Src and Syk kinase attenuation of HIV infection

A recent paper in The Journal of Immunology (2007, p2862) (no DOI?) concerning the role which two kinases (Src and Syk) play in the early steps of HIV infection, is interesting in a number of ways. Firstly, it reveals how these kinases might attenuate the infection early-on and second, it shows how a drug might have a completely unexpected adverse effect.

HIV, in most cases, enters the body by hitching a ride in dendritic cells (DCs) which are located in the mucosal lining of exposed tissues. The virus doesn’t infect these cells, it merely binds to cell surface receptors (eg. the mannose receptor or DC-SIGN) and is internalized into endolysosomal vacuoles. Migration of the DCs to the lymph nodes where the virus is passed on to CD4⁺ T-cells initiates the actual infection.

Tremblay and co-workers found that if inhibitors of the Src or Syk kinases, PP2 and piceatannol, were present during the transfer of the virus from the DCs to the CD4⁺T-cells (in cell culture) that the overall amount of virus that was present, afterwards, was higher than in their absence. The same result occurred if they used siRNA to knockdown the kinases. After additional experiments it was apparent that the kinase-inhibitors were causing the DCs, themselves, to become infected.

One might conclude from these experiments, that under normal circumstances the kinases “protect” the DCs from becoming infecting, allowing them to act as passive transporters of the virus to the lymph-nodes. How this occurs can only be speculated. One idea offered by the authors is that the inhibitors cause the virus to be shuttled to the cytoplasm of the DCs instead of the endolysosomal vacuole, whereupon it infects the cell.

I am probably thinking too hard about this but…a question not addressed in this paper, is what would happen to a person who is at high risk of becoming infected with HIV if they were on a drug regimen that included a Src or Syk inhibitor? Would there chances of becoming infected increase?

 

Word of the week..idiosyncratic

As in idiosyncratic drug reaction…an unpredictable adverse drug reaction that occurs in a very small subset of patients. With the recent withdrawal from the market of a number of drugs in the last few years, the pharmaceutical industry has begun to focus its attention on finding ways to avoid this problem. If you want to witness a very spirited discussion (debate) amongst your colleagues simply mention idiosyncratic drug reactions and people will quickly divide up into groups based on what they believe causes idiosyncratic reactions and the value of the assays most research organizations are introducing to evaluate the risk of the event occurring.

The most accepted theory  (the “hapten hypothesis”) suggests that reactive drug metabolites covalently bind to proteins and act as haptens to effect an immune response to the drug and host-protein. There are a few problems with this theory one being that it fails to explain why the immune response only occurs in a very small subset of individuals. In order to explain the low frequency other researchers have invoked the “danger hypothesis”, which states that in order to activate the immune system towards a hapten it has to be accompanied by some sort of physical injury. But here again there are problems, the primary one being the the “danger hypothesis” is not well accepted by immunologists since it goes against the primary dogma of immune “self-tolerance” (Check out the review in Science by one of the proponents of this theory, Polly Matzinger). Last but not least, Werner Pichler has introduced the “PI” theory, which ignores reactive metabolites and covalent binding altogether and focuses on the parent drug. According to the PI theory the drug binds directly to the T-cell receptor, stimulating the cellular immune system.  T-cell receptors varyf from one individual to the next depending on genetics and other factors which would explain why only a small set of people would have receptors that bind to the drug. This hypothesis suggests that the immune reaction is caused by ‘off target’ binding of the drug and as such should be countered by increasing selectivity for the desired target and not by reducing metabolic stability.

Rather than adding my own opinion to this debate, I wanted to address a more fundamental issue…the definition of the word idiosyncratic. Unfortunately, based on the use of the word in the context of describing an unpredictable drug reaction that occurs with low frequency, most people assume that it means an unusual event. However, the true definition has more to do with the person…’idio’ comes from the Greek word idios which means “one’s own” or “personal”… and the definition of the word is more along the lines of  “characteristic, habit, mannerism, or the like, that is peculiar to an individual”

Actually, I prefer this definition, since I tend to think of idiosyncratic drug reactions as being a problem that resides with the affected patient, not necessarily with the drug. It is also the reason that I like Pichler’s PI theory since it addresses the patient’s susceptibility to an adverse drug reaction, rather than looking at the drug’s propensity to cause the reaction. The problem with focusing on the drug is that most, if not all drugs, will form reactive metabolites if they are metabolized in the body, therefore you can only reduce risk by removing those compounds that exhibit a certain level of covalent binding. The accepted level of covalent binding is subjectively determined and usually reflects the measure of risk one is willing to tolerate. With this paradigm you wind up tossing out a lot of potentially useful drugs, particularly if one is very conservative.

Unfortunately, we are not at the point where we are able to predict which patients will have a problem and it will be very difficult to get there. There is a lack of data due to the small number of patients affected and the inability to examine the issue prospectively in the clinic. Therefore, I have a few ideas/suggestions for speeding up research in this area;

1) Clinical data, be it from academia or pharmaceutical companies, should be pooled so that a sufficiently large database can be produced. This will help in probing for links in genetics or patient-histories with the frequency of the adverse drug reaction.

2) Would it be possible to expand the genetic diversity of the pre-clinical species that the drug is tested in? Although, more than one species of animals is employed in tox and safety studies, these are all derived from homogeneous colonies, so in reality the drug has only “seen” 2 to 3 different genotypes. Is it cost prohibitive to set up a panel of genetically diverse rats or dogs for screening?

3) Apply the same principle of genetic diversity for selecting patients for clinical trials, if possible, in order to increase the chance that a problem will be detected in early trials.

Finally, a renin inhibitor makes it to market

Very recently approved…Novartis’ Tekturna^® (aliskiren) is the first new treatment for hypertension to reach the market in over 10 years. However, the story on how this drug made it market goes back even further. An interesting review was published last year in The Lancet, which believe it or not actually contains chemical structures.

As an indication of how old (or young depending on your perspective) I am, renin was a very “hot” target when I was interviewing for my first job a number of years ago. It seemed like every company I visited had a high profile renin program. Back then renin was not just a target, rather it representative a whole paradigm for discovering drugs, namely the combination of ”peptidomimetics” and computer aided drug design. The idea was to exploit peptides as leads which and then be convert them into bona fide drug like compounds by tweaking the backbone. Renin was a prime example of this approach.

Of course, renin had been contemplated as a target for treating hypertension since the 1950’s (Skeggs et al. J. Experimental Medicine 1957, 106, 439) but it took some time to figure out how to develop an oral drug.

The aspartyl protease, renin, lies at the beginning of the renin-angiotension system (RAS) that ultimately, results in the synthesis of the peptide angiotensin II (AII). AII acts at the kidneys to increase Na⁺resorption, vasoconstriction and water retention, resulting in increased blood pressure. As shown below, cleavage of angiontensinogen to angiotensin I (AI) is the first step in the cascade, after which angiotensin converting enzyme (ACE) converts AI into AII via a second proteolytic reaction.

 

Over the years a number of agents have been developed that inhibit various steps of the process. For example, b-blockers hamper the release of renin from the kidneys, ACE-inhibitors prevent the conversion of AII to AI, while AII-antagonists block the action of AII at the kidneys. While these drugs have found wide use in the treatment of hypertension, each has its own set of issues.

In addition to preventing the cleavage of AI to AII, ACE-inhibitors also block the proteolytic inactivation of bradykinin and substance P, leading to two common side-effects…a dry cough and in some cases angioedema while AII-antagonists show limited receptor subtype activity.  Because renin is upstream form these events, a therapeutic which targeted this enzyme should be free form these side effects and limitations.

From the beginning, the primary problem in this area was not the lack of leads but the compounds that were active against the enzyme were large, peptide like compounds…very “non-drug” like according to the most lenient standards. This was no accident since the majority of these compounds were designed using the peptide substrate as a starting point. Examples of 1st generation compounds are, shown below. CGP-29287 (the first compound to demonstrate oral activity), A72517 (zankiren), A64662 (enalkiren), Ro 42-5892 (remikiren), U-71038 (ditekiren)…etc.

Novartis (Ciba-Giegy at the time), discovered aliskiren in the early 90’s but licensed it out to Speedel since it it appeared to expensive to make. Speedel was able to develop a cost-effective method for producing the compound and examined the compound all the way through to phase III, at which point Novartis exercised its “call back” option and filed the compound for approval.

Interestingly enough, Roche appears to be pursuing a similar pathway, in that they also have handed over their renin program to Speedel. The exact structure(s) have not been released but one of Roche’s advanced compounds had been remikiren.

Evidently, for renin, patience and perserverence has finally paid off. It will be interesting to see if other companies decide to jump in this area as well.

Idiosyncratic adverse drug reactions and renin

The two topics are only linked by the fact that they both have been on my mind this week. Novartis recently received approval to market a renin inhibitor…a major accomplishment if you are familiar with this target. I am in the process of writing up a small piece on this…expect it soon.

I am playing with the idea of using “idiosynsyncratic”, as in idiosyncratic drug reactions, as the word of the week. I realize it is a topic that most of us have become very familiar with over the last few years. It is really just a chance to add my two cents to the discussion concerning this issue.

Word of the week…Urticaria

If your field of study is inflammation or if you try to keep on top of drug safety issues such as drug hypersensitivity you might have come across this term.

Simply put, urticaria is another word for hives (not The Hives) or skin rash which is sometimes one of the symptoms of drug-hypersensitivity (ie. a drug induced allergic response) . This type of reaction is usually triggered by mast cells that are located in the connective tissue of skin, gut, and respiratory and gastrointestinal tract. Mast cells can be stimulated to release histamine and other immune enhancing agents by a number of mechanisms, either through antibody (IgE) binding or direct exposure to offending agents, although the former mechanism is what most people are familiar with. However some drugs elicit a mast cell response by direct stimulation and avoid the immune system altogether. There are a number of drugs which fall into this category including opiates, codeine, amphetamines, atropine, muscle relaxants, pentamidine….

Nobel Proze Winner Alan MacDiarmid Dies

Professor Alan MacDiarmid (1927-2007)

I was sad to see in the latest issue of the University of Pennsylvania Alumni magazine that chemistry professor, and Noble prize winner (2000, chemistry), Alan MacDiarmid died on February 7, 2007.  MacDiarmid is best known for co-discovering conducting polymers…a complete accident if you read his biography.

MacDiarmid taught undergraduate inorganic chemistry, at Penn, and anyone who had taken the course (including myself) will remember him for his passion for chemistry and his exceptional talent for teaching. Its been a while, but I can still remember his class…he would show up, cigarette, ashtray and chalk in hand (this was 1985 and there was no such thing as a “smoke free” building)…each class would begin with a written quiz…MacDiarmid required his class to know the periodic table from memory…it’s been over 20 years but I can still make it through most of the non-transition elements. Although, I went on to pursue studies in organic chemistry I credit him as one of the individuals that played a role in my development as a scientist. 

“Science as Art” the passage below was copied from MacDiarmid’s autobiography:

Seeking the Great White Bird of Absolute Truth

The dependency of any one person’s research on the labors of scores of earlier scientific pioneers is illustrated very beautifully by a few sentences of this variation from a book by Olive Schreiner, written at the turn of the century, entitled, “The Story of an African Farm.” I would like to share with you this adapted portion.

The story concerns a young hunter who, in his youth, heard about the great white bird of “absolute truth” which lived at the very top of a high mountain far in the east. He had spent all his life seeking it without success - and now he was growing old.

The old thin hands cut the stone ill and jaggedly, for the fingers were stiff and bent. The beauty and strength of the man were gone.At last, an old, wizened, shrunken face looked out above the rocks. He saw the eternal mountains still rising to the white clouds high above him.The old hunter folded his tired hands and lay down by the precipice where he had worked away his life.I have sought,” he said, “for long years I have labored; but I have not found her. By the rough and twisted path hewn by countless others before me, I have slowly and laboriously climbed. I have not rested. I have not repined. And I have not seen her; now my strength is gone. Where I lie down, worn out, other men will stand, young and fresh. By the steps that I, and those before me, have cut, they will climb; by the stairs that we have built, they will mount. They will never know those who made them, their names are forgotten in the mists of time. At the clumsy work they will laugh; when the stones roll, they will curse us; but they will mount, and on our work they will climb, and by our stair! They will find her, and through us!”The tears rolled from beneath the shriveled eyelids. If truth had appeared above him in the clouds now, he could not have seen her, the mist of death was in his eyes.…

Then slowly from the white sky above, through the still air, came something falling … falling … falling. Softly it fluttered down and dropped on to the breast of the dying man. He felt it with his hands --

 it was -               

 - a feather.

Novel mechanism of HIV-RT inhibition by NNRTI

An interesting paper published recently in Angewandte demonstrating that a compound which binds to an allosteric site on an enzyme can behave like a competitive inhibitor.

Non-nucleoside reverse transcriptase inhibitors are structurally and mechanistically unique compared to the more traditional nucleoside inhibitors and are included as a component of one of the most recommended therapies for HIV. The story behind this class of compounds is interesting, but best left for another time, needless to say they are distinguished from the nucleosides in thier ability to bind to an allosteric site on RT and act as non-competitive inhibitors (they reduce k_cat not K_M). One such compound is TNK-651 (or HEPT) which is a pure noncompetitive inhibitor. Based on this Maga et al. made a library of pyrimidine based compounds which included compound 1  (K_i= 8 nM) and similar analogues but found that they were competitive with the nucleotide substrate (apparent K_Mincreased with increasin concentrations of 1).

The interesting part came when they saw that these compounds were sensitve to the same resistance mutations as TNK-651. These mutations are located around the allosteric binding site and lead to resistance to TNK-651 and other NNRTIs. The fact that these mutations also lead to resistance to 1 indicates that it is binding at the allosteric site not at the active site. In order for this to work you have to assume that binding of 1 to the allosteric site causes a significant conformational distortion in the substrate nucleotide binding site such that its affinity is dramaticlly lowered. Of course in order for the compound to be truely competive the opposite should be true as well, binding of the substrate should distort the allosteric pocket and reduce the binding of affinity of 1.

I don’t claim to be an expert on enzyme/inhibitor kinetics so I don’t know if any other proposal could explain thier data. I would be interested to know if there are alternative interpretations.

Word of the week; Heinz Bodies

I thought I might try something new…provide a word/phrase of the week that deals with some aspect of medicinal chemistry or pharmacology…drawing upon my own experience to include subjects that one will likely encounter at some point in their career in  drug discovery.

First up is “heinz bodies”

Sooner or later one will come across a compound in a program that has the potential for causing hemolysis of red blood cells. In some cases hemolysis is easy to detect in vivo since the animals will exhibit clinical signs of stress/discomfort and blood will be evident in the urine. On the other hand, subclinical hemolyis might go undetected during acute or short term exposure. Nonetheless, in some cases it might be possible to detect subclinical hemolysis depending on the mechanism (typically oxidation of hemoglobin (haemoglobin to the English)), using “heinz bodies” as a biomarker.  Drugs such as quinidine, quinine and isoniazide have been associated with oxidative damage to hemoglobin, either directely or indirectly. Oxidative damage to hemoglobin causes the percipitation of “cellular” components in the red blood cell resulting in small inclusions within the “cell” body..these are heinz bodies. I put in a picture below which I copied from the Crookstone Collection of Medical Slides 

 

 Heinz Bodies

KCR1 blocks drug HERG binding

Individualized medicine to reduce the risk of adverse side effects of certain drugs will hopefully find a place in drug development in the near future. Had this approach been available in the past there would be a number of drugs that would not have been pulled from the market due to unacceptable side effects in a small percentage of patients. In the past decade HERG blockage has arisen as a major concern, which has probably stopped a number of drugs from being developed.

In a recent paper in J. Biol. Chem. expression of the protein KCR1 (K+ channel regulatory) tends toprotect HERG from being blocked by dofetilide.  There is a “gain of function” polymorphism that is observed in the gene that codes for this protein which leads to a lower risk of acquired drug induced QT prolongation. Therefore, drugs associated with QT prolongation might be safe for people having this polymorphism.

Talk like doctor

I recently learned a new word…. “coprophagia” to describe a certain habit of animals (dogs in particular) which can lead to an interesting PK plot (Plasma concentration versus time). This term turned up in the report our ADME group wrote describing the oral PK of a compound that was given to dogs. If you see two nearly equal Cmax’s separated by a period of a few hours you too might have observed “coprophagia”. Interestingly enough, when I “Googled”this term, I noticed that eBay was offering coprophagia at reasonable price.