IISc’s potent molecules show promise for TB therapy

SEM photo of Mycobacterium tuberculosis bacteria. - Photo NIAD-Optimized

The two molecules were able to prevent biofilm formation and even disrupt biofilms that had formed.

Scientists at the Indian Institute of Science (IISc) Bengaluru have developed two new, potent molecules that can severely impact the survival of mycobacteria, including Mycobacterium tuberculosis that causes TB. The results were published in the journal Antimicrobial Agents and Chemotherapy.

Unlike most antibiotics that target the bacterial metabolism by aiming at the cellular components, the novel molecules inhibit the stress response pathway of mycobacteria. The stress response pathway is crucial for bacteria to survive during hostile conditions such as lack of nutrients and the presence of antibiotics, to name a few. So any inhibition of this pathway will lead to its death.

The master regulator of stress pathway in the case of mycobacteria is (p)ppGpp (Guanosine pentaphospahte or Guanosine tetraphosphate). Though a molecule that inhibits the (p)ppGpp formation has already been synthesised, the efficacy is not much. “Very high concentration of Relacin molecule is needed to inhibit the pathway and, therefore, the efficacy is low. So we synthesised two new molecules — acetylated compound (AC compound) and acetylated benzoylated compound (AB compound) — by bringing about a modification in the base of the Relacin molecule,” says Prof. Dipankar Chatterji from the Division of Biological Sciences at IISc and the corresponding author of the paper.

“We found both the molecules to be very good inhibitors of stress response. The two compounds affected the rate of synthesis of (p)ppGpp and also reduced the cell survival,” he says. Laboratory studies showed that the two molecules were not toxic to human cells and were able to penetrate the human lung epithelial cells.

IMG_0508

Inhibiting (p)ppGpp synthesis would target the survival of the bacteria, says Dr. Kirtimaan Syal.

“We found our compounds were targeting the Rel gene. The Rel gene makes Rel protein, which in turn synthesises (p)ppGpp. When the Rel gene is knocked out, the long-term survival of Mycobacterium smegmatis decreases,” says Prof. Chatterji.

“The Alarmone molecule “(p)ppgpp”, a modified nucleotide, is ubiquitous in bacteria and absent in humans. Inhibiting (p)ppgpp synthesis would specifically target the survival of bacteria without having any effects on humans,” says Dr. Kirtimaan Syal from the Division of Biological Sciences, IISc and the first author of the paper.

Earlier studies have shown that when the rel gene is deleted, the long-term survival ability under stress was lost; the M. tuberculosis bacteria was unable to persist in mice and unable to form tubercle lesions in guinea pigs.

“The major reason for prolonged treatment of TB is the bacterium’s ability to persist in dormant form, which is tolerant to most antibiotics used in the treatment regimen. So inhibition of (p)ppGpp-mediated persistence could help in shortening the treatment regime, dealing with the emergence of multiple drug resistance and treatment of chronic infections, Dr. Syal says.

Inhibiting biofilm

Under hostile conditions, bacteria tend to form biofilms, which protect the bacteria from stress and induce tolerance to antibiotics. Recent studies have shown that tuberculosis bacteria that cannot form a biofilm cannot survive inside the host. Evidences have shown that at the time of infection, the M. tuberculosis display a biofilm-like phenotype and this helps the bacteria to survive inside the host.

Studies carried out by the researchers showed that both the molecules were able to inhibit biofilm formation by M. tuberculosis and M. smegmatis and also disrupt the already formed biofilm. “The biofilm formed by TB bacteria is very dangerous. The ability of the molecules to destroy the biofilm and even prevent its formation is a very important achievement,” says Prof. Chatterji.

Since there are very few antibiotics that target the stress response pathway of the bacteria, the two molecules offer great promise. “The next step is to test the molecules on animals. We have not thought about it. It will also be interesting to see if the bacteria develop resistance against these molecules,” Prof. Chatterji says.

Published in The Hindu on April 15, 2017

Indian researchers find a new bacterial target for drug development

Anshika Andaleeb Richa-Optimized

(From left) The study by Anshika Singhal, Andaleeb Sajid and Richa Misra helped understand how bacteria form biofilm.

Indian researchers have found a new target that can potentially be used for developing new antibiotics that will be effective against many bacteria. The new target is made of two proteins — which form a complex that is responsible for the formation of biofilm — that perform very important functions and are critical for bacterial ability to successfully infect humans. The results were published in the journal Biofilms and Microbiomes.

Bacteria form biofilms, a kind of matrix, during infection in plants and animals. Biofilm shields the bacteria from antibiotics and help bacteria to survive harsh conditions such as extreme temperature or stress. Now a study by Indian researchers has found the molecular signaling events that play a crucial role in biofilm formation in Bacillus anthracis, the causative agent of anthrax.

Till now, all attention has been on developing antibiotics that target disease-causing bacteria and not the biofilm itself.
One of the basic questions that scientists have been trying to answer is how and when bacteria decide to form biofilm. “One possibility is that bacteria has sensors on the surface which senses some signal and helps in biofilm formation,” says Andaleeb Sajid from the Institute of Genomics and Integrative Biology (IGIB), Delhi and one of the authors of the paper.

“It was serendipity. Our lab was working on signaling in bacteria and we were studying PrkC and similar proteins. When PrkC protein is deleted, Bacillus bacteria are unable to form biofilm. So we started studying the mechanism by which PrkC protein controls biofilm formation,” she says.

IMG_0485

Gunjan Arora says the GroEL-PrkC complex could be a target for developing new drugs.

“Our hypothesis is that PrkC senses some signal and transmits it from outside to inside the cell. This signal goes to other proteins like GroEL. PrkC adds phosphate group (phosphorylate) to different proteins. The mystery to biofilm formation lies with one chaperone protein called GroEL. The addition of phosphate to this tiny machine initiates a course of events within bacterial cells leading to complex biofilm formation,” Dr. Sajid says.

The team found several proteins receive signals from PrkC protein. Using cutting edge genetics, molecular biology and proteomics techniques, they confirmed that GroEL was regulated by PrkC.

“From other unrelated bacteria we already had a clue that GroEL has a role in biofilm formation. We looked at the molecular level and found six amino acid residues where phosphate was getting added to the GroEL protein. Through a series of steps, we ascertained how important phosphorylation was for proper functioning of GroEL,” says Gunjan Arora from IGIB and the first author of the paper.

“We wanted to know if the bacteria has any other compensation mechanism to form biofilm in the absence of PrkC. So we made PrkC mutant bacteria to produce more of GroEL. The bacteria were able to form biofilm even in the absence of PrkC. This experiment helped us understand that PrkC is the influencer and GroEL is key to biofilm formation,” Dr. Arora says.

Both PrkC and GroEL perform very important functions and are critical for bacterial ability to successfully infect humans. “We think GroEL-PrkC complex could be a target for developing new antibiotic that will be effective against many bacterial pathogens such as the ones that cause MRSA, TB and pneumonia. One strategy to tackle drug resistant bacteria will be to develop multi-drug regimen that combines traditional antibiotics with candidate drugs that can block bacterial signaling and prevent biofilm formation,” Dr. Arora says.

Published in The Hindu on March 26, 2017

Drug discovery for GPCR signalling made easy by IIT Kanpur researchers

arun-photo

The work by (from left) Ashish Srivastava, Punita Kumari and Dr. Arun Shukla changes the way people will look at drug discovery for GPCR signalling.

Discovering new drugs that bind to G Protein-Coupled Receptors (GPCRs), which are central to almost every physiological process in our body such as vision, taste, immune response and cardiovascular regulation, has become easier, thanks to research by a team led by Dr. Arun K. Shukla from the Department of Biological Sciences and Bioengineering, Indian Institute of Technology (IIT) Kanpur.

Nearly 50 per cent of prescription drugs currently available in the market for the treatment of blood pressure, heart failure, diabetes, obesity, cancer and many other human diseases target GPCR receptors. All these drugs bind to their respective receptors and either activate or stop their signalling. The work by Dr. Shukla’s team has shown that the regulation of these receptors by these drugs can be simpler than generally thought — it can be mediated by engaging only the end of the receptor, which is called the tail of the receptor. The results were published in the journal Nature Communications.

Receptors found on the cell surface receive signals and transmit them to inside the cells. A part of the receptor is embedded in the cell membrane and the other part protrudes outside the membrane and inside of the cell. The part of the receptor that protrudes outside the membrane changes its shape whenever a stimulus in the body binds to itm. In response to this change in the outside part of the receptor, a corresponding change happens in the shape of the receptor that is positioned inside the cell. This change in the shape of the receptor positioned inside the cell allows it bind to other proteins called effectors. These effectors cause specific effects in the cell, referred to as cell signalling, which leads to physiological changes in our body.

For example, a hormone in the blood called angiotensin binds to its receptor and activates the effector protein inside the cell causing an increase in blood pressure.

In people with normal blood pressure, specific type of proteins called arrestins, which are effector proteins of GPCRs, bind to the receptor and pull it inside the cell (a process called receptor endocytosis). This prevents the angiotensin from binding to the receptor, thereby help in controlling the blood pressure.

In the case of people with high blood pressure, the prescribed drug binds to the receptor. So even if angiotensin is present on the surface of the cell, it cannot bind to the receptor and start the signalling process that increases blood pressure.

“We were interested in understanding how different receptors interact with effectors and how the receptors recognise the stimuli,” says Dr. Shukla. “We looked at the interaction of a receptor, which is a target for heart failure drugs, with its specific effectors, namely arrestins. When arrestins bind to the receptor, they arrest or disrupt the receptor signalling.”

“The text book understanding is that arrestins have to simultaneously bind at two sites — the tail of the receptor and the core of the receptor — for the drug to become effective in pulling the receptor inside the cell [to prevent the stimuli from binding to the receptor and start signalling],” says Dr. Shukla. “Through specific engineering of the receptor we basically disrupted one of the two binding sites, namely the core of receptor. We found that even without the second site, the arrestin was able to pull the receptor inside the cell by binding just to the tail of the receptor [which is the other binding site].”

There is a key region in the core which the team genetically deleted thereby making the core of the receptor ineffective.

“Whenever researchers are designing a drug to stop GPCR signalling, they look for a drug that simultaneously triggers the binding of arrestins to both the sites in the receptor. Our work changes the way people will look at drug discovery for GPCR signalling,” he says. “The drug has to trigger binding of arrestin to just at the tail of the receptor to arrest the signalling. Researchers can now design simple drugs to accomplish this.”

Published in The Hindu on January 1, 2017

ICGEB researchers adopt a novel approach to drug discovery

amit-optimized

(From left) Manmohan Sharma, Dr. Amit Sharma and Manicham Yogavel used a conserved enzyme family in parasites for drug discovery

Researchers from the International Centre for Genetic Engineering and Biotechnology (ICGEB), Delhi have found a novel route to discover new drug targets and potential drugs for parasites that cause several diseases such as Loa loa nematode (roundworm) and Schistosoma mansoni platyhelminths (flatworm). The results were published in the journal PLOS Neglected Tropical Diseases.

Both these parasites cause a major health burden particularly in African countries. There are limited treatment options and there is the usual threat of drug resistance. There is little interest in developing drugs for these diseases by pharmaceutical companies; they are hence called neglected tropical diseases.

Instead of blindly screening molecules, which takes a long time and is expensive, a team led by Dr. Amit Sharma from the Molecular Medicine Group at ICGEB looked at Aminoacyl-tRNA synthetases (aaRSs) of the two parasites.

The aaRSs are vital enzymes that decode genetic information and enable protein translation. “The reason why we chose the tRNA synthetase enzyme family is because it is conserved (genomic similarity) in malaria and other parasites including L. loa and S. mansoni,” says Dr. Sharma, the corresponding author of the paper. The novel approach of looking at the conserved region of the parasites is direct, quicker and cheaper.

The aaRSs enzyme family has 20 members and each one of the enzymes contributes to protein synthesis. Even if one of the 20 enzymes is missing then protein synthesis cannot happen. “We have elaborated all the critical aaRs enzymes that contribute to protein synthesis,” he says.

In a next step, the team picked up one of the enzymes and validated it as a drugable target. For that purpose, the enzymes were recombinantly produced and their activities were studied. “Cladosporin, a very potent compound that targets the malaria parasite in both blood and liver stages, seems to inhibit the enzymes of the L. loa and S. mansoni with high potency,” Dr. Sharma says.

The researchers studied the crystal structure of the enzyme with cladosporin. This revealed how tightly the drug binds within the active site of the enzyme. The researchers could understand the active sites of the enzyme and how the drug inhibits their enzyme activity.

“Once we have solved the crystal structure of the enzyme we were able to make an atomic map of all the interacting atoms of both the drug and enzyme. The crystal structure helps us to identify the drug pockets in the enzyme where the drug binds,” Dr. Sharma says.

So once the genome sequence of any parasite is known then it becomes possible to look at the same interacting atoms in the highly conserved tRNA synthetase enzyme family and assess whether the drug will be able to inhibit the enzyme activity. “In the days before the genome sequence was available this approach could not have been used,” he says.

Cladosporin promise

Cladosporin is a very potent compound that targets the malaria parasite. In this study the researchers have shown that the drug compound is a “very potent inhibitor” of essential enzymes in L. loa and S. mansoni as well. The proof-of-concept data shows that it is possible to use the compound to target the tRNA synthetase enzyme family of other parasitic worm diseases.

Since bioavailability of cladosporin compound is a major issue, derivatives of the compound have to be developed for use as an effective drug. “Drug discovery for malaria is being done based on derivatives of this compound. So these derivates can be tested on other parasites too. It will save a lot of time and resources,” he says.

Published in The Hindu on December 6, 2016

When pills make us worse: Monitoring the adverse effects of medicines

Basu

By Debaleena Basu

A healthy 21-year-old youth was admitted to a hospital for an elective surgery. All preoperative physiological parameters were normal. On the surgery day, the commonly used drug bupivacaine was administered to induce spinal anaesthesia. Following the injection, he immediately went into shock. His blood pressure dropped, pulse weakened, and his breathing became abnormally rapid. Efforts made to reverse the situation failed and he succumbed within a day.

This case report is one of the five mentioned in a May 2014 study published in the Indian Journal of Critical Care Medicine, which documented casualties arising from adverse reactions to drugs (ADRs) in an Indian hospital. Adverse drug reactions refer to undesirable effects of a drug beyond its expected therapeutic function and often have serious consequences.

Drug toxicity has become a major concern for healthcare providers globally and is reported to be among the top ten causes of death. It poses an economic as well as public health burden on nations. With more and more new medicines being introduced in the market, active monitoring of the side effects of drugs has become the need of the hour.

One may ask why there are any adverse effects at all! Aren’t all licensed medicines safe? In the above case, a commonly used anesthetic, bupivacaine was administered. Discovered in 1957, the chemical bupivacaine has undergone multiple clinical trials to estimate its safety and efficacy. Today, it is a frequently used drug, and is also a part of the World Health Organisation (WHO) model list of essential medicines, a compilation of necessary medicines required for a basic healthcare system. Yet, the usage of such a tried-and-tested, frequently applied drug, took a tragic turn, resulting in an unexpected and untimely death. ADRs are on the rise due to various factors, a major one being the inherent shortcomings of the drug discovery and approval process.

Mice4. Photo Sathees Raghavan-Optimized

Information collected during animal trials help in deciding the start of clinical trials on humans.

New promising chemical compounds discovered in the lab go through a rigorous drug development process to establish the basic properties such as the chemical makeup, toxicity and stability of the compounds. Pre-clinical tests, also known as animal trials, involve testing on animals, and the information gathered helps in deciding the start of clinical trials on humans.Clinical trials with human volunteers are conducted in phases, with each approved phase progressively testing the safety and efficacy of the experimental drug in larger cohorts of people. However, even at the end of all the three phases of the trial (Phase-I, II & III), the effect of a potential drug is studied only in a select group of typically 5,000 volunteers or less.

That a drug, which is 100 per cent selective and specific in its therapeutic action and has no adverse side effects in the body, is at best a mythical one. The clinical trial mechanism is there to only allow potential drugs that maximize benefit to be released into the market. Even with the current improved standards of clinical trials, every possible side effect of the drug may not show up during evaluation.

For one, rare adverse effects may not get manifested in the limited patient pool tested in clinical trials. Additionally, clinical trials only report adverse reactions that appear within the limited duration of the trial. Also, children, pregnant women, and the elderly are typically not included, or under-represented in clinical trials, and thus, the safety of the drug in these cases remains unknown until its release.

The only way to find out about such adverse reactions for drugs being currently used by the public is by being vigilant for negative side effects, and accordingly, regulating the drug’s description and distribution.

Monitoring adverse drug reactions

India is a developing country having a large drug consuming population with substantial ethnic variability. It is the third largest producer of pharmaceuticals by volume in the world and new medicines are continually flooding the market. It is crucial that adverse drug reactions are identified as early as possible to ensure the well being of the populace at a reasonable cost.

The WHO established its Program for International Drug Monitoring in response to the thalidomide disaster. The WHO international collaborating center at Uppsala promotes drug monitoring at the country level. 

Following a number of unsuccessful attempts, the current nation-wide Pharmacovigilance program of India (PvPI) was initiated in 2010 in collaboration with the WHO’s drug monitoring program.

Pharmacovigilance refers to the detection, assessment and prevention of adverse drug reactions. The program is overseen by the Central Drugs Standard Control Organization (CDSCO), under the aegis of the Ministry of Health and Family Welfare.

Healthcare professionals have most important role to play in pharmacovigila nce. William McBride, an Australian doctor, published the now famous case report in The Lancet in 1961, suspecting a causal link between thalidomide intake and serious foetal deformities, which ultimately lead to the banning of the drug. He is considered by many to be the first ‘pharmaco-vigilante’.

The physician is the one who has knowledge of the patient’s medical history, and is the first one to suspect an ADR, thus it is crucial for any pharmacovigilance program to recruit as many doctors as possible. Under the PvPI, 179 hospitals and medical institutes, both government and private, have doubled up as ADR monitoring centers.

When an ADR is suspected, any medical personnel (doctors, nursing staff, interns) can report it. Patients experiencing side effects can also submit reports about the drug. Reports from the individual monitoring centers are collected and analyzed at the national coordinating center for PvPI, the Indian Pharmacopoeia Commission (IPC) at Ghaziabad. If the ADR data is found to be robust and ‘valid’ after review, the IPC recommends regulatory interventions to the central drug regulatory authority. It also communicates the risks associated to healthcare professionals and the public through quarterly newsletters. Additionally, the IPC contributes the reports to the global ADR database (Vigibase) maintained at WHO’s Uppsala Monitoring Center.

Pharmacovigilance in action

Many of us do not look through the package inserts of medicines, containing the list of possible side effects in miniature font. Each side effect is included only after careful evaluation of test results by expert committees. The interest of the pharmaceutical companies lies in keeping the list small, while that of patients’ is in knowing about all possible reactions.

insert

Package insert. – Photo: CDC

The drug carbamazepine is a commonly prescribed anticonvulsant medicine for epilepsy, and falls under the WHO’s recommended essential medicine list. The PvPI received 1887 reports (over a period of three years) of adverse drug reactions associated with carbamazepine from the nationwide ADR monitoring centers, out of which 119 were life threatening or fatal. Furthermore, a certain genetic variant commonly found in the Indian population (HLA-B* 1502), was found to be strongly associated with severe ADRs.

Based on the reports submitted by doctors to the PvPI database, the IPC recommended a label change to include the risk factors. The central drug regulatory body (CDSCO) has recently issued a directive for all manufacturers to revise package inserts of carbamazepine after reviewing the recommendations, in a bid to sensitize healthcare providers to the observed risks.

The road ahead for PvPI

India’s current pharmacovigilance program has made notable progress in the past few years. According to a PvPI update, India is the first Asian country to have more than 1.0 lakh individual case safety reports in the Uppsala center’s global database, making it among the top 10 countries to have submitted reports in 2015. However India’s contribution at 2 per cent was far behind that of two other Asian countries, China (8 per cent) and Korea (11 per cent).

The national PvPI coordination center, IPC, is set to become the first WHO Collaborating Centre for Safety of Medicines and Vaccines in the South-East Asia region. The Uppsala center gives a completeness score to every national program, indicating the how adequate the reports submitted are. India has got an impressive 0.94 out of 1, placing PvPI among the top-rankers and pointing towards the high quality of adverse effect reports submitted to the WHO.

Sten Olsson, program expert at the Uppsala monitoring center, has lauded India’s fledging pharmacovigilance program, stating: “Although India has a long way to go in the area of pharmacovigilance, it has created a momentum in ADR monitoring. The World Health Organization’s Uppsala monitoring center is now looking to make India a hub for pharmacovigilance training”.

However, for a country of 1.252 billion, the current scale of PvPI is alarmingly small. Drug monitoring needs to grow at an accelerated pace to quickly ensure patient safety. Adverse drug effects are grossly underreported, mainly due to the limitations of resources and lack of awareness among healthcare professionals.

The PvPI has steered continuing medical education programs that aim to promote ADR reporting and greater participation in pharmacovigilance. To encourage the ADR reporting process, the PvPI has set up a toll free number (1800-180-3024) and a mobile application. And in a welcome step, IPC has started collaborating with the Indian Medical Association, aiming to pool in more medical practitioners into the PvPI. After all, medical professionals remain the most critical stakeholder in any pharmacovigilance program, as they are the ones who control both detection and reporting of ADRs.

Increasing outreach

Currently, the PvPI database has insufficient indigenously generated reports and is dependent on data submitted to the global database by other nations to make conclusive recommendations for many drugs. PvPI outreach efforts are directed towards developing the national database towards self-sufficiency.

Pharmaceutical companies have their major interest in developing and marketing the drug.  However, attempts have been made to rope them into drug monitoring as well. In March 2016, the country’s drug regulatory body through a gazette notification directed pharmaceutical companies to set up in-house pharmacovigilance systems to monitor ADRs arising from the use of drugs manufactured or marketed by them, in accordance with global standards. It is noteworthy that various pharmaceutical companies have already initiated the process and have contributed 18.80 per cnet of the ADR reports submitted to PvPI in the year 2015.

It is heartening that after multiple attempts, the current drug-monitoring program of the country is growing at a steady pace. Late to join the global drug-monitoring program actively, much work lies ahead for the PvPI to scale up operations to properly serve the needs of the nation. The program must aim to go beyond making recommendations, and make pharmacovigilance a regular practice for healthcare and allied professionals, starting from including it in the medical training, to formulating a set of comprehensive standard guidelines, similar to Good Pharmaceutical Practices (GVP) of the European Medicines Agency.

Monitoring adverse effects of drugs is indispensable for making informed and safe medical decisions in today’s age, and though the pharmacovigilance program of India is young and developing, it is hoped that with adequate financial and infrastructural support, it will grow to become an integral part of the healthcare system of the country.

 

(Debaleena Basu is currently pursuing her doctoral degree in Neuroscience at  the Indian Institute of Science, Bengaluru. She is a science-writing enthusiast and contributes to the ClubSciWri, an info-blog belonging to the ‘Career Support Group’, a networking, discussion and resource-sharing platform for scientists. She has also written for other portals.)