drug research

Before the First Dose

Posted on by Elise Johnson

Kierkegaard observed that one of humanity’s enduring tensions is that while life can only be understood backwards, it must be lived forwards. It’s a truth medicine knows intimately: in the treatment that worked until it didn’t, the resistance that arrived without warning, the moment a doctor has to tell a patient that the drug that was helping has stopped. Not because anyone made a mistake, but because the critical knowledge that would have mattered arrived too late, if at all.

A recent paper from the National Cancer Institute is, in a small but meaningful way, science’s pursuit of that elusive foresight: an understanding that emerges early enough, for once, to change what happens next.

The Elegant Idea

For decades, chemotherapy has worked by brute force, flooding the body with toxins designed to kill rapidly dividing cells. The problem is that rapid division isn’t unique to cancer. Hair follicle cells, gut lining cells and immune cells also divide rapidly, which is why patients lose hair, lose energy and become susceptible to infection. Chemotherapy targets a behavior, but the drug has no way to tell a healthy cell from a cancerous one.

Antibody-drug conjugates (ADCs) change that. Instead of targeting what cancer cells do, they target what cancer cells are. Cancer cells tend to display certain proteins on their surface in far greater numbers than healthy cells do. The antibody is engineered to seek out those proteins specifically. It navigates to its target, binds and waits for the cell to do what cells routinely do: pull it inside. Once there, the cell’s own digestive machinery (the lysosome) breaks down the chemical tether holding the toxin to the antibody, releasing the toxin to kill the cell from within. More than a dozen ADCs have received FDA approval in recent years, and the field is evolving fast.

What the Cell Does Next

But cancer cells don’t simply accept their fate. Even when an ADC delivers its payload perfectly—the antibody finds its target, the cell pulls it inside, the lysosome cuts the tether—a pump embedded in the cell membrane can grab the released toxin and throw it back out before it causes damage.

The delivery worked. The package got ejected anyway.

These pumps—ATP-binding cassette transporters, or more plainly, efflux pumps—are a normal feature of cell biology. Their job is cellular housekeeping, clearing out unwanted or toxic substances before they cause damage. Under the pressure of drug treatment, cancer cells do what life has always done under pressure: the ones best equipped to survive do. The same mechanism that has shaped living things for billions of years now works against the treatment. Not all cancer cells are identical, and the ones that happen to produce more pumps survive while others don’t, gradually shifting the tumor toward resistance.

Continue reading “Before the First Dose”

The Breakthrough Was There All Along

Posted on by Elise Johnson

If you’ve ever played The New York Times game Connections, you know the feeling. You’re staring at a grid of words, knowing the solution is there, but unable to see how the pieces fit together. All you can do is work with the words in front of you. There are no extra clues, no new information coming. The only option is to shuffle, to look at the same information in a different arrangement until patterns begin to appear.

Nothing about the problem changes. Then something about how you see it does.

In 2014, a third-year medical student named David Fajgenbaum checked himself into the emergency room mid-exam. He felt off. By the time anyone understood why, he was in the ICU with multiple organ failure from a disease so rare it wasn’t taught in medical school: Castleman disease. The only approved drug didn’t work. A priest came to his bedside and read him his last rites. He was 25.

Fajgenbaum survived that relapse, and four more after it. As he recounted in a recent episode of NPR’s Radiolab, he understood that chemotherapy was keeping him alive without curing him, and that waiting for a new drug to be developed (a process that typically takes 10 to 15 years and billions of dollars) wasn’t an option he had. So he did something unusual. He started asking his doctors to save his blood samples, and he ran experiments on himself.

What he found was that a specific signaling pathway in his immune system, mTOR, was in overdrive. When he searched the existing pharmacological literature for something that could block it, he found an answer that had been sitting in pharmacies for 25 years. Sirolimus, a drug approved in 1999 to prevent organ transplant rejection, had never been used for Castleman disease. The biology of his disease hadn’t changed. The drug had always existed. The connection simply hadn’t been made.

He took it. It worked. He has been in remission for over a decade.

The detail worth holding onto isn’t the drug or the disease. It’s the instinct. Fajgenbaum didn’t wait for new knowledge to arrive. He looked differently at what already existed.

Continue reading “The Breakthrough Was There All Along”

Paving New Ways for Drug Discovery & Development: Targeted Protein Degradation

Posted on by Riley Bell

The Dana-Farber Targeted Protein Degradation Webinar Series discusses new discoveries and modalities in protein degradation.

In this webinar, Senior Research Scientist, Dr. Danette Daniels, focuses primarily on proteolysis-targeting chimeras, or PROTACs. A variety of topics are covered including the design, potency, and efficacy of PROTACs in targeted protein degradation. Watch the video below to learn more about how PROTACs are shifting perspectives through fascinating research and discoveries in targeted protein degradation.

Learn more about targeted protein degradation and PROTACS here.

Increasing Drug Research and Development Efficiency Using a 4-point Screening Method to Determine Molecular Mechanism of Action

Posted on by Michele Arduengo, PhD

Fig 4. Four point MMOA screen for tideglusib and GW8510. Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature. Experiments run with 10μM GSM peptide, 10μM ATP, and buffer. Minute preincubation (30 min) was preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer. ATP was mixed to initiate reaction. No preincubation contained inhibitor, GSM peptide, ATP, and buffer. The reaction was initiated with TbGSK3β. Reactions were run at room temperature for 5 min and stopped at 80°C. ADP formed was measured by ADP-Glo kit. Values are mean +/- standard error. N = 3 for each experiment and experiments were run in duplicates. Control reactions contained DMSO and background was determined using a zero time incubation and subtracted from all reactions. Black = 30 min preincubation Grey = No preincubation.
Four point MMOA screen for tideglusib and GW8510.
Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature.  Black = 30 min preincubation Grey = No preincubation.
The first small-molecule kinase inhibitor approved as a cancer therapeutic, imatinib mesylate (Gleevec® treatment), has been amazingly successful. However, a thorough understanding of its molecular mechanism of action (MMOA) was not truly obtained until more than ten years after the molecule had been identified.

Understanding the MMOA for a small-molecule inhibitor can play a major role in optimizing a drug’s development. The way a drug actually works–the kinetics of binding to the target molecule and how it competes with endogenous substrates of that target–ultimately determines whether or not a a candidate therapeutic can be useful in the clinic. Drugs that fail late in development are extremely costly.

Drug research and discovery for neglected tropical diseases suffer from a lack of a large commercial market to absorb the costs of late-stage drug development failures. It becomes very important to know as much as possible, simply and quickly, about MMOA for candidate molecules for these diseases that are devastating to large populations.

One such neglected topical disease is Human African trypanosomiasis (HAT, also known as sleeping sickness). Continue reading “Increasing Drug Research and Development Efficiency Using a 4-point Screening Method to Determine Molecular Mechanism of Action”

Mass Spectrometry Application: Antibody Quantitation for Preclinical PK studies

Posted on by Gary Kobs

Isoform_Antibodies_LinkedInTherapeutic monoclonal antibodies (mAbs) represent the majority of therapeutics biologics now on the market, with more than 20 mAbs approved as drugs (1–3). During preclinical development of therapeutic antibodies, multiple variants of each antibody are assessed for pharmacokinetic (PK) characteristics across model systems such as rodents, beagles and  primates. Ligand-binding assays (LBA) are the standard technology used to perform the PK studies for mAb candidates (4). Ligand-binding assays (LBAs) are methods used  to detect and measure a macromolecular interaction between a ligand and a binding molecule. In LBAs, a therapeutic monoclonal antibody is considered to be the ligand, or analyte of interest, while the binding molecule is usually a target protein.

LBAs have certain well-documented limitations (5). Specific assay reagents are often not available early in a program. Interferences from endogenous proteins, antidrug antibodies, and soluble target ligands are potential complicating factors.

Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS)-based methods represent a viable and complementary addition to LBA for mAb quantification in biological matrixes. LC–MS/MS provides specificity, sensitivity, and multiplexing capability.

A recent reference (6) illustrates an automated method to perform LC–MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard,and anti-human Fc enrichment. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. The interesting finding of the difference between ELISA and LC–MRM-MS data indicated that those two methods can provide complementary information regarding the drug’s PK profile.

Literature Cited

  1. Mao, T. et al. (2013) Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry. Anal.Chem. 85, 4239–46.
  2. Weiner, L. M. et al. (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 10, 317–27.
  3. Nelson, A. et al. (2010) Development trends for human monoclonal antibody therapeutics. Nat. Rev. Drug Discovery. 9, 767–74.
  4. DeSilva, B. et al. (2003) Recommendations for the Bioanalytical Method Validation of Ligand-Binding Assays to Support Pharmacokinetic Assessments of Macromolecules. Pharm. Res. 20, 1885–00.
  5. Ezan, E.et al. (2009) Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodies. Bioanalysis 1, 1375–88.
  6. Zhang, Q. et al. (2014) Generic Automated Method for Liquid Chromatography–Multiple Reaction Monitoring Mass Spectrometry Based Monoclonal Antibody Quantitation for Preclinical Pharmacokinetic Studies. Anal.Chem. 86, 8776–84.