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Drug Assays

Target (In)validation

Here’s a short review in ACS Medicinal Chemistry Letters on target validation. As the author (Ramzi Sweis of AbbVie) says, it’s easy for medicinal chemists to lose track of that being part of their role:

Too often, medicinal chemistry teams blindly accept the biological underpinnings of a new program as sound and are unaware that their ensuing efforts at compound optimization may confirm or debunk the link between the biological target and the disease. In many instances, this event occurs very early in the discovery process, at the level of preclinical interrogation.

The number of targets that you can be sure are good ones, before anyone’s actually made a drug to hit them, is not large. You rarely get signals like PCSK9, where you can point at a human population with exactly the phenotype you’re trying to cause (and even then, there’s always the worry that your therapy might not recapitulate that “human knockout” mutation in every respect). There are a few targets kicking around out there that have never really been “drugged” but that everyone’s still pretty sure would be great things to affect (p53 and/or cMyc in cancer, for example), but the list is short. Otherwise, unless someone else has already blazed that trail, you’re going to be finding out not only “Is this compound going to hit the target”, but “Is this target worth hitting in the first place”.
Here’s some good advice:

The infrastructure behind any drug discovery program should be stress-tested at a very early stage. Fear of obtaining suboptimal results from a suboptimal compound should not be a reason to defer such system testing. It is not uncommon for medicinal chemists to challenge their primary assay and note when occasional aberrations in output are observed. This feedback to biologists is quite helpful in establishing the durability of the primary assay. Accordingly, this philosophy should extend to downstream assays as well.

That fear is a very real one. “Oh, if you try to test using just the chemical matter we have now, you’ll probably kill the project for no good reason”. That’s not a silly argument; it really is possible to go in too early with something that’s too nonselective or too non-potent. But it can also be used to delay the day of reckoning, to avoid getting an answer that no one wants to get. You have to be honest with yourself and be ready to hear the bad news, and not keep telling yourself that you’re waiting for a better compound before asking the hard questions. Otherwise, you’ll have a compound that’s been optimized in every direction and is ready for the clinic before you know if you should have been doing all that work in the first place. Some readers may just possibly have had such an experience in their careers.
But as this paper goes on to say, even trying out your compound in vivo and seeing the effect you wanted may not be enough. That’s a big step, for sure, but you’ll be a lot more confident if you can hit the system with more than one chemical series. (This, in my experience, is a rare luxury). It’s a rare project that won’t charge ahead at this point (and a rare management team that won’t complain if they don’t).

The certainty of target validation/invalidation is far from absolute. It is akin to a lawyer’s task of establishing judgment beyond a reasonable doubt to a jury. A highly effective medicinal chemistry team is commonly lauded for intense focus. However, this focus should not preclude a critical assessment, from the broader perspective of drug discovery, of the compounds being used to make project go/no-go decisions. . .

If you don’t have that wonderful situation of multiple chemotypes, the paper suggests, then you should make the most of what you have – take some structurally similar compounds into the in vivo model, but ones that don’t have activity in the primary assay. These negative controls might surprise you. That’s also good advice, but can be a tough sell with some biologists. But from what I’ve seen, and from a look over the history of drug research in general, I’d say that skimping on the negative controls is both one of the most common shortcuts and one of the ones you’ll be most likely to wish you hadn’t taken.
Human nature is not built for scientific research. We have innate curiosity, no doubt about it, and we have pattern-recognition and narrative-forming abilities (and how). But what doesn’t come naturally is organized suspicion of our own convictions. That has to be learned, and relearned. In this line of work, the tuition can get expensive.

14 comments on “Target (In)validation”

  1. Anonymous says:

    Biologist to Chemist: “Don’t try to tell me how the biology works, just make the bloody drug, and leave the biology to me”.

  2. slcimmuno says:

    speaking of that hard-to-hit target, a Holy Grail pathway, p53, Kevetrin (thioureidobutyronitrile) has been show in humans — not mice — to activate wild type p53 and degrades oncogenic mutated p53, per this just released ASCO abstract (Dana Farber/Cellceutix research).
    “Kevtrin was shown to activate wild type p53 and degrade mutant p53. Since Kevetrin activates both transcriptional-dependent and transcriptional-independent pathways to promote apoptosis through wild type p53 activation and degrades oncogenic mutant p53, Kevetrin can function as a major inducer of apoptosis in many types of tumors independent of p53 mutation status.”
    pretty exciting stuff. not there yet, Ph I, but kevetrin may offer patients some real hope.

  3. anonao says:

    Derek, you forgot to close the italic html tag.

  4. Dupedbymenon says:

    #2. Go away cellceutux, this is a blog visited by actual chemists. The notion that a structure like “kevetrin” is a specific inhibitor of anything is laughable. How do I know? I made diverse analogs of this ridiculous compound, all showed the same activity.
    S-alkyl isothiocyanates were long ago shown to be nothing more than non specific inhibitors of oxidative phosphorylation. Something to spray on crops as a fungicide…
    Go promote your ridiculous pump and dump somewhere else.
    If Danishefsky really is on your SAB, then why does he say he doesn’t even know you?

  5. Cellbio says:

    Good advice. Worked on a program that did not start out challenging preformed concepts of the targets role and therefore the expected pharmacology. Screening paradigm was filter based: select only those less than x in assay Y etc, so ‘failures’ never went forward into next assay. We turned it upside down by screening nearly 3,000 compounds in extensive cell assays, and followed that with in vivo testing to show that not only did the ‘on target’ activity vary, but so did the animal pharmacology. Our filters had selected for a common tox profile in rodents and early clinical failure when other similar structures were on-target but without notable tox. Chemotype seemed to have little to do with variation in pharmacology (similar structures w/distinct profiles, distinct chemotypes with similar profiles), but in this mature program, structures were not varying wildly so not a question explored in great depth.

  6. dupedbymenon says:

    Oh, sorry, now you say it is a pathway activator. At first it was a “specific” kinase inhibitor with only millimolar activity.
    What will it be next?

  7. Vader says:

    “But what doesn’t come naturally is organized suspicion of our own convictions.”
    This was strongly selected against among our ancestors, I suspect, because it was rather unhelpful while organizing the next mammoth hunt.
    There are still social settings where it is unhelpful.
    Science is not one of them. Perhaps this explains why science has a particular appeal to the not very socially skilled. (Like myself.)

  8. Magrinho says:

    Ironic to see a Kevultron promo show up for this particular blog topic.
    “And if you order now, you will get a Kevultron golf caddy at no extra cost.”

  9. slcimmuno says:

    @4 @6
    so i guess Dana Farber and Dr Shapiro, the head of their early drug development program, are similarly duped. but then again, i’m no chemist so maybe ive been duped too.
    a couple cites for you-Kevetrin may just be that drug many cancer patients have been waiting for.
    “An Essential Passenger with p53”
    Regrettably, we have no general strategy for targeting tumour-suppressor loss, and nowhere is a creative solution to this challenge more urgently needed than in the context of the tumour suppressor p53. Alteration of the p53-encoding gene TP53 is the most common genetic event observed among 12 of the most common cancer types (42% of tumours)3. Yet despite definitive biological characterization over decades of study, there is no drug that acts directly on the p53 pathway. [see Kevetrin]
    p53 is a transcription factor that activates the expression of cell-cycle checkpoint genes such as CDKN1A in response to DNA damage, thereby arresting cell proliferation. In cancer, alteration of TP53, either by mutation or deletion, promotes cell proliferation and survival. Mutant p53 lacks a capacity for transcriptional activation, and so direct-acting small molecules would need to have a compensatory, corrective function. Such molecules have proved elusive. [see Kevetrin] Hemizygous deletion of TP53 results in reduced abundance of p53, establishing a rationale for stabilizing remaining p53 by targeting the protein MDM2 — a ubiquitin-ligase enzyme that promotes p53 degradation5. Derivatives of the small molecule nutlin-3a, which disrupts the MDM2–p53 interaction, have recently transitioned to human clinical investigation. But there remains a pressing need for therapeutics that target p53 loss. [see Kevetrin]
    Drugging the Undruggable (Clinical Oncology News, April 2013)
    The intracellular molecules—p53, Myc and Ras—have long been deemed unfeasible targets for therapy, but drugging them would be a boon for cancer research and patient care. Aberrations in p53, Myc and Ras represent some of the most common events in human cancer, and experiments show that restoring normal function to p53, Myc and Ras has strong, highly selective anti-tumor activity.
    However, “these [intracellular] proteins don’t seem to have good binding sites for conventional drugs, which is why we call them undruggable,” said Sir David Lane, PhD, the chief scientist at the Agency for Science, Technology and Research in Singapore, and winner of the UK Lifetime Achievement in Cancer Research Prize, who gave a keynote talk at the meeting.
    “These are the three horsemen of the cancer apocalypse,” said James Bradner, MD, a medical oncologist at the Dana-Farber Cancer Institute and an assistant professor at Harvard Medical School in Boston, in a phone interview. “We can’t yet prescribe even one drug that targets these most common contributors to cancer pathogenesis.” [see Kevetrin]

  10. Anonymous says:

    Have been a few biological meetings, I found them useful. Fully agree that most of claimed targets are not the right targets!

  11. Anonymous says:

    but the knockout proved this was a valid target…..and your compounds are the issue

  12. lynn says:

    Negative controls! Who’d a thunk it?
    “If you don’t have that wonderful situation of multiple chemotypes, the paper suggests, then you should make the most of what you have – take some structurally similar compounds into the in vivo model, but ones that don’t have activity in the primary assay. These negative controls might surprise you. That’s also good advice, but can be a tough sell with some biologists.” Maybe, but it’s often a tough sell with chemists, too. They are often not in love with providing enough of the “inactive” material to carry out those controls. The scientific method is critical for all of us.

  13. B.Izzy says:

    Thats my question as well…why would Dr. Shapiro and Dana Farber be involved in a trial with Kevetrin when dupedbymenon obviously solved the riddle years ago with the chemistry set his folks bought him?..thank the lord for “real chemists”…hope those morons at Harvard start pedalling faster!

  14. cdsouthan says:

    Here is a recent example of apparent de-validation for BACE and T2D

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