Identification and Biochemical Studies on Novel Non-Nucleoside Inhibitors of the Enzyme Adenosine Kinase

Authored by Jae Park, Gayathri Vaidyanathan, Bhag Singh & Radhey Gupta

Department of Biochemistry & Biomedical Sciences, McMaster University

Adenosine Kinase is an enzyme that adds a phosphate to adenosine.

Adenosine is one of four bases in DNA, and when it has three phosphates attached, becomes the energy currency of the cell, called ATP.

I nodded at him and joined the line of mostly women and a few kids. A few frisks later, we walked into a hall filled with plastic playroom furniture. A door opened and officers escorted a line of men into the room.

I didn’t remember what Dawkins looked like. He’d had his back turned to me in court. Always in a frumpy gray sweatshirt, a tattoo on his neck.

Dawkins arrived. He looked like his dad (Winston, always impeccably dressed in a three piece suit and fedora), but younger and taller. I pumped his hand thinking melodramatically, hands that’ve killed twice. He was again in gray, this time a standard issue jumpsuit, torn at the crotch. He had seven tattoos, none from prison, he assured me. Tattoos acquired in prison are a sign of gang affiliation.

I asked him at one point why he was carrying a steak knife in his pocket at the age of 14? For his first murder, Dawkins had stabbed the neighborhood bully in the neck, severing a major artery and killing him.

He smiled his first smile. It lighted up his face, revealing for an instant something deeper, a shade of the personality he had been hiding. His lightly bearded cheek dimpled.

Then it closed up. He launched into his lie, repeating it again and again with increasing pitch and crescendo. He talked in circles. He touched my knee to reinforce his point. My body strained against the light touch.

I was disappointed. I’d expected an ideal character for my narrative, but the perfect is often the exception rather than the rule. He was what he was, perfectly human.

I asked him about his second murder. He said he didn’t do it. His lawyer had said he was a nasty piece.

Monoclonal antibodies light up rat tissue culture

I wrote about the cancer drug ipilimumab last year, which has been hailed as miraculous by some. The drug has been through Phase 3 clinical trials and results will be presented on June 6 at the annual meeting of the American Society of Clinical Oncology (ASCO).

Meanwhile, following announcements of the miracle cure in three men having prostate cancer, ipilimumab has also been show to be modestly effect against lung cancer, according to the company.

Ipilimumab works by targeting an inhibitor of the immune system called CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and subsequently boosting the response of the killer T-cells. It belongs to the highly promising monoclonal antibody field of therapeutics.

Quote from a press release sounds like the company is excited:

“We are excited by the potential of immuno-oncology, an entirely new paradigm in the treatment of multiple types of cancer in which a patient’s own immune system is activated to fight cancer cells,” said Elliott Sigal, M.D., Ph.D., executive vice president, chief scientific officer and president, Research and Development, Bristol-Myers Squibb.

“We are leading the way with ipilimumab, the most advanced investigational compound in our immuno-oncology portfolio, in testing this new paradigm and we look forward to presenting results from the ipilimumab clinical development program at this year’s American Society of Clinical Oncology Annual Meeting.”

But while the promise of the basic science is cool, it’ll be difficult to draw conclusions about the real-world application of the drug until the results are formally presented and peer reviewed.

The chicken farm in Food, Inc. reminded me of a poultry farm in a village in Karnataka, India. The chickens in that farm were healthy and caged in separate coops. It was a clinical operation; the neatest farm of any variety I’ve visited in India.

In Food, Inc., the fat birds (they have excess breast tissue) totter about on their feet, unable to carry their own weight before collapsing.

With a burgeoning population and the challenges that agriculture will face in the coming years with changing climate and water scarcity, the question of how to feed everyone is becoming increasingly relevant. The answer doesn’t have to be factory farming controlled by a small number giant corporations. It could be a network of smaller farmers using the best of technology to increase productivity despite smaller land sizes, according to an agricultural expert Gerald Nelson from the International Food Policy Research Institute. He was speaking at a conference in Washington, DC last week.

But to ensure that the control and choice of technology and farming remains in their hands, greater public investment in agriculture is critical. Food, and knowledge of food, needs to move back into the public sphere. The farm to table movement is important, not because it argues for a return to an ancient, unsustainable agriculture, but because it calls for greater transparency.

Not to say the private sector hasn’t made critical advances over the past few years as governments have slashed agricultural spending. But their responsibility to their shareholders makes profit the overarching ideal. They plan with the short-term motive of money in mind without seeing a grand master plan for the planet or the human race.

“You know what’s in the sewage?” asks the beefy engineer at the Woodward Avenue water purification plant in Hamilton, a small city in Ontario once famous for its steel. These days, it is more known for its non-achievements: steel mill layoffs; Ti-Cats football fanatics who shout “Oskie-Wee-Wee! Oskie-Wa-Wa! Holy mackinaw! Tigers…ha! Ha! Ha!” even as their team repeatedly loses; and an underdeveloped downtown core with an overdeveloped pigeon problem.

But Hamilton is also a city of waterfalls and streams, rivers and harbors, all a natural home for Chlamydia. As, of course, is sewage.

“Corn,” the engineer answers himself seriously. “Corn doesn’t get digested. It passes through the intestine intact.”

He hands me five liters of the waste water.

Under a darkening sky, I drive back to my research laboratory at the McMaster University hospital and lug the jugs of water upstairs, past the red zone where just-born infants rest peacefully in plastic incubator cages, under individual yellow bulbs.

Rather like plants.

Children are at great risk of infection by Chlamydophila pneumoniae, which causes pneumonia. Its cousin Chlamydia trachomatis is infamous for causing the sexually transmitted disease Chlamydia, which can result in blindness. Prachlamydia and Simkania cause respiratory illness. Waddlia causes abortion in cows.

I run through the list, memorized for my upcoming thesis defense. The next sentence in the presentation goes so:

New species of the bacterium are still being found, in places as far-flung and extreme as Antarctic salinity lakes. Wherever they are found, they are pathogenic. And yet, their presence in our environment, in our water sources—rivers, ponds, and lakes—has been largely ignored.

My job is to figure out a way to detect them that lab technicians can employ easily, an amazingly difficult task as I’ve found out over the past four months. I have trudged through forests and swamps and manmade reservoirs located next to deserted hiker’s trails and country roads and highways. I have watched the Red Hill Expressway, intended to cut travel time between Hamilton and Toronto in half, grow from a muddy road to a cemented monster, all the while collecting water from the Red Hill Creek. The different water sources have distinctive fauna and flora that comes to life under a microscope.

A single drop of water is a universe unto itself, inhabited by creatures of reduced proportions. Magical shapes drift in and out under 40 times magnification—translucent amoebas, paramecia, rotifers, sun animalcules (“little animal”) with hair-like flagellum around them like a halo, and other strangely beautiful blobs. These are the visible creatures.

My target is the inhabitant of a world of less than a single micrometer—invisible.

Invisible is what I feel as I pass into the purple zone of the hospital. Fluorescent bulbs cast shadows in the deserted hallway. Graduate students have fled their labs to quell their boredom in alcoholic beverages. I am alone in my quest for what I have come to view as an ethereal creature, my yeti. It exits, but has eluded me repeatedly. I do not know if this is because of human error, or because the water samples lack Chlamydia.

I do not know which answer I would prefer.

So filtering sewage is what it comes down to.

–

Every day, for the past four months I have performed the same set of experiments.

One. Collect sample. Spin sample in a centrifuge at 500 times the speed of gravity. This weighs down all the debris that can be thrown away.

Two. Take the “soup”—the resulting sample—and filter using paper with pore size of 1000 micrometers under strong vacuum. All but the biggest microorganisms will go through.

Three. Filter through a paper with one micrometer-sized pores. Only the smallest organisms will pass through.

Four. Add phenol and chloroform, chemicals that will cause the beautiful, mysterious living blobs to burst open, spilling out their genetic secrets—DNA and RNA—into the water.

Five. Amplify regions of DNA where the four bases—adenine, guanine, cytosine, and thymine—have come together in a sequence that, out of all the organisms in the world, are present only in the Chlamydial genome. The sequence is a signature as unique as a thumbprint, which, after many steps inside the nucleus of the bacterium, results in the “signature protein” CT429. No one knows the function of this protein, one of the thousands that allow these bacteria to grow and multiply and infect.

Six. Amplify regions of DNA that correspond to Heat shock protein 70, a protein present in most organisms on Earth. It helps fold other proteins into shapes that must be painfully conserved for proper function. Misfolded proteins can result in disease—dementia and Lou Gherig’s, for example.

Seven. Separate amplified DNA fragments according to size and charge using agarose gel electrophoresis.

If the heat shock protein amplifies but the Chlamydia protein doesn’t, it means the water sample contained organisms of many varieties, but not Chlamydia.

Or it could mean that there is a glitch in the experimentation.

This uncertainty is ever present in the lab, I have found. Why did something not work? Or why did something work? Was the result an outlier? Could it be possible that the wrong sequence got amplified due to primer mismatch? Perhaps the AGTCCCT primer, instead of attaching to TCAGGGA, attached to a region of the DNA that goes TCACGGA. Now the wrong region is amplified, a size of 600 base pairs instead of the 400 base pairs that I am expecting.

Or perhaps, serendipitously, the wrong region is also 400 base pairs long, creating the illusion of the perfect match. Voila! The protein is found! Chlamydia exists!

But clone the region into a vector and re-amplify. Sequence. The protein sequence is not the same as for CT429. The wrong protein got amplified. Chlamydia doesn’t exist.

–

I pour the sewage into 1000 ml centrifugation bottles. The lab vibrates as the ancient machine spins. I bounce slightly on my toes in rhythm with the rocking cultures of bacteria grown by my colleagues.

This is a world of objectivity, ruled by the gods of precision and accuracy. But much as life itself, the world of experimentation is ruled by uncertainties, with a result true to perhaps a 90th percent of certainty, or 95th percent. There are no final answers.

But there is always the quest.

The court room was packed in respect for the large sums of money — $100 million, $2.5 million etc.–under discussion. Nothing like white-collar crime to attract the media in New York. I left my cell phone and camera at the office and rode up to the court from the clerks office with a PR rep for Raj. He had a whole retinue of lawyers, media reps and hangers-on.

The Galleon founder was asking the judge to reduce bail. Words flew in measured tones as the defense adopted a submissive stance. The white-haired lawyer John Dowd seemed like he didn’t have a good response to give to the question: why reduce the bail to $25 million?

The obvious answer is that it’s just too much. $100 million dollars. I wonder at which million our comprehension of the extent of that sum breaks down. But this argument lacks logic. Dowd struggled to give a better one. It seems that Rajaratnam had only scrounged up $2.5 million in cash, and paid the rest in securities and property.

The prosecution (comprising 3 from the DA’s office and one FBI agent) was confident in its arguments. They revealed new evidence saying that they had found a senstive IM transmitting information about Polycom (a California based tech company) between Raj and an accomplice. The IM said: “don’t proceed on PLCM until I give further guidance.”

At the end of the hearing, the judge refused to reduce the bail but allowed Raj travel privileges within the United States. Everyone got up and the courtroom cleared to judge once again, stories of more normal and less expensive criminal behavior.

I traveled back down in an elevator filled with chatty Raj hangers-on who quickly shushed themselves upon my arrival.

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Since Darwin, evolution has been in vogue. Most scientists take it on principle that accumulation of mutations in DNA over million of years leads to new life forms.

But a question that has intrigued researchers for some time is whether organisms can go back to their ancestral forms. Is evolution is reversible? Conventional wisdom—known in the sphere of evolutionary biology as “Dollo’s law” after pre-eminent dinosaur researcher Louis Dollo—says no. A recent study published in the journal Nature has elegantly confirmed that evolution is a one-way street by studying the process at the molecular level.

As evolution occurs, the changes are so intricate that it becomes nearly impossible for the organism to go back to its original form. Freshwater fishes that live in a dark cave will lose their eyesight over generations. Even if a landslide creates an opening in the cave and lets in some sunshine, it is highly unlikely that the eye will reform.

Dollo’s theory has remained mostly unchallenged, except for a few works that say that evolution is reversible. In 2003, a team of scientists said that they’d found a species of snail that had regained its ability to coil into a loop after having lost the trait in previous generations.

“But their methods were unreliable,” said Boris Igic, a professor of biology at the University of Illinois in Chicago who was not affliated with the study. The snails could either have gone back to their original genetic makeup, or they could have gained new proteins that give it the old coiling.

The problem was that there was no real test to prove these theories. Evolution of organisms takes millions of years, making it difficult for scientists to make any direct observations.

Joseph Thornton, a biology professor at the University of Oregon, and his colleagues went around the problem by examining a single protein that helps humans and vertebrates cope with stress.

Millions of years ago, a fish existed that lacked bones. It is the ancestor to most life forms on earth today. That fish contained a small protein called the glutocorticoid receptor, which became active in the presence of two distinct hormones.

Over the course of the next 40 million years, the receptor evolved and became more specific such that it activated in the presence of only a single hormone—cortisol. It had accumulated 37 changes, but only seven were necessary to make it into the new receptor.

The researchers wanted to find out whether evolution could reverse at the level of the protein. To do so, they reversed the seven changes.

“But to our surprise, we got a dead receptor when we reversed,” said Thornton.

They found that the only way to make the protein reverse completely was to make five extra changes. These five fine-tuned the receptor, but did not give it a new function.

The probability of all five of these changes getting reversed is highly unlikely since they don’t confer a new advantage to the organism. They act like brakes that have to be removed to make evolution a two-way street.

Once the scientists fixed these five, they found that making the seven key changes reversed the protein to the ancestral form. They called the five brakes “ratchets” that prevented reverse evolution.

“This is not to say that the ancestral function cannot be re-acquired,” said Igic. But the function will come from forward evolution rather than a reversal. When whales evolved from a four-legged terrestrial ancestor, they evolved new proteins that resulted in fins. It was a reversal in function toward an ancestor of tetrapods that could swim, but in biochemistry, it was a movement forward.

Natural selection can take numerous paths during evolution but once those paths are chosen, reversal is highly unlikely. This is the first study of its kind, but Thornton does not expect this to be a rare case.

That an experiment at the molecular level can deliver a decisive conclusion about higher-order evolution is testament to the elegance of life.

“Everything that makes us who we are is stored in DNA,” said Ortlund, a biochemistry professor at Emory University, and co-author of the study. “Changes at the macroscopic scale have to start at the molecular level.”

The indigenous people of the Ryukyu Islands have, it seems, the elixir of old age.  They live on average for 110 years.

They longevity has been tied to their eating habits, and termed the Okinawa diet by commercial interests.  The islanders consume only 1 calorie per gram of food, primarily through green and yellow vegetables, grains and very little sugar.  Essentially, they are eating a nutrient-rich, low-calorie diet that triggers a stress response within their cells.

The years of low calorie consumption leads to a long, healthy life, say the proponents of the diet.  A recent article by Dr. Sandy Westerheide and colleagues in the journal Science provides support for this theory, although in organisms several orders of magnitude less complex than humans.

The nematode Caenorhabditis elegans responds similar to human cells in times of calorie restriction.  Within the millions of cells that make up the tiny nematode, there are specific pathways that act in concert to keep the organism going.

In response to stress such as reduced calorie intake, the cells adapt by changing their pathways, according to Dr. Westerheide.  They do so in tiny increments that, in the way of life, have amplified results—in this case, increased longevity.

The key players in the pathway that get changed are highly conserved proteins that are the master controllers of the microscopic world of the cell.

They are called transcription factors.  They tell the cell whether to grow, divide, fight invaders, specialize, or commit suicide.  Their functions are as ubiquitous in humans as the nematode, although small differences in sequence may exist.

Dr. Westerheide focused on two proteins, the enzyme sirtuin 1 (SIR1) that increases the production of the second protein, heat shock factor 1 (HSF1).  Both increase in concentration within cells when the body consumes fewer calories.

The ultimate result is an increase in HSF1 production.  This protein works entirely by binding to DNA, and sending signals that promote growth.

In aging individuals, there is less SIR1, and in the absence of this protein, HSF1 can no longer bind DNA.  There is no growth.  Instead, there is aging, and eventually, death.

But in an individual who meticulously controls his or her calorie consumption, a stress response is triggered.  These responses, learnt early in evolution, are protective mechanisms that allow cells to remain alive in the face of harsh conditions—thermal stress faced by single-celled bacteria on hot sulfur springs, oxygen toxicity faced by the lung cells of scuba divers, and calorie restriction faced by cells in Ryukyu islanders.

In the nematode, the stress response increases the levels of SIR1.  This leads to increased binding by HSF1 to DNA, leading to growth signals even in an aging cell.

That is the secret of the long life of the healthy nematode.  Whether HSF1 has a part in the response in higher organisms remains to be seen, but SIR1 looks important.

And given that cancer cells have strong stress responses, Dr. Westerheide’s work indicates a role for HSF1 in forming tumors, something that has been hinted at already by other researchers.

Small proteins have big roles in the elixir of the Ryukyu islanders.