, further evaluation of these compounds indicated weak pharmacokinetic properties and a relatively low maximum tolerated dose. On the basis of an ADME analysis, a new series of compounds, the arylazanylpyrazolones, has been synthesized, and structure-activity relationships were determined. The SAR results showed that the pyrazolone ring is critical to cellular protection. The NMR, IR, and computational analyses suggest that phenol-type tautomers of the pyrazolone ring are the active pharmacophore with the arylazanylpyrazolone analogues. A comparison of experimental and calculated IR spectra is shown to be a valuable method to identify the predominant tautomer.

Substituted pyrazolones require N2 hydrogen bond donating ability to protect against cytotoxicity from protein aggregation of mutant superoxide dismutase 1.

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal neurodegenerative disease. Although the cause remains unknown, misfolded protein aggregates are seen in neurons of sporadic ALS patients, and familial ALS mutations, including mutations in superoxide dismutase 1 (SOD1), produce proteins with an increased propensity to misfold and aggregate. A structure activity relationship of a lead scaffold exhibiting neuroprotective activity in a G93A-SOD1 mouse model for ALS has been further investigated in a model PC12 cellular assay. Synthesis of biotinylated probes at the N(1) nitrogen of the pyrazolone ring gave compounds (5d-e) that retained activity within 10-fold of the proton-bearing lead compound (5a) and were equipotent with a sterically less cumbersome N(1)-methyl substituted analogue (5b). However, when methyl substitution was introduced at N(1) and N(2) of the pyrazolone ring, the compound was inactive (5c). These data led us to investigate further the pharmacophoric nature of the pyrazolone unit. A range of N(1) substitutions were tolerated, leading to the identification of an N(1)-benzyl substituted pyrazolone (5m), equipotent with 5a. Substitution at N(2) or excision of N(2), however, removed all activity. Therefore, the hydrogen bond donating ability of the N(2)-H of the pyrazolone ring appears to be a critical part of the structure, which will influence further analogue synthesis.

Chiral cyclohexane 1,3-diones as inhibitors of mutant SOD1-dependent protein aggregation for the treatment of ALS.

Cyclohexane 1,3-diones were identified as a class of molecules exhibiting a protective effect against mutant SOD1 induced toxicity in PC-12 cells, but an optimized analogue had little or no effect on life extension in the G93A SOD1 mouse model for amyotrophic lateral sclerosis (ALS). Additional testing showed that these compounds were inactive in neurons and further analogue synthesis was carried out to identify compounds with neuronal activity. Starting from two racemic derivatives that were active in cortical neurons, two potent analogues (1b and 2b) were resolved, which were protective against mutant SOD1 induced toxicity in PC-12 cells. Both compounds were found to be active in cortical neurons and presented good ADME profiles in vitro. On the basis of these results, an ALS mouse trial with 1b was carried out, which showed slightly greater life extension than the FDA-approved ALS drug riluzole, thereby validating cyclohexane 1,3-diones as a novel therapeutic class for the treatment of ALS.

Cyclohexane 1,3-diones and their inhibition of mutant SOD1-dependent protein aggregation and toxicity in PC12 cells.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2-3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identified and classified as cyclohexane-1,3-dione (CHD) derivatives. A concise and efficient synthetic route has been developed to provide diverse CHD analogs. The structural modification of the CHD scaffold led to the discovery of a more potent analog (26) with an EC(50) of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efficiently penetrate the blood-brain barrier. However, compound 26 did not exhibit any significant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cells, potentially due to poor selective cell penetration. Further structural modification of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73) were synthesized, which had significantly enhanced cortical neuronal cell permeability, as well as similar potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel therapeutic candidates for ALS.

ADME-guided design and synthesis of aryloxanyl pyrazolone derivatives to block mutant superoxide dismutase 1 (SOD1) cytotoxicity and protein aggregation: potential application for the treatment of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. The arylsulfanyl pyrazolone (ASP) scaffold was one of the active scaffolds identified in a cell-based high throughput screening assay targeting mutant Cu/Zn superoxide dismutase 1 (SOD1) induced toxicity and aggregation as a marker for ALS. The initial ASP hit compounds were potent and had favorable ADME properties but had poor microsomal and plasma stability. Here, we identify the microsomal metabolite and describe synthesized analogues of these ASP compounds to address the rapid metabolism. Both in vitro potency and pharmacological properties of the ASP scaffold have been dramatically improved via chemical modification to the corresponding sulfone and ether derivatives. One of the ether analogues (13), with superior potency and in vitro pharmacokinetic properties, was tested in vivo for its pharmacokinetic profile, brain penetration, and efficacy in an ALS mouse model. The analogue showed sustained blood and brain levels in vivo and significant activity in the mouse model of ALS, thus validating the new aryloxanyl pyrazolone scaffold as an important novel therapeutic lead for the treatment of this neurodegenerative disorder.

Pyrimidine-2,4,6-trione derivatives and their inhibition of mutant SOD1-dependent protein aggregation. Toward a treatment for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons, leading to muscle weakness, paralysis, and death, most often from respiratory failure. The only FDA-approved drug for the treatment of ALS, riluzole, only extends the median survival in patients by 2-3 months. There is an urgent need for novel therapeutic strategies for this devastating disease. Using a high-throughput screening assay targeting an ALS cultured cell model (PC12-G93A-YFP cell line), we previously identified three chemotypes that were neuroprotective. We present a further detailed analysis of one promising scaffold from that group, pyrimidine-2,4,6-triones (PYTs), characterizing a number of PYT analogues using SAR and ADME. The PYT compounds show good potency, superior ADME data, low toxicity, brain penetration, and excellent oral bioavailability. Compounds from this series show 100% efficacy in the protection assay with a good correlation in activity between the protection and protein aggregation assays. The modifications of the PYT scaffold presented here suggest that this chemical structure may be a novel drug candidate scaffold for use in clinical trials in ALS.

Identification of compounds protective against G93A-SOD1 toxicity for the treatment of amyotrophic lateral sclerosis.

The underlying cause of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder, remains unknown. However, there is strong evidence that one pathophysiological mechanism, toxic protein misfolding and/or aggregation, may trigger motor neuron dysfunction and loss. Since the clinical and pathological features of sporadic and familial ALS are indistinguishable, all forms of the disease may be better understood and ultimately treated by studying pathogenesis and therapy in models expressing mutant forms of SOD1. We developed a cellular model in which cell death depended on the expression of G93A-SOD1, a mutant form of superoxide dismutase found in familial ALS patients that produces toxic protein aggregates. This cellular model was optimized for high throughput screening to identify protective compounds from a >50,000 member chemical library. Three novel chemical scaffolds were selected for further study following screen implementation, counter-screening and secondary testing, including studies with purchased analogs. All three scaffolds blocked SOD1 aggregation in high content screening assays and data on the optimization and further characterization of these compounds will be reported separately. These data suggest that optimization of these chemicals scaffolds may produce therapeutic candidates for ALS patients.

Arylsulfanyl pyrazolones block mutant SOD1-G93A aggregation. Potential application for the treatment of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. Mutations in copper/zinc superoxide dismutase 1 (SOD1) have been implicated in the pathophysiology of this disease. Using a high-throughput screening assay expressing mutant G93A SOD1, two bioactive chemical hit compounds (1 and 2), identified as arylsulfanyl pyrazolones, were identified. The structural optimization of this scaffold led to the generation of a more potent analogue (19) with an EC(50) of 170nM. To determine the suitability of this class of compounds for further optimization, 1 was subjected to a battery of pharmacokinetic assays; most of the properties of 1 were good for a screening hit, except it had a relatively rapid clearance and short microsomal half-life stability. Compound 2 was found to be blood-brain barrier penetrating with a brain/plasma ratio=0.19. The optimization of this class of compounds could produce novel therapeutic candidates for ALS patients.

A cell wall-active lipopeptide from the fungus Pochonia bulbillosa.

Bioassay-directed fractionation of a fermentation of Pochonia bulbinosa, culture 38G272, led to the isolation of a series of structurally novel, prospective cell wall-active lipopeptides. The main component of this suite is 1, a linear hexapeptide with a delta-hydroxymyristic acid amide substituted N-terminus. The structure was deduced using high-field microsample NMR, Fourier transform mass spectrometry, and microscale chemical degradation. The potent cell wall activity and synthetically accessible structure of 1 make it a potential lead for further investigation.