Will PTP1B be a new therapeutic option for T2DM and Obesity?


Insulin and Lectin are two hormones which play crucial roles in energy storage and peripheral energy uptake. Resistance to these hormones is a hallmark for both Type 2 Diabetes Mellitus (T2DM) and obesity1. Nearly 220 million people are living with diabetes and WHO projected the death due to diabetes would double between 2005 and 20302. The global epidemic nature of T2DM and obesity demand the need for new therapeutic approaches. PTP1B stands out in the PTP family as a negative regulator of insulin signal transduction pathway through its ability to dephosphorylate and inactivate the insulin receptor3. PTP1B also negatively regulates the leptin signalling pathway by dephosphorylating JAK2 (a phosphorylated tyrosine kinase) in the hypothalamus. Leptin is a key hormone directly associated with obesity that regulates food intake and energy expenditure4,11.

sri-1

Fig.1: PTP1B dephosphorylates the insulin receptor during its biosynthesis in the endoplasmic reticulum (ER)4

Natural products like prenylated xanthones, flavonoids, bromophenols, phenolic acids, cumarins, terpenes, steroids were isolated and tested to have potent PTP1B inhibition of 1.6-30.0 µmol/L5. These natural products have opened the gate to many small molecule inhibitors for PTP1B. However, carboxylates and phosphonates with nanomolar inhibition have failed to progress to the clinic due to poor membrane penetration6.

Many studies have postulated that existing anti-diabetic drugs belonging to the thiazolidinediones (TZDs) family, commonly known as glitazones (rosiglitazone7, pioglitazones etc.), could inhibit PTP1B8. Early competitive PTP1B inhibitors based on Vanadium compounds (vanadates and pentavanadates) have significant therapeutic values in human but they are not specific8,11. Series of benzofurans, benzothiophenes and phosphonates have been synthesised and patented as potential PTP1B inhibitors6,8. Unfortunately, none of them has made Phase II clinical trials9. Ertiprotafab from Wyeth, the first PTP1B inhibitor, was halted in Phase II due to data inconsistencies10.

Selectivity6,11 and bioavailability12 are the main challenges in developing potent PTP inhibitors. The PTP1B catalytic site shares structural motifs of other enzymes of PTP family and is highly charged. Consequently, highly charged inhibitors with very strong PTP1B inhibition lack biovailability11. Achieving selectivity against TC-PTP13 is the most challenging task for medicinal chemists. Many clinical trials, patent applications and drugs validation experiments proves that PTP1B has emerged as a novel target for the management, treatment of T2DM. However, the journey in the search for selective PTP1B inhibitors has been facing many pitfalls.

Interestingly, current phase II trial on Trodusquemine14 and PTP1B antisense oligonucleotides (IONIS-GCGRRX) by Ionis pharmaceuticals15 could be an alternate therapeutic option for T2DM and obesity.  At least one of the clinical candidates in Phase II studies will receive green signal from FDA in few years’ time

Blog by Srinivasan Natarajan

References

  1. D.Popov, Biochemical and Biophysical Research Communications, 2011, 410, 377-381

www.sciencedirect.com/science/journal/0006291X/410/3

  1. WHO diabetes fact sheet available from

http://www.who.int/mediacentre/factsheets/fs312/en/

  1. S.Koren and I.G Fantus., Best Practice & research Clinical Endocrinology & Metabolism, 2007, 21(4), 621-640

https://www.ncbi.nlm.nih.gov/pubmed/18054739

  1. J.Montalibet and B.P.Kennedy., Drug Discovery Today: Therapeutic Stragies, 2005, 2, 129-135.

www.sciencedirect.com/science/article/pii/S1740677305000069

  1. C.S JIANG, L.LIANG and Y GUO, Acta Pharmacologica Sinica (2012) 33: 1217–1245

https://www.ncbi.nlm.nih.gov/pubmed/22941286

  1. H.Cho Vitamins and Harmones, V 91, chapter 17, 2013, Elsevier Inc.,

https://www.ncbi.nlm.nih.gov/pubmed/23374726

  1. The antidiabetic drug rosiglitazone was withdrawn from European market since 2010 due to the increased risk of heart attack

https://en.wikipedia.org/wiki/Rosiglitazone

  1. Review by A.K Tamarkar and A.K. Rai, Expert Opin. Ther. Patents, 2014, 24(10), 1-15.

https://www.ncbi.nlm.nih.gov/pubmed/25120222

  1. Review by M.L.Tremblay et al., Crit.Rev.Biochem.Mol.Biol., 2013, 48(5), 430-445

https://www.ncbi.nlm.nih.gov/pubmed/23879520

  1. DV. Erbe et al. Molecular Pharmacology, 67(1), 69-77.

http://molpharm.aspetjournals.org/content/67/1/69

  1. S.Koren, I.G. Fantus, Best Practice & Research Clinical Endocrinology & Metabolism, 214, 621-640.

http://www.sciencedirect.com/science/article/pii/S1521690X07000814

Tonks N.K et al., Nature Chemical Biology, 2014, 10, 558-568

http://www.nature.com/nchembio/journal/v10/n7/abs/nchembio.1528.html

  1. H.Oh et al., Molecules, 2015, 20, 11173-11183

www.mdpi.com/1420-3049/20/6/11173/pdf

  1. T.Tcell PTP (TC-PTP) which linked to the development of several inflammatory disorder including type 1diabetes, Crohn disease and rheumatoid arthritis (J.Am.Chem.Soc.,2009, 131(36), 13072-79)

http://pubs.acs.org/doi/pdf/10.1021/ja903733z

  1. K.A. Lantz et al. Obesity, 2010, 18(8), 1516-1523.

https://www.ncbi.nlm.nih.gov/pubmed/20075852

  1. Phase II for IONIS-GCGRRX completed in January 2017. See the company website http://www.ionispharma.com/pipeline/

also see https://www.ncbi.nlm.nih.gov/pubmed/25197025  for anti -sense oligos for PTP1B

 

 

 

 

Making ‘Marvelous Medicines’ at the Brighton Science Festival


As part of the Brighton science festival the Sussex Drug Discovery Centre (SDDC) has participated for the last two years by doing an activity at the bright sparks event. The bright sparks weekend is aimed at 7-14 year olds and is the festival’s flagship event where there are over 50 stalls, stands and shows across the two days.

The SDDC activity aimed to do a journey though drug discovery called Making Marvelous Medicines. We did this by creating 4 stations of activity lasting 50 minutes. The 4 stations included: What are proteins, Designing Drugs, Synthetic Chemistry and Testing Drugs.

What Are Proteins ?

This station aimed to introduce the concept of proteins, by explaining their role as tiny machines doing millions of jobs that allow us to function healthily.james-1

To illustrate the “power of proteins” the enzyme catalase was used in liver to degrade hydrogen peroxide in our bubbly volcano experiment. An animated video of the motor protein kinesin was also used, and the use of ATP as protein food to give them energy was also introduced.

 

Designing Drugs

Now we introduced a ATP “eating” protein from tuberculosis, using molymods the children built molecules to fit into the ATP binding pocket of this protein. We then introduced a human ATP binding protein and tested the selectivity of the molecule.

Synthetic Chemistry

Participants were introduced to the concept of a chemical reaction using baking as an analogy: different reagents (“ingredients”) can be combined in order to make new chemical products e.g. a new medicine. The young people carried out their own reaction to make their “drug”  – we used the popular “golden rain” precipitation reaction between potassium iodide and lead nitrate https://www.youtube.com/watch?v=zJkv0HiqApo.

Gasps of awe and wonder came from the young scientists (and the adults with them) when two seemingly colorless solutions formed a bright yellow solid when mixed together.  The student demonstrators also recrystallized the products the children had formed in order to explain the importance of purifying medicines that are made in a lab.

  Testing Drugs

In the final stall, we aimed to simulate a biochemical assay to test our drugs effectiveness. This assay tuned blue for negative and pink for positive. The actual reagents used for this were Phenolphthalein and Thymolphthalein.

james-6

Feedback from parents and children was really positive and the audience grasped the key concepts. Parents were often just as interested as the children. At the end some children left comments on the white board.

james-7

Blog written by James Noble [PhD Student]

 

 

 

 

 

Synthesise a CNS drug that can cross the blood brain barrier?


Central nervous system (CNS) drugs include analgesics, sedatives, and anticonvulsants, with drugs being used to treat the effects of a wide variety of medical conditions such as Alzheimer’s disease, Parkinson’s disease, and depression. More than 1 billion people globally suffer from a CNS disease, with one in five Americans taking at least one psychiatric drug. In the US and Europe combined, the overall cost of the economic burden of CNS diseases is estimated to be more than $2 trillion, with that figure expecting to triple by 2030 (1). Whilst most pharmaceutical companies are patient centric, these figures are financially appealing. However, development of therapies for CNS diseases has lagged behind that for other therapeutic areas. CNS drugs can take more than 20 months longer to develop than other drugs, with attrition rates greater than 50%. These failures can be attributed to a number of reasons such as inadequate dosage to hit the therapeutic target, high placebo effect, high patient dropout rate, inaccuracies of preclinical disease models, and incomplete understanding of brain disease mechanisms. (1)

One of the challenges of working on a CNS drug discovery project is for the drug to traverse the blood-brain barrier (BBB). The BBB protects the brain from most pathogens, sheltering it from the systemic circulation. It also prevents most large molecule neurotherapeutics and more than 98% of all small molecule drugs reaching the brain from the bloodstream, the tight junctions of the endothelial cells lining brain capillaries restricting paracellular movement of substances across the BBB. The BBB serves roles other than that of blocking circulating substances from entering the CNS. It also facilitates and regulates the entry of many substances that are critical to CNS function and secretes substances into the blood and CNS. These extra-barrier functions allow the BBB to influence the homeostatic, nutritive, and immune environments of the CNS and to regulate the exchange of informational molecules between the CNS and blood. (3)

High attrition rates of preclinical and clinical drug candidates led Wager et al (4) to design a tool based on key physicochemical properties (clogP, clogD, molecular weight, topological polar surface area, hydrogen bond donors, and pKa) that would enable multiparameter optimisation (MPO) of druglike properties to accelerate the identification of drug candidates with optimal pharmacokinetic and safety profiles. After nearly 8 years of using this tool at Pfizer, Wager et al have reported a reduction in the number of compounds submitted to exploratory toxicity studies and an increase in the survival of the CNS MPO candidates through regulatory toxicology into first in human studies. (5) The tool has also been used outside of Pfizer to reduce attrition and improve compound quality in the design phase.

An understanding of the barrier and extra-barrier aspects of BBB physiology is also critical to developing drugs that can access the CNS. A recent CNS paper by Patel et al (6) discusses several key approaches for brain targeting including physiological transport mechanisms such as adsorptive-mediated transcytosis, inhibition of active efflux pumps, receptor-mediated transport, cell-mediated endocytosis, and the use of peptide vectors. Drug-delivery approaches comprise delivery from microspheres, biodegradable wafers, and colloidal drug-carrier systems (e.g., liposomes, nanoparticles, nanogels, dendrimers, micelles, nanoemulsions, polymersomes, exosomes, and quantum dots). These alternative approaches look promising.

The Canadian company Angiochem is using a physiological approach to gain entry across the BBB. They have engineered ANG1005, an Angiopep-2 paclitaxel conjugate to gain entry into the brain by targeting lipoprotein receptor-related protein (LRP-1), which is one of the most highly-expressed receptors on the surface of the BBB.  Once inside the brain, ANG1005 enters tumour cells using the same receptor-mediated pathway through LRP-1, which is upregulated in various cancer cells including malignant glioma and metastatic cancers in the brain. (7) Phase II data presented in October 2016 shows ANG1005 has demonstrated clinical benefit, both intracranially and extracranially in pre-treated breast cancer patients with recurrent brain metastases. (8)

Blog written by Kamlesh Bala

References

(1)www.parexel.com/files/4314/4113/4032/Venturing_Into_a_New_Era_of_CNS_Drug_Development_to_Improve_Success.pdf

(2) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC539316/

(3) https://bmcneurol.biomedcentral.com/articles/10.1186/1471-2377-9-S1-S3

(4) ACS Chem Neurosci. 2010 Jun 16;1(6):435-49

(5) ACS Chem. Neurosci. 2016, 7, 767−775

(6) Patel, M.M. & Patel, B.M. CNS Drugs (2017).

(7) http://angiochem.com

(8) http://angiochem.com/angiochems-ang1005-shows-clinical-benefits-and-prolonged-survival-breast-cancer-patients-brain

 

A powerful small molecule; believing in GABA


rosemary-1

GABA (γ-aminobutyrate) is the major inhibitory neurotransmitter in the mammalian brain. It contributes to the all-important excitatory-inhibitory balance of neuronal communication. Dysfunction of the GABAergic system is implicated in disease, such as epilepsy, insomnia, schizophrenia and anxiety disorders. The history of GABA, from when it was first discovered to when its power as an inhibitory neurotransmitter was accepted, is an interesting one. It is wrapped up in personal accounts of the scientists involved, and looking back at the winding path of acceptance is fascinating.[1],[2],[3]

The story starts with the discovery of a ninhydrin-reactive material in mouse brain tumours by Eugene Roberts and Sam Frankel in 1949.[4] GABA had been known since 1910, of course, and had been found in bacteria and fungi and potatoes.[5] Once the structure of this ninhydrin-reactive material was confirmed to be GABA by Sidney Udenfriend, Roberts scribbles “the brain is like a potato!” in his notebook, and sets about proving its biological significance. [6],[7]

Roberts and Frankel’s work was published in 1950 for a conference at which Jorge Awapara also reported the presence of an “unidentified amino acid” found only in the brain.[8] By the time of the conference Awapara had also recognised it was GABA. At the conference a room-shortage led these two into close proximity, and Awapara and Roberts apparently agreed that Roberts would continue working on metabolism of GABA, and Awapara would continue working on the metabolism of taurine.7b

Six years pass, and scientists were still questioning whether the presence of GABA in the brain meant it had a role in signal conductance.[9] It should be noted that the debate over whether synaptic transmission was electrical or chemical had only just reached its chemical conclusion.[10] Ernst Florey had found Factor I, a chemical of unknown composition, extracted from mammalian brain and which inhibited the crayfish muscle system amongst others.[11] With help from Merck scientist Alva Bazemore, they purified over 45 kg of cow brain, and, using over 450 L of acetone they managed to yield crystalline Factor I. It was GABA, obviously.[12] There were still things to clear up, however, as some of their earlier work on the spinal cord of cats was initially unrepeatable by David Curtis, a fact now anecdotally put down to the allosteric modulation by the barbiturates also used in the experiments, or contamination of glycine in Florey’s earlier samples.11, [13]

Evidence against GABA being a neurotransmitter was accruing. The fact that GABA was so widespread in the brain was a worry for many in the field. “It’s just a metabolic wastebasket” remarked one great neurochemist.7b Strychnine was known to inhibit transmission in the spinal cord, and yet did not compete with GABA, which was another point of confusion at the time. With hindsight we know it was the as-yet-undiscovered glycine receptors in the spine causing havoc here.

Some scientists remained convinced there was more to the story, and Eugene Roberts was one of them. “Stop bringing your GABA solutions to our labs,” pleaded biologists, daily. “They take up room, and we’re not going to test them.”

In 1959 a notable conference was convened to hear the latest research and clear up the confusion. This proceeding (the 1st Interdisciplinary Conference on GABA) at a nearby pub (City of Hope Research Institute) was attended by many of the leading people in the field. Excitement was in the air as scientists from Australia, Canada, England, France, Hungary, Japan, the United States and the former Soviet Union bustled together, spilling pints and talking Neuroanatomy. Most researchers had a top night, and left feeling like they had made life-long friends and collaborations from all over the world.

GABA itself, however, fared badly. It was not going to join the echelons of the neurotransmitters quite yet. Alongside everything else it was seen not to have a rapid, enzymatic degradation pathway, unlike the newly-discovered acetylcholine neurotransmitter. This was thought to preclude it from involvement in rapid on-off communication. GABA was a metabolite with some generalised depressant nature, but that was all.[14]

It was not until nearly ten years later that unequivocal proof was published by Krešimir Krnjević and Susan Schwartz in 1967 that GABA was shown to be the major inhibitory neurotransmitter.[15] Intracellular recordings, as well as pharmacological tests revealing selective block by picrotoxin and bicuculline, clearly demonstrated GABAergic transmission.[16] After this proof, there was an exponential growth in GABA research. Writing in 1974, Eugene Roberts says ‘In the 25 years since the discovery of GABA … the status of the compound has passed from that of a biochemical curiosity and physiological enigma to that of a major inhibitory transmitter.’[17] At fifty years GABA receptors are shown to have decisive roles in many diseases, and they become big targets in drug discovery, including big-hitting benzodiazepines and general anaesthetics.[18] At 67 years, there are a huge 74,000 references containing ‘gamma-aminobutyric acid’ on Scifinder Scholar.[19]

The mixture of personal and peer-reviewed literature accounts of how GABA came to be accepted is what makes it fascinating. It is useless to deny that the direction of any scientific progress is open to contemporary influences and personal preferences. Perhaps it just reminds us that, as Carl Sagan writes: “Nature is always more subtle, more intricate, more elegant than what we are able to imagine.” And that means the path to understanding this transmitter will never stop winding.

Blog written by Rosemary Huckvale

POST SCRIPTS

  1. Dr Eugene (Gene) Roberts died last year on 8th November 2016 at the age of 96. His obituaries can be found here.
  2. This blog hopes to relate only a small portion of the history surrounding the establishment of GABA as a major inhibitory transmitter. There will have been other important contributions not mentioned here.

REFERENCES

[1] Johnston, G.A.R, GABA Australis, some reflections on the history of GABA receptor research in Australia, Pharmacological Research, article in press, 2016.

[2] Avoli, M., Krnjević, K., The Long and Winding Road to Gamma-amino-butyric acid as neurotransmitter The Canadian Journal of Neurological Science, 43:219-226, 2016.

[3] Krnjević, K. How does a little acronym become a big transmitter? Biochemical Pharmacology, 68:1549-1555, 2004.

[4] a) Roberts, E. and Frankel, S. γ-aminobutyric acid in brain. Federation Proceedings 9:219, 1950. b) Roberts, E. and Frankel, S. γ-aminobutyric acid in brain: its formation from glutamic acid. Journal of Biological Chemistry 187:55-63, 1950.

[5] Steward FC, Thompson JF, Dent CE. γ-Aminobutyric acid. A constituent of the potato tuber? Science 110:439-440, 1949.

[6] Udenfriend, S. Identification of gamma-aminobutyric acid in brain by the isotope derivative method. Journal of Biological Chemistry 187:65-9, 1950.

[7] Roberts, E.. Gamma-aminobutyric acid, Scholarpedia, 2(10):3356, 2007. B) Roberts, E., The History of Neuroscience in Autobiography Volume 2, Edited by Squire, L.R, Academic Press, 350-395, 1999.

[8] a) Awapara, J. Detection and identification of metabolites in tissues by means of paper chromatography. Federation Procedings, 9:148, 1950. b) Awapara, J., Landua, A.J., Fuerst, R., and Seale, B. Free gamma-aminobutyric acid in brain. Journal of Biological Chemistry 187:35-9, 1950.

[9] Roberts E. Formation and utilization of γ-aminobutyric acid in brain. Edited by Korey S.R., and Nurnberger J.I., Progress in neurobiology. 1. Neurochemistry. New York: Hoeber-Harper, 1956;11-25.

[10] Eccles, J.C., The Synapse: From electrical to Chemical transmission, Annual Review of Neuroscience, 5:325-39, 1982.

[11] For overview see Florey, E., GABA: history and perspectives, Canadian Journal of Physiology and Pharamcology, 69(7):1049-56, 1991.

[12] Bazemore, A.W., Elliott, K.A.C., Florey, E., Isolation of Factor I, Journal of Neurochemistry, 1:334-9, 1957.

[13] Johnston, G.A.R, GABA Australis, some reflections on the history of GABA receptor research in Australia, Pharmacological Research, article in press, 2016.

[14] Bowery, N.G, Smart, T.G, GABA and glycine as neurotransmitters: a brief history. British Journal of Pharmacology, 147:109-119, 2006. And references therein.

[15] a) Krnjević, K. & Schwartz, S. Is γ-aminobutyric acid an inhibitory transmitter? Nature, 211:1372, 1966. b) Krnjević, K. & Schwartz, S. The action of γ-aminobutyric acid on cortical neurones. Experimental Brain Research, 3, 320–326, 1967.

[16] Curtis, D.R., Duggan, A.W., Felix, D., Johnston, G.A.R., GABA, Bicuculline and central inhibition., Nature, 226:1222-1225, 1970.

[17] Roberts, E., γ-aminobutyric acid and nervous system function – a perspective, Biochemical Pharmacology, 23:2637-2649, 1974.

[18] GABA in the Nervous System: The view at Fifty Years, Edited by Martin, D.L and Olsen, R.W., Lippincott Williams & Wilkins, 2000.

[19] Accessed Jan 2017.


A brief introduction to enzyme inhibitors -Nonspecific, Irreversible and reversible (competitive, uncompetitive and non-competitive) inhibitors


Enzyme inhibitors are molecules that bind to the enzyme and reduce the catalytic activity of enzymes. There are many types of inhibitors, including nonspecific, irreversible or reversible (competitive, uncompetitive and non-competitive inhibitors).

Non-specific inhibitors can inhibit multiple enzyme targets by forming the aggregate. One mechanism for this nonspecific interaction is the formation of colloidal aggregates by self-association of low molecular weight compounds in aqueous solutions. These aggregates tend to sequester protein, presumably by partially unfolding it and thus inhibiting its function.[1]

Irreversible inhibitors usually react with the enzyme by forming covalent bonds; thus dissociating very slowly from its target enzyme.[2, 3] There are some important drugs act as irreversible inhibitors to the enzyme. One example is penicillin, as a first discovered antibiotic, can covalently bind to the enzyme transpeptidase by mimic the normal substrate the D-Ala-D-Ala peptide. It is therefore preventing the synthesis of bacterial cell walls and killing the bacteria.[2] (Fig.1-2) It is very useful to identify if inhibitors are irreversible or not. One method is through dilution, namely measuring the recovery of enzymatic activity after a rapid and large dilution of the enzyme-inhibitor complex. [3, 4] (Fig.3)

tina-1

Figure 1. Transpeptidation Reaction. An acyl-enzyme intermediate is formed in the transpeptidation reaction leading to cross-link formation. Figure is from the following published study: Reference 2.[2]

tina-2

Figure 2. Conformations of Penicillin and a Normal Substrate. The conformation of penicillin in the vicinity of its reactive peptide bond (A) resembles the postulated conformation of the transition state of R-d-Ala-d-Ala (B) in the transpeptidation reaction. Figure is from the following published study: Reference 2.[2]

tina-3

Figure 3. Hypothetical time-courses for activity recovery after rapid dilution of enzyme-bound inhibitor into a solution containing saturating substrate concentration. C, Control sample having no inhibitor present; R, Reversible inhibitor bound as E$I complex at zero-time; SR, Slowly-reversible inhibitor bound as E$In complex at zero-time, but must isomerize through one or more intermediate states to form E$I complex, which then releases inhibitor; I, Irreversible inhibitor bound as E–I complex at zero-time. The actual time-scale for inhibitor release will depend to the transit time for isomerization of enzyme-inhibitor complexes to that enzyme species from which inhibitor is released. Figure is from the following published study: Reference 3.[3]

In contrast with irreversible inhibitors, reversible inhibitors have a rapid dissociation to the enzyme and bind non-covalently. [2, 3] Reversible inhibitors contain three types of inhibitors, competitive, uncompetitive and non-competitive, depend on whether the inhibitors compete with substrate binding to the enzyme or binding the enzyme-substrate complex.[3] (Fig.4) Competitive inhibitors can compete with substrate to bind the active site of the enzyme and this binding state can be relieved by increasing the substrate concentration. On the contrary, noncompetitive inhibitors can bind to the enzyme simultaneously with the substrate and this inhibition cannot be overcome by increasing the substrate concentration. Uncompetitive inhibitors can only bind to the enzyme-substrate complex, in which a binding site is formed.

tina-4

Figure 4. Three types of reversible inhibitors: A. competitive; B. noncompetitive; C. uncompetitive inhibitors. Figure is from the following published study: Reference 3.[3]

These three types of inhibitions can be determined by Michaelis-Menten kinetics. Since the competitive inhibition can be overcome by a sufficiently high concentration of substrate, Vmax can be maintained in the same value and Km value (Km = [S] at Vmax/2) is increased in the presence of a competitive inhibitor. Noncompetitive inhibitors inactive the enzyme, lower the concentration of functional enzyme, so Vmax is declined and Km is unchanged. Uncompetitive inhibitors work best when substrate concentration is high, both Vmax and Km values are therefore decreased.[2, 5](Fig.5)

tina-5

Figure 5. A. Competitive inhibition is characterized by an increase in Km for the substrate and no change of Vmax. B. Non-competitive inhibition is characterized by no change in the Km value and a decrease in Vmax. C. Uncompetitive inhibition is characterized by a decrease in the apparent Km value and a decreased Vmax. Figure is from the following published study: Reference 5.[5]

Among three types of reversible inhibitors, uncompetitive inhibitors are rare, but some potent drugs have been regarded as uncompetitive inhibitors to the enzyme.(Table.1) Many enzyme-substrate complexes are extremely short-lived in the enzyme-catalyzed reactions, so uncompetitive inhibitors have much less opportunities to bind the target than other two types of reversible inhibitors. [3] One example is about the first uncompetitive inhibitor-blebbistatin of myosin, binding at a site other than the nucleotide- or actin filament-binding sites, which opens and closes during the contractile cycle. Blebbistatin can stabilize the metastable or ‘transition’ state of myosin, representing a long-live complex of myosin with ADP and inorganic phosphate. [3, 6]

tina-6

Table 1. Several clinically useful uncompetitive inhibitors. Table is from the following published study: Reference

Blog by Tina(Xiangrong) CHEN


References

  1. Habig, M., et al., Efficient elimination of nonstoichiometric enzyme inhibitors from HTS hit lists. Journal of biomolecular screening, 2009. 14(6): p. 679-689.
  2. Berg, J.M., J.L. Tymoczko, and L. Stryer, Biochemistry New York. NY: WH Freeman, 2002.
  3. Purich, D.L., Enzyme kinetics: catalysis and control: a reference of theory and best-practice methods. 2010: Elsevier.
  4. Copeland, R.A., Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists. 2013: John Wiley & Sons.
  5. Kenakin, T.P., Chapter 6 – Enzymes as Drug Targets, in Pharmacology in Drug Discovery. 2012, Academic Press: Boston. p. 105-124.
  6. Allingham, J.S., R. Smith, and I. Rayment, The structural basis of blebbistatin inhibition and specificity for myosin II. Nature structural & molecular biology, 2005. 12(4): p. 378-379.

 

 

Understanding Tuberculosis and the challenges ahead


Recently, I had the opportunity to meet one of my colleagues from Sussex University whose area of expertise and research is based in tuberculosis (TB), Dr. S. Wadell. This heightened my curiosity on the disease and I wanted to know more.

Thus I have chosen the paper in Expert Opinion on Drug Discovery by Anuradha, Kumar et al. [1].

TB is caused by the bacteria Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle and badgers, where it still an area of ardent discussion and where there is still lot of work to do [2]

TB is highly likely to spread in areas of poor sanitation, poor access to drugs and where patients have to be confined; it is treated with cocktails of up-to-4 antibiotic drugs over periods of time greater than 6 months.

Far from being an under-controlled disease as I initially thought, TB bacteria still possess a threat to humans due to:

  1. The nature of bacteria: stick-shaped cell with a highly lipophilic-rich cell membrane, slow growing and highly infectious organism which has to be dealt in a controlled environment.
  2. Its physiology: present in an active/ latent form, existing in multiple physiological states (explaining the length and combination of antibacterial drugs required for an effective treatment)
  3. The lack of animal models due to the difficulty to replicate the disease as it occurs in humans.
  4. Location: the bacteria can be found intracellular (eg. Macrophages) or extracellular in granulomas; or cavities, individually or as aggregates. Adding a second cell membrane to be crossed and making drug delivery and distribution a challenge.
  5. Drug-resistance as it has followed in other bactericidal-type diseases. Sometimes appears associated to HIV infection.
  6. Difficulty to identify the biological environment, small target space and mechanism of action
  7. Traditional approaches targeting biochemical assays have failed due to screening libraries of compounds based in Lipinski’s rule of 5 with a low number of compounds meeting the clogP > 5 requirement is matched [3].

In contrast, the whole-cell screening, although more successful, has highlighted promiscuous targets like MmpL3 and QcrB alerting that the development of new drugs will have to answer the need to have  multiple-target active compounds.

The use of TB infected macrophages has been used as valid source of new inhibitors, either from synthetic origin as well as based in natural products [4] – open the door to chemicals no covered in commercial screening libraries. This approach has later derived in obtaining semi-synthetically analogues from Rifamycin, where the common structure (inside the blue box) is kept and highlighted are the modifications in Fig 1.

jose

   Fig 1 Current therapeutic drugs derived from Rifamycin family discovered in the 1950s and its semi-synthetic analogues.

The synthesis of active analogues from natural products can be challenging but it has recently highlighted some potential leads to be further investigated [5]. Therefore, new active compounds could follow from the isolation of active substances based in their physiochemical properties (eg. high lipophilicity) from sub-fractions of natural product mixtures [6].

The authors emphasise that current techniques using metabolomics and genomics could be valuable tools in the design and understanding the synthesis of new active compounds. The use of Diversity-orientated synthesis (DOS) is pointed as a way to generate and explore the untargeted chemical space [7].

Clear progress and innovation from new emerging techniques have led to a better understanding in fighting the disease. The lack of compounds with specific physicochemical properties as well as suitable animal models, how to penetrate the TB lipophilic cell membrane, shorter and more efficient patient treatments and which targets to focus on are going to be the areas where it is anticipated more researching resources will be deployed.

Blog written by Jose Gascon

References:

[1] Anuradha Kumar, Somsundaram Chettiar & Tanya Parish, Expert Opinion in Drug Discovery, 2017, Vol

12, 1, 1-4

[2] https://www.theguardian.com/science/2012/nov/11/alice-roberts-bovine-tb-badgers ;

http://www.tbfreeengland.co.uk/faqs/

[3] Koul, A et al  The challenge of new drug discovery for tuberculosis Nature, 2011, Jan 27:469

(7331):483-490. DOI:10.1038/nature09657

[4] Cragg, GM et al  Natural products: a continuing source of novel drug leads. Biochim Biophys Acta,

2013, 1830, 6, 3670-3695.  DOI:10.1016/j.bbagen.2013.02.008

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