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Q&A: Novel Macrocyclic Drugs for cancer Treatment – Exploring “beyond rule-of-five (bRo5)” chemical space

Dr. Spandan Chennamadhavuni, Emory University

Q: Increasing the Mw what is happening with this compound’s solubility?
A: Solubility of the drug candidate depends on many physicochemical properties such as Mol. Weight, cLogP, Polar surface area, no. of hydrogen bond donors and hydrogen bond acceptors etc. Therefore, it is possible to make drug molecule more soluble even if the molecular weight is high (major example in the literature is Cyclosporine).

Q: Can macrocycles be designed to induce PPI rather than to inhibit? Are there any successful examples?
A: We could imagine bifunctional small molecules that could interact with two different proteins could be used to bring two proteins together. (Stabilize protein-protein interactions). Typically due to very nature of macrocycles (occupying multiple extended binding sites), they are typically used for inhibiting protein-protein interactions. There are few examples of natural product based macrocycles that were used to induce PPIs (For example Swinolide A, Rhizopodin and Lobophorolide etc.) Please see review article “Expert opinion on drug discovery, 2017, vol. 12, no. 9, 925–940” for more details. 

Q: How did you select the binding pocket? Because the peptide binds to the large surface area then what could be the rationale to select that binding pocket?
A: There was no co-crystal structure of macrocycle bound to our PPI target. Our crystallography collaborators tried to get X-ray crystals for number of years. We opted to use FTMap, a computational method to find the druggable “hot spot” region on the protein surface. Our macrocycle design strategy was to have conformationally either flexible or rigid carbocycles with small substituents in the periphery. Therefore. The idea was those small substituents would occupy the binding hot spots and carbocycle would keep the small substituents in place. Our hypothesis was that we need to occupy those shallow binding pockets on the protein surface to cause PPI inhibition.

About FTMap In brief, Binding hot spots, protein sites with high-binding affinity, can be identified using X-ray crystallography or NMR by screening libraries of small organic molecules that tend to cluster at such regions.

FTMAP, a direct computational analog of the experimental screening approaches, globally samples the surface of a target protein using small organic molecules as probes, finds favorable positions, clusters the conformations and ranks the clusters on the basis of the average energy. The regions that bind several probe clusters predict the “binding hot spots”, in good agreement with experimental results. Small molecules discovered by fragment-based approaches to drug design also bind at the hot spot regions. To identify such molecules and their most likely bound positions, we extend the functionality of FTMAP ( to accept any small molecule as an additional probe. In its updated form, FTMAP identifies the hot spots based on a standard set of probes, and for each additional probe shows representative structures of nearby low energy clusters. This approach helps to predict bound poses of the user-selected molecules, detects if a compound is not likely to bind in the hot spot region, and provides input for the design of larger ligands.

Q: How do you explain oral bioavailability of macrocycles despites their physio-chemical properties (high MW, high TPSA, ClogP)?
A: Please refer to our Nature Chemical Biology article (Nat Chem Biol. 2014 Sep; 10 (9):723-31. doi: 10.1038/nchembio.1584.) The factors that effects oral bioavailability of macrocycles
Macrocycles have a number of inherent advantages that improve their prospects for achieving oral bioavailability, even when their physical properties lie outside the traditional Rule-of-5 chemistry space. Macrocycles could adopt three-dimensional conformations that overcome barriers to permeability. If we take the example of cyclosporine, it is orally available due to “Intramolecular Hydrogen Bonding” capability of the molecule that would allow the molecule to permeate through the cell despite of its high molecular weight.

Q: Is virtual conformer generation for macrocycles more challenging than regular compounds? Are there steps that require more care or treated differently?
A: Synthesis of macrocyclic library takes huge resources thus generating virtual combinatorial library of macrocycles is definitely a proper approach. We do need to filter virtual macrocycle library through our physicochemical properties filter to find “drug-like” macrocycles. At this stage, we generated ensemble of conformations for each macrocycles. It is not challenging as it is done in silico. We need to be careful in setting the energy parameters correct to get reasonable number of conformations. For example, if the macrocycles has too many rotatable bonds, then we would get hundreds of conformations for each molecule. Nevertheless, we choose 3-4 distinct conformations per macrocycle to use them in our docking studies.

Q: Are there some macrocyclic libraries publicly available?
A: None of the macrocyclic libraries is publicly available. However, we routinely collaborate with academic and Industry all around the world to screen our compounds. If you want to screen our proprietary macrocyclic library or any other compounds from our library in your HTS assay, you need to fill out this questionnaire:
We will review and arrange a time to speak in more detail about a potential collaboration and initiate preparation of our material transfer agreement. For further details, please reach out to Prof. Lauren Brown and Prof. John Porco at CMD.

Q: All the macrocyclic compounds you have synthesized (or shown) contain a trans-double bond. Did you also make the corresponding cis-compounds?
A: We did all synthetically feasible structural modifications to our macrocycles including isomerizing trans-double bond to cis to facilitate macrocylization via Thorpe–Ingold effect. We also did N-methylation, further functionalization of macrocycles to synthesize structurally diverse and complex macrocycles etc. 

Q: Have you considered using thioacetones to incorporate sulfur atom into the cycles?
A: We certainly considered thiolactones. The results from that study would be published in due course. There are certain stability issues associated with thioesters in a macrocycles that would be discussed in the due course. 

Q: What is the solubility of these macrocyclic compounds overall in general?
A: Our macrocyclic library has reasonable solubility. We routinely send out compounds in the 96 well plate for HTS assays. We did not see any difficulties in conducting assays thus far. 

Q: APE1 inhibitors seem to have µM affinities: would it be sufficient to effectively inhibit the PPI in cells, in animal models and ultimately in human patients?
A: As you could imagine, getting micro molar affinity for PPI targets (traditionally undruggable targets”) is itself is fundamental a proof of concept discovery that shows we could use macrocycles and/or bRo5 compounds for inhibiting PPI targets. We needed to make lot more progress in designing cell permeable, orally available macrocyclic drugs that would show efficacy in animals and humans. We are certainly making big strides in that direction. 

Q: Does degree of conformational rigidity of the macrocycle affect cell growth inhibition?
A: Yes, Conformational flexibility/rigidity has lot of influence on its cell permeability. Whether you would want to synthesize a conformationally restrained macrocycle or flexible macrocycle that would depends on the target of our interest. 

Q: How to address the macrocycle synthetic limitation for increasing scaffold diversity and rapid generation of analogues in a medicinal chemistry context?
A: It is a very difficult question to answer. Our goal is to discover and develop methods for synthesizing structurally complex and diverse macrocycles in a straightforward combinatorial fashion to explore diverse chemical space, carbocycles size, stereochemically diversity etc. It is the challenge that every medicinal chemist face, while doing any synthesis project. 

Q: Do you foresee the feasible strategies for macrocycle drug scale-up challenges?
A: We certainly anticipate issues in scaling-up macrocycle synthesis. We are trying to address now. There are several pharmaceutical drugs that are currently in the market that went through regular process development process and bulk scale production of those macrocycles is already established. Some macrocycle industrial production require semi-synthesis from a natural source. Nevertheless, I still feel that process chemistry of bRo5 compounds still in its infancy. 

Q: How to optimize the macrocyclic linker for drug-likeness properties (permeability, solubility, metabolic) without detriment to activity?
A: I think, the best approach is do regular structure based rational design of macrocycles for the biological target of interest to find the “Hit”. We could think of incorporating drug-like properties during hit to lead optimization step. I do not think there is a set protocol for optimizing rationally. Its try, fail, learn and repeat method for the time being. 

Q: What is the highest yield ring cyclization method(s)? I think you said the mactrolactamization had a yield of 19%.
A: Sorry for the mispronunciation. Our best yields are 99% not 19%. 

Q: What is optimal macrocycle size for achieving oral bioavailability? Comparing calculated physicochemical properties for the macrocyclic drugs with their route of administration reveals that orally administered macrocycles are smaller in size and more lipophilic than parenteral macrocycles, as further indicated by their lower polar surface area (PSA) and fewer hydrogen bond donors (HBDs)
A: According to our study, we think 12 to 15 membered macrocycles would be optimal. However, it also depends on the PPI target that we are working with. 

Q: What field do you use for the structural analysis? R-field? Born Solvation etc.?
A: We used standard docking methods using Schrodinger suite and/or autodock vina for our docking studies. We used OPLS force field and Born solvation settings. As I was involved in virtual library generation of millions of compounds, our collaborators did the docking studies. I hope this answers your question. 

Q: How stable are the macrocycles metabolically? Are the cycles easily breakable?
A: We are still working on finding the metabolic stability and cell permeability of the synthetic macrocycles. The results would be published in due course. These macrocycles are certainly chemical stable and not breakable. We routinely do QC on the samples before sending them out for HTS and we did not observe any noticeable degradation in most of the macrocycles over time.

Q: What is the route of administration for the 80-100 FDA approved drugs that are bRo5?
A: Most of the non-oral macrocycles and bRo5 compounds are administrated parenterally (route other than digestive tract) administration: subcutaneous (SC/SQ), intraperitoneal (IP), intravenous (IV), intradermal (ID), and intramuscular (IM). It depends on the disease it is getting used for. 

Q: Can you please talk about the use of DNA Encoded Libraries (DELs) to find novel small molecule molecular glues that do not follow the rule of 5? I.e. what are empirical, non-rational methods to discover small molecules that do not appear drug like?
A: This is such a broad question that would be very hard to answer. Please look at the technology developed at Ensemble Therapeutics ( and their publications. They develop DELs of macrocycles. If you do not want to use rational drug design to find the bRo5 compounds, then only option would to do high-throughput screening of several thousands of bRo5 compounds that are already available through various sources. However, chances of finding something interesting would be very slim. 

Q: Is Macugen (Pegaptanib) included in the 100 list of FDA approved drugs, which do not follow the rule of five? Alternatively, any other RNA drug?
A: Please find the poster for Beyond Rule of Five (bRo5) Drugs.