May disordered protein cause serious drug side effect?

Insomnia is a sleep disorder characterized by the inability to fall asleep, in 2011 the 12-20% of the general adult population suffer insomnia, with more predisposition in the USA. The abnormal sleep cycle results in other symptoms like, lack of motivation, mood disturbances, loss of memory, tiredness, deficiency of energy, headaches and some gastrointestinal disorders.
The most common treatment for insomnia is medication with benzodiazepine and non-benzodiazepine drugs. The benzodiazepine drugs enhance the effect of the GABA (g amino butyric acid) at GABAA receptor, resulting in a sedative, hypnotic, muscle relaxing, sleeping inducing and anxiolytic effects. Non benzodiazepine drugs include Non-benzodiazepine drugs include zolpidem tartrate (Ambien®), sodium butabarbital (Butisol®) and eszopiclone (Lunesta®). The major disadvantages of these drugs are the multiple side effects: tolerance, dependence and memory impairment.
In the article by Weng and Calvin ( they proposed a possible drug design strategy that might reduce the side effects by considering disordered proteins related with insomnia.
The sleep-related protein/receptor can be classified into the clock complex and the hypnotic-related receptor (HRR). The clock complex controls the circadian rhythm activity; genetic mutations in this complex had showed dysfunctions in the perception of sunset and sunrise, leading to sleep disorder. This complex is formed by several proteins, like BMAL-1, CLOCK, CRY1/2, CKI/II and PER1/2/3.
The HRR, includes dopamine receptors (D2/D3), GABAA, histamine receptor (H1/H2), melatonin receptor, muscarinic receptor M1 and orexin receptor 1/2.
In the study they analyzed the structural properties of clock complex and HRR by computational techniques and they found that the majority of the clock complex proteins share a highly disordered property, located in the middle of the sequence, implicated the flexibility of the whole complex. Over the 30% of CLOCK, PER1/2/3, BMAL-1, M1, melatonin receptor and CKI are disordered, the percentage of structural disorder in HRR is much lower that the clock complex.


The clock complex showed more disordered rate in the middle of the sequence, implicated the flexibility of the whole complex. They suggest HRR it might be a better drug target for new insomnia therapies due to the small possibility of affecting the flexibility and stability of the complex. They mention that some investigators had give strong evidence of structure lead to side effects, because the there are limited binding sites in nature.
They suggest not to screen or base studies on the rigid structure of disordered proteins, because these proteins do not exist in a fixed structure, and even a small disordered region can change the conformation, flexibility and stability of the protein. Many chemists screen the databases by using the rigid structure of the protein, not considering the flexible movement and the cryptic allosteric sites (CAS). CAS do not exist in the native state but they might be present in the transition state, being possible positions for drug targeting.
Also a coarse-grained model might be a good method to map out the transition pathway of disordered proteins transformation, being able to predict possible cryptic allosteric sites.
They mention that cognitive behavioral therapy is another useful treatment for insomnia, this therapy not always work for all patients. Finally the traditional Chinese medicine (TCM) seems to be a good potential treatment for insomnia, however is not enough evidence or being nontoxic, due to the highly ingredients extracted from herbs, they suggest that more clinical studies are required. Further studies and research is needed for identifying a novel highly folded protein, related with the clock complex or the sleep related receptors.

Schizophrenia drug targeting negative symptoms remains as elusive as ever with the demise of Roche’s bitopertin

Schizophrenia is a major disorder of brain function, prevalent in 1% of the population worldwide and estimated by the World Health Organisation to be the fifth leading worldwide cause of disease. Symptoms of schizophrenia can be categorized as positive and negative; positive symptoms can include hallucinations, delusions and disordered thoughts and speech, while negative symptoms include apathy, poverty of speech, anhedonia (inability to experience pleasure) and social withdrawal. In general, positive symptoms are treated fairly well by antipsychotics but there is an unmet need for drugs that are able to treat the negative symptoms and these largely untreated symptoms remain a huge barrier to the resumption of a fully functional, “normal” life for affected individuals. Being unable to do so results in a huge emotional and societal burden, from the costs of the diseases management to the impact on caregivers and resources with an annual estimated cost in the UK alone of around £12 billion. Additionally, an inability to function and contribute to society often leads to withdrawal and depression, leading to a suicide rate of 4-5% in the schizophrenic population.
In January of this year, Roche announced that two Phase III clinical trials of bitopertin (RG1678, see here), a glycine transporter 1 (GlyT1) inhibitor, failed to achieve their primary end-point in the treatment of the negative symptoms of schizophrenia – severely reducing hopes that this might be the first drug to target currently untreatable negative symptoms (see here). At that time, four additional Phase 3 studies were continuing. However, in their quarterly report last month, the company stated that an additional Phase 3 study looking at sub-optimally controlled symptoms, such as hallucinations and delusions, also failed its primary endpoint (see here)In the light of these results, the company terminated two of the remaining three studies leaving just one study in sub-optimally controlled symptoms active.

And so once more a much-touted, potential breakthrough CNS therapeutic has essentially failed, prompting the obvious question of why? Well, the Phase III studies were triggered by a Phase II 8-week study of bitopertin in 2010, which demonstrated a significant improvement in negative symptoms compared to placebo, as measured by the PANSS negative symptom factor score (see Figure), although in retrospect, the narrow therapeutic window, with efficacy at 10, 30 but not 60 mg doses, might have been a cause for concern. However, this is not the first time (and unfortunately it will probably not be the last time) that Phase 2 efficacy does not translate into Phase 3 efficacy for a novel CNS therapeutic (with Merck’s NK1 antagonist being perhaps the most spectacular recent example). And at least Roche had positive Phase 2 data rather than the “Intriguing preliminary data” in a negative Phase 2 study that was the basis for Lilly to commence Phase 3 studies with their mGlu2/3 agonist pomaglumetad methionil (see here).
This once positive outlook of bitopertin can have only intensified the disappointment not only of Roche but also a neuroscience community in general. From a mechanistic point of view, the Roche data also calls into question the efficacy of therapeutically modulating glutamatergic pathomechanisms, thought to underpin schizophrenia and complement the dopaminergic hypothesis the underlies the positive symptoms. Roche’s rationale was to target the glycine transporter, GlyT1, and inhibit it with bitopertin in an effort to raise the synaptic levels of glycine and thereby increase the activity of NMDA receptors, for which glycine is a co-agonist along with glutamate. This is based on increasing evidence that implicates dysfunction in glutamatergic signaling pathways, specifically a hypofunction in NMDA receptor signaling, in the pathogenesis of schizophrenia and the appearance of negative symptoms and is also the basis of several other therapeutic approaches, including modulation of the mGluR2 and mGluR5 receptors. While it would be unwise to attempt to directly activate NMDA receptors, indirectly modulating NMDA receptor activity by blocking the reuptake transporter of glycine, has been an attractive and less risky alternative. An alternative approach to treating the negative symptoms of schizophrenia is being pursued by AbbVie and EnVivo who are both targeting nicotinic acetylcholine receptors and it is to be hoped that one or both of these drugs makes it to market to reenergize the beleaguered neuroscience therapeutic area.
Figure taken from Roche’s December 2010 pipeline update (see here).

DESs: New ionic fluids

You will surely have heard of ILs or ionic liquids, which were discovered more than a century ago, but have you hear of the new variety of ionic fluids called Deep Eutectic Solvents or DES?
I had certainly not come across DES until this recent joint publication by Spanish and Scottish research groups. ( 201400889/abstract)
A Deep Eutectic Solvent is a type of ionic solvent with special properties. It is composed of two or three cheap and safe components that together form an eutectic mixture, with a melting point much lower than either of the individual components. They were first described in 2003 when Abbot and co-workers reported on a low melting mixture of a 1:2 mole ratio of choline chloride (2-hydroxyethyl-trimethylammonium chloride [ChCl]) and urea. Since then many different mixtures have been described. In most cases a DES is obtained by mixing a quaternary ammonium salt with metal salts or a hydrogen bond donor (HBD) that has the ability to form a complex with the halide anion of the quaternary ammonium salt (Scheme 1). Thus the components of such solvents are low in cost, biodegradable and low in toxicity and synthesis of DESs is 100% atom economic, easy to handle and no purification is required and it is easily recycled. Moreover, in comparison to common organic solvents they are less volatile and not flammable making them ideal green alternative media.
carol 1

Since their preparation, DES have found applications in a variety of synthetic procedures such as brominations, polymerizations, dehydrations, cycloadditions, hydrogenations, condensations, NaBH4-reductions and in Heck and Stille coupling reactions. However it is this latter publication that firstly reports the successful coexistence of Grignard or organolithium reagents and green solvents within the same solution using Deep Eutectic Solvents.

We are all familiar with Grignard reactions and you don’t need to be a chemist to have heard of them. As a synthetic chemist you will have definitely carried out one of these reaction, if not more, and you will know that addition of Grignard reagents (or organolithiums) to carbonyl groups requires the use of aprotic dry solvents under inert atmosphere and at temperatures ranging from 0˚C to –78˚C and therefore the use of glycerol (Gly) or even water would definitely not be your choice of solvent. Hevia and co-cowerkers, however, report chemoselective addition of Grignard reagents (Table 1) and organolithiums (Table 2) to ketones using the eutectic mixtures 1ChCl/2Gly, 1qChCl/2EG and 1ChCl/2H2O at room temperature and in air



It is of interest to point out that the addition reaction of the Grignard reagents or organolithium is orders of magnitude faster than their protonation by water, ethylene glycol (EG) or glycerol (Gly) present in the DES and that the reactions are completed immediately (2-3s) suggesting a kinetic activation of the alkylating reagents. The authors speculate that the ammonium salt ChCl present in the DESs employed may have a further role than as a component of the DES mixture. Although not isolated in their work, they believe that an anionic magnesiate (from the Grignard reagents) and a dianionic halolithiate (from the organolithium) species are formed that have and enhanced nucleophilic power which favor the addition reaction in DES over the competing protonation process.
Although choline chloride is not a chemical we currently stock in the lab I am tempted to order it and have it ready for my next Grignard reaction so that I can test this simple methodology, which avoids use of Schlenk techniques and low temperatures. Are you not tempted to try it too?