Category Archives: drug discovery

Practical Fragments blog has reviewed our paper!

Our latest fragment-based drug discovery paper against the p97 ATPase has been noticed and reviewed favorably by the widely-read Practical Fragments blog.

Here is an excerpt from that review:

“The protein p97 is important in regulating protein homeostasis, and thus a potential anti-cancer target. But this is no low-hanging fruit: the protein has three domains and assembles into a hexamer. Two domains, D1 and D2, are ATPases. The third (N) domain binds to other proteins in the cell. All the domains are dynamic and interdependent. Oh, and crystallography is tough. Previous efforts have identified inhibitors of the D2 domain, but not the others. Not to be put off by difficult challenges, a group of researchers at the University of California San Francisco (UCSF) led by Michelle Arkin and Mark Kelly have performed fragment screening against the D1 and N domains, and report their adventures in J. Biomol. Screen.

Cancer immunotherapy and the role of tryptophan


cancer immunotherapy drug 1-MT



In the body, L-tryptophan is catabolized by an enzyme called Indoleamine 2,3-dioxygenase (IDO) to form a class of molecules known as kynurenines.  These compounds have been shown to be immunosuppresive, preventing inflammation and T-cell mobilization.  Additionally, depletion of cellular stores of L-tryptophan also appears to induce down-regulation of the  immune response.

What does this have to do with cancer immunotherapy?  Interestingly, cancer actively hijacks the IDO pathway to promote immune system suppression and tolerance to tumor cell antigens by overexpressing IDO in the tumor, at host cells in the immediate area of the tumor, and at tumor-draining lymph nodes where T-cells could normally become activated against tumor antigens.

Think of it like a beekeeper using smoke to keep the bees calm as the keeper removes honey from the hive.   By upregulating the expression and activity of the IDO pathway, tumors effectively “hide” from the immune system while they grow out of control in the host tissue.  But this exploitation of the body’s own immune regulation by cancer also presents a weakness that can leveraged in the fight against tumor progression.

Inhibiting IDO to enable tumor recognition

Enter 1-methyl-DL-tryptophan (1MT), pictured above.  1MT is known to be an inhibitor of IDO that works presumably by mimicking the natural substrate (although I believe this has not been shown explicitly).   IDO inhibition by 1MT has been shown to work in combination with chemotherapy approaches to limit tumor progression in mouse models.

Adding 1MT to chemotherapy treatments allows the host immune system to mediate a response to the tumor cells, especially in the presence of dying tumor cells undergoing apoptosis and releasing antigen.  By taking away tumor-induced immune tolerance, 1MT inhibition of IDO allows the T-cell system to recognize, attack and destroy cancer cells in synergy with chemotherapy.

Early  clinical trials involving 1MT appear to be ongoing, with work being done by NewLink Genetics in Ames, IA.




FTMap: fast and free* druggable hotspot prediction

*free to academics

FTMap is a useful and fast online tool that attempts to mimic experimental fragment-screening methodologies (SAR-by-NMR and X-ray crystallography) using in silico methods.   The algorithm is based on the premise that ligand binding sites in proteins often show “hotspots” that contribute most of the free energy to binding.

Often, fragment screening will identify these hotspots when clusters of different types of fragments all bind to the same subsite of a larger binding site.   In fact, x-ray crystallography studies of protein structures solved in a variety of organic solvents demonstrate that small organic fragments often form clusters in active sites.

In the FTMap approach, small organic probes are used for an initial rigid-body docking against the entire protein surface.  The “FT” of FTMap stands for the use of fast Fourier transform (FFT) methods to quickly sample billions of probe positions while calculating accurate energies based on a robust energy expression.

Following docking of each probe, thousands of poses are energy-minimized and clustered based on proximity.  The clusters are then ranked for lowest energy.   Consensus sites (“hot spots”) are determined by looking for overlapping clusters of different types of probes within several angstroms of each other.   If several consensus sites appear near each other on the protein surface, that is a strong indication of a potentially druggable binding site.

Isotopic labeling of proteins in non-bacterial expression systems

As therapeutic proteins gain importance alongside traditional small molecule drugs, there is increasing interest in using NMR methods to examine their structure, dynamics, and stability/aggregation in solution.

Modern heteronuclear NMR of proteins relies on isotopically-labeled samples containing NMR active nuclei in the peptide backbone, sidechains, or both.

Although isotopic-labeling of recombinant protein is typically carried out in E. Coli expression systems, many biotherapeutic proteins must be expressed in eukaryotic systems to insure proper folding and/or post-translational modifications.   In practice, this means overexpression in either yeast, insect or mammalian cells.

Increased interest in attaining labeled protein samples for analysis by NMR is leading to better commercial availability of isotopically-labeled expression media and improved vectors for overexpression in non-bacterial systems.

Comprehensive reviews of state-of-the-art protocols and procedures for expression of isotopically-labeled proteins in non-standard systems are available here: yeast, insect cells, and mammalian cells.



Virtual screening capability for under $5K?

Many early stage companies may be missing out on the value that docking can provide at the validated hit and hit-to-lead stages of development, where structure/activity relationships (SAR) can help guide chemistry development of lead compounds.

While docking large HTS libraries with millions of compounds may require specialized CPU clusters, docking of small libraries (i.e., thousands of compounds) and SAR compounds from experimental assays is readily achievable in short time frames with a relatively inexpensive Intel Xeon workstation.

Following an initial investment in the workstation and software, follow-on costs are minimal (e.g., electricity, IT support and data backup). Turnaround times may be faster than with CRO services.  Also, sensitive IP data is also protected by being retained onsite and not transmitted over the internet.

Equipment / cost breakdown:


AutoDock Vina (non-restrictive commercial license)       cost: free

Accurate (benchmarked against 6 other commercial docking programs)

Compatible with AutoDock tools

Optimized for speed (orders of magnitude faster than previous generation)

Parallelized code for multi-core systems

AutoDock Tools (non-restrictive commercial license)      cost: free

PyMol Incentive (commercial license)                                        cost ~$90 / mo

Visualize docking results, free plugin can allow Vina to be run within PyMol GUI

Fedora Linux                                                                                              cost: free


HP Z620 Workstation (stock configuration)                          cost: $2999

2 GHz (6 Core) Intel Xeon E5-2620 2GHz

USB keyboard and mouse                                                                  cost: $50

Dell Ultrasharp 27” LED monitor                                                  cost: $649

1TB USB HD for data backup                                                          cost: $150

IT support for initial setup ~ 4 hours                                           cost: $400

Total initial capital expenditure:                                                  ~$4350



10 Common Mistakes in Fragment Screening

There is an excellent review paper from Dan Erlanson and Ben Davis that came out last year detailing some of the more common mistakes and artifacts that can arise in fragment-based screening campaigns (so-called “unknown knowns”).  I encourage readers to go read the original paper.  I have summarized some of the key points below:

1) Not checking compound identity to make sure what you think you purchased is what you actually have.

2) Low-level impurities in compound stocks can cause problems at the high concentrations used in fragment screens.

3) DMSO, commonly used to store fragments in plates, can act as a mild oxidant and is also hygroscopic.

4) Pan-assay interference compounds (PAINS) are common in many libraries and are found to give false positives to many targets.

5) Reactive functional groups in fragment hits can cause covalent binding or aggregation of the target.

6) Many fragments can show binding or inhibition while acting as aggregators rather than reversible binders.  Including a small % of detergent can help eliminate these kinds of fragments from giving positive signals.

7) STD-NMR is very sensitive to weak binders, but because it relies on a relatively fast disassociation rate for the ligand, tight binders (<1 uM) can be missed by this method.

8) X-ray crystallographic structures are often taken as the “truth” when they are in fact a model of an electron density.  Fragments can often be modeled into the density in incorrect orientations or in place of solvent atoms.

9) SPR methods are very sensitive to fragment binding, but can be confounded by non-specific binding of fragment to the target or chip, as well as compound-dependent aggregation.

10) Fragment hits should be validated by more than one method before embarking on optimization.  They should also be screened for being aggregators by DLS or other methods.

Gilead’s innovative approach to Hep C drug, Sovaldi

Hepatitis C virus (HCV) is a single-stranded RNA virus that infects an estimated 180 million people worldwide.

In 2013, Gilead received FDA approval for a new HCV drug, Sovaldi (sofosbuvir), that inhibits viral replication by targeting the virus’s NS5B polymerase.  Sovaldi has shown a very high cure rate (nearly 100% HCV suppression and sustained virological response) in clinical trials of previously untreated patients and has fewer side effects than pegylated-interferon and ribavirin therapies.

Sovaldi is a methyluridine-monophosphate prodrug: it is metabolized in the body back into methyluridine-triphosphate, which acts as a potent substrate mimic and inhibitor of the NS5B polymerase.

What is interesting about Sovaldi is the approach the scientists took to getting the inhibitor into the cell, relying on phosphoramidate prodrug  technology that had been effectively used to develop anti-HIV drugs, but had never been applied before to this class of anti-HCV drugs.

During development, the researchers decided that they needed to deliver the charged methyluridine-monophosphate (rather than the neutral methyluridine)  into the cell on the basis of two key observations:  1) the methyluridine triphosphate is the active compound against HCV NS5B polymerase, while the methyluridine alone is inactive (owing  to very low conversion to monophosphate in vivo) and 2) the methyuridine monophosphated derivative can be anabolized in the cell back to the potent triphosphate form by an endogenous uridine-cytidine monophosphate kinase.

The active uridine triphosphate (6) can be created when 4 is metabolized to methyluridine-5′-monophosphate. Compound 5 is not phosphorylated and is inactive in cells.


The phosphoramidate prodrug technology had never been applied to HCV inhibition until Solvadi.

The idea behind  phosphoramidate prodrug technology is to create a membrane-soluble neutral prodrug derivative that can be metabolized in the liver by carboxylesterase-mediated cleavage and subsequent steps back to the monophosphate form.

The researchers applied the approach and after a significant amount of  SAR investigation and PK/PD studies around the chemical composition of the phosphoramide substituents, they concluded that the structure of compound shown above was the optimal structure to deliver the methyluridine-monophosphate to the liver.

The result is a new generation of highly effective HCV therapeutics with few side effects that can make a significant difference in the lives of patients living with HCV.


Tackling challenging targets with Chemotype Evolution

Carmot Therapeutics, a small company located in San Francisco’s Mission Bay, has developed a very innovative drug discovery technology, called Chemotype Evolution (CE), that relies on fragment-based discovery but is different from traditional FBDD and HTS approaches in important ways.

The first important innovation is that CE relies on a “bait” molecule as a starting point for screening.  The bait can be a known ligand, cofactor, or inhibitor.  The bait is then derivatized with a linker moiety that allows it to become chemically bonded with every fragment in a proprietary library.  This process generates a screening library that contains thousands of bait-fragment hybrids.

The most powerful aspect of CE is the ability to iterate over chemical space, allowing access to an exponential number of possible fragment-bait hybrids.

These hybrids are then screened against the target for binding using either biophysical or biochemical screening techniques in a high-throughput plate format.
The most powerful aspect of CE is the ability to iterate over chemical space, allowing access to an exponential number of possible fragment-bait hybrids.  The method can be iterated with new “baits” derived from the best fragment hits of the previous round.  Thus, instead of having 7,000 fragments in your library, after 3 iterations you access 7,000^3 possible combinations (343 billion possible compounds), selecting only the most target-relevant chemotypes at each stage.

Schematic of the Chemotype Evolution process through 3 iterations. Note that at any point after each iteration, the hit molecules can be taken into hit-to-lead optimization.

The CE approach is similar in concept to the “tethering” approach pioneered at Sunesis, but differs in the fact that no protein engineering of cysteine residues needs to be performed.  The bait molecule performs the role of the engineered cys, providing a “handle” that binds to the target and selects for complementary fragment binders.

Carmot Therapeutics just embarked upon their first major industry collaboration with the January 2014 announcement of a partnership with Amgen

Carmot Therapeutics just embarked upon their first major industry collaboration with the January 2014 announcement of a partnership with Amgen to use CE technology against two challenging targets.  Identifying leads and developing hits will be carried out jointly between the companies, while clinical trials will proceed at Amgen.  I think Carmot is definitely a company to watch given its innovative and potentially paradigm-shifting discovery technology and increasing interest from big pharma.




Why isn’t pharma making blockbuster antibiotics?

It seems intuitive that there would be a large market for new, highly-effective antibiotics.  Doctors are warning publicly about the waning effectiveness of today’s antibiotics owing to over-prescription and increased drug resistance.

The linked article even mentions that a course of action could be to provide government incentives to the industry to make new antibiotics.  But where the market creates a profit potential, why would government incentives be necessary in the first place?

I had never heard a suitable explanation for this situation until recently, in a conversation, the following theory was advanced:  if new wonder drugs are developed, they will be “held back” by doctors seeking to establish last-line-of-defense antibiotics, and will therefore not be heavily prescribed, dramatically limiting profitability.

Does the above explanation make sense?  Is there more to the story?  Share your thoughts in the comments below.




Rethinking drug action: activating an ion channel to treat Cystic Fibrosis

In my first “Rethinking Drug Action” post, I described how researchers are seeking activators of PARK9, a protein that is mutated in Parkinson’s Disease.  In a similar manner, Ivacaftor, a new drug for Cystic Fibrosis (CF), shifts the paradigm from treating CF symptoms to therapeutic treatment of the underlying cause of the disease: defects in the activity of the CFTR ion channel owing to genetic loss-of-function mutation.

The molecular structure of Ivacaftor (Kalydeco).
The molecular structure of Ivacaftor (Kalydeco).

In this case the mutation is the rare G551D variant (4-5% of all CF patients) that makes CFTR non-responsive to ATP-dependent channel opening.  The more common delta-F508 CFTR mutation is thought to prevent membrane expression of CFTR through misfolding, and indeed, clinical trials showed that ivacaftor alone had no effect on patients with this mutation.

Ivacaftor, a new drug for Cystic Fibrosis (CF), shifts the paradigm from treating CF symptoms to therapeutic treatment of the underlying cause of the disease

However, for patients with the G551D mutant, where CFTR does reach the membrane but is less active than WT, the drug is very efficacious.  In a clinical trial, patients who received ivacaftor were 55% less likely to experience pulmonary exacerbation (defined as a worsening of lung function owing to infection or inflammation) after 48 weeks on the drug.  Other markers of CF were also improved during this period.

The exact mechanism of action of ivacaftor is not known. Interestingly, however, ivacaftor enhances spontaneous ATP-independent activity of both G551D-CFTR and WT-CFTR to a similar magnitude.  In a recent PNAS paper, researchers propose that ivacaftor affects both WT and G551D in the same way, namely by shifting the equilibrium from the closed (C2) state towards the open2 (O2) state, in essence, “wedging” CFTR open.

Proposed mechanism of CFTR gating from the PNAS paper cited below.  Ivacaftor is thought to stabilize the O2 form over the C2 form.
Proposed mechanism of CFTR gating from the PNAS paper cited above. Ivacaftor is thought to stabilize the O2 form over the C2 form.

In the same paper, the researchers propose that the CFTR transmembrane domains (TMD) may be the site of binding for the drug.  In support of this, they note that the drug is relatively hydrophobic and is measured to increase gate opening times regardless of whether it is applied from the cytoplasmic or extracellular side, suggestion membrane permeation and binding to the TMDs.

In a clinical trial, patients who received ivacaftor were 55% less likely to experience pulmonary exacerbation

More studies are needed to prove this mechanism, but it will be very interesting to see how this paradigm-shifting new drug works on the molecular level.  In addition, other compounds are in development that aim to enhance the folding and membrane expression of the more common DF508 mutation.  Perhaps combination therapy with new compounds for DF508 and ivacaftor together will aid those CF patients who currently are not helped by ivacaftor alone.