Mini-library approach delivers an arsenal in drug development pipeline

Dr. Naganna Nimishetti, Chief of the Small Molecule Drug Discovery Division (SMDD) and Dr. Dun Yang, CEO/CSO of Anticancer Bioscience, a precision therapeutics company headquartered in Chengdu, China, explain how the company is identifying bioactive hits using libraries of less than 100 compounds.

During the drug discovery process, approximately one million compounds need to be tested before discovering and developing one marketable drug1. Such a high attrition rate, coupled with the fact that an estimated 6,000 or more druggable targets are yet to be explored, generates pressure for building more extensive libraries and boosting the capacity of high-throughput screening (HTS) to increase the odds for identifying lead compounds. However, if fewer compounds could be tested without compromising the probability of success, timelines and cost would be significantly reduced. This would also enable more small start-up companies and academic laboratories to participate in the drug development process, since they are typically limited by lack of access to large compound libraries and HTS facilities.

Scientists at the J. Michael Bishop Institute of Cancer Research (MBICR) and its commercialisation partner,  Anticancer Bioscience (ACB), have described the first novel general utility new scaffold-drug fragment (GUNS-DF) library that includes less than 300 synthesised compounds. Demonstrating their utility in drug discovery, ACB has shown that within this library, ten compounds have been successfully identified with low nM activity for inhibiting a potential MYC-synthetic lethal target in cell-based assays. Direct targeting of MYC has been a challenge for decades owing to its “undruggable” protein structure, with a lack of a drug-binding pocket. This result highlights the advantage of this proprietary GUNS-DF library technology platform in uncovering the next generation of anticancer compounds.

Addressing the unmet need in drug discovery

Rather than establishing one extensive library before kicking off any screening effort, ACB’s approach was to build a series of mini libraries of 60-80 compounds for HTS and then expand the compound diversity of the libraries on demand during the lead optimisation process. These libraries are called ‘general utility new scaffold-drug fragment’ libraries, with the acronym “GUNS-DF”. The first library was constructed using principles of medicinal chemistry that anticipate potent inhibitors with enhanced drug-like properties. One key feature of this and future planned GUNS-DF libraries is a patentable lead-like core scaffold that permits size expansion and diversity augmentation during lead optimisation. All compounds in the library are new chemical entities (NCEs) to enable patentability and structures predict drug-like properties to reduce the odds of attrition and enable patient-friendly, oral availability for the clinical setting. NMR and HPLC/LCMS validation has been used to ensure the high purity of compounds in the ACB GUNS-DF library.

ACB’s approach is to design several new core scaffolds to mimic each of the high-frequency elements ACB identified in FDA-approved drugs or investigational new drugs in clinical trials. Each core scaffold acts as an intermediate that can be connected to 2-4 sets of side chains through distinct conjugation reactions at different positions (Figure 1). Extension of these new scaffolds with commonly utilised side-chains can rapidly enable the creation of a pilot library harbouring a variety of drug-like compounds to assay for bioactivity. Once a hit is identified from the pilot library, the screening effort automatically generates structure-activity relationship (SAR) data that can inform a pharmacophore and guide the synthesis of additional related compounds. This facilitates the discovery and optimisation of potent molecules that are predesigned to have drug-like properties. GUNS-DF libraries are constantly expanding and evolving as ACB researchers move through iterative screening and optimisation processes against different targets associated with multiple screening projects that are being conducted simultaneously (Figure 2).

The small strategic sizes of GUNS-DF libraries

Theoretical studies have revealed that as the ligand-receptor match becomes complex, the probability of finding a detectable interaction for a random oversized ligand rapidly approaches zero. Therefore, screening smaller compounds is expected to deliver more hits. Indeed, the hit-rate for fragment-based screening is typically 3-10%, much higher than the 1 out of 1 million rate with HTS of large compound libraries. Another reason for opting for smaller compounds as a starting point is that compounds with smaller sizes better accommodates mass increases during the lead optimisation process. These properties underpin the successful development of fragment-based screening as a viable alternative to HTS of larger libraries with more complex structures.

Already being used in many kinase inhibitor programs, the fragment-based drug discovery (FBDD) approach has the potential to identify novel fragment-sized hinge binders and allow specific evolution of a hit fragment into a potent inhibitor of a target protein. One well-known kinase-privileged fragment, 7-Azaindole, for example, has been incorporated into many kinase inhibitors as a hinge-binding motif by making two hydrogen bonds with the kinase hinge region2. Despite advantages such as this, FBDD has two significant intrinsic shortcomings. First, FBDD typically uses compounds with a molecular weight of less than 200 Da. They are likely to have low affinity binding to their receptors and this demands much more sensitive assays than those generally used in HTS. Second, the successful execution of FBDD requires the availability of structural information for the drug target.

Unlike FBDD, ACB’s GUNS-DF compounds have a molecular weight of around 300-350 Da, allowing identification of hit compounds using methods used in traditional HTS assays. However, like FBDD, the reduced size of GUNS-DF library compounds also increases the chance of identifying a hit compound. In our hands, the hit rate is further enhanced by phenotypic screening. ACB has used the GUNS-DF libraries to screen for compounds that can elicit phenotypes of interest by interfering with any component in a pathway or a complex.  A hit rate of about 3% has been achieved in one cell-based phenotypic screening assay. This means screening 60-80 compounds is often sufficient to identify at least one hit compound. In our opinion, the GUNS-DF library approach achieves a good balance, limiting shortcomings inherent in both FBDD and HTS, allowing high hit rates as well as adequate potency.

Creation and proof-of-concept screening with the HJ series, GUNS-DF library

As mentioned, hit compounds arising from HTS are not always suitable as lead compounds for modification simply because of their large molecular weight. Therefore, there is a need for synthetic fragment-like libraries, in which all compounds are NCEs and have good lead-like properties. It is this necessity that inspired the ACB’s GUNS-DF library model.

“In this approach, we started with a high frequency element (HFE) found in FDA-approved drugs and modified it into a new chemical entity (NCE) to enable patentability.” said Dr. Nimishetti. “We engineer the NCE core scaffold to harbour multiple conjugation sites that can facilitate the synthesis of library compounds through a variety of linkers. Within two steps, we generated a key construction intermediate, ‘hydroxy, ester and amine-containing scaffold’, and use it for the synthesis of final library compounds in one more step. All compounds we synthesize are NCEs that have good lead-like properties and conform to Lipinski’s rule of five.”

For initial proof-of-concept, ACB has used the patent-pending ‘HJ’ series, mini- library in a phenotypic screen to identify synthetic lethal compounds attacking tumours that overexpress the MYC oncoprotein. Overproduction of MYC occurs in 50-70% of human malignancies and is correlated with poorly differentiated and very aggressive cancers, which currently lack a potent targeted therapy. Since MYC has proven difficult to inhibit, researchers at ACB are enthusiastic about developing synthetic lethal therapies to treat MYC-overexpressing cancer. Dr. Dun Yang, CEO of ACB, and his Nobel laureate mentor Dr. J. Michael Bishop, have previously identified a synthetic lethal interaction between MYC and disablement of the chromosomal passenger protein (CPP) complex3. Building on that expertise, ACB has developed a mechanism-informed phenotypic screen to identify novel antimitotic agents that mimic the disablement of the CPP complex, therefore having potential as MYC-synthetic lethal agents4. Equipped with a prior understanding of the versatile functions of the CPP complex, its role in orchestrating karyokinesis and cytokinesis and its upstream modifying pathways, the screening assay scores for phenotypes associated with CPP disfunction. ACB has applied this synthetic lethal screening assay for MYC tumours to its HJ series of compounds to guide the identification and optimisation of leads.

Lead optimisation and target deconvolution

The MYC-synthetic lethal screening was performed in isogenic cell lines engineered for an image-based HTS of defined cellular phenotypes. The visualisation technology enabled systemic analysis of alterations at the cellular, sub-cellular and molecular level. While HTS of synthetic compound libraries typically assays tens of thousands of compounds to identify a hit/lead compound, the GUNS-DF technology allows us to explore less than 100 compounds to identify a hit compound using our image-based approach.

ACB was also able to quickly identify the relevant target protein within one week of hit identification. Timely target deconvolution then facilitated our medicinal chemists in the use of computation-based drug design by modelling the protein-ligand interaction. Identification of the drug target also enabled a precise look at whether target inhibition is correlated with the expected cellular consequences during the lead optimisation.

Jian Huang, a project leader from the SMDD group, said: “We integrate lead optimisation and expansion of the library since the synthesis of additional compounds during the lead optimisation increases the diversity of our library. As a result, we synthesised less than 300 compounds to obtain more than ten potent compounds with low nM activity for inhibition of the intended cellular target. We achieved this within half a year, and this achievement could be partially attributed to the fact that the primary screening process generated adequate SAR data that was essential to guide our next run of synthesis. In addition, quick identification of the drug target by our Discovery Oncology division and the availability of the structural information around the drug target also helped us a lot in lead optimisation.”

From the HJ series, ACB has identified multiple lead-like compounds that exhibit synthetic lethality with the MYC oncogene. The low nM anticancer activity of several HJ compounds has been confirmed in vitro in both 2D and 3D assays against a diverse set of human cancer cell lines, including lung, colorectal, cervical and mammary cancer cell lines.

Identification of the molecular target for small molecule inhibitors, i.e. target deconvolution, can be challenging. By performing mechanism-informed phenotypic screening, ACB was able to quickly identify the target for its HJ compounds. The cellular activity of the HJ lead compounds in vitro is as robust as that seen with inhibition of Aurora B kinase, disablement of which is known to elicit synthetic lethality in cells over-expressing MYC. However, the target for these novel compounds of the HJ GUNS-DF series is different from the well-known MYC synthetic lethal targets Aurora kinase A or B.  Public disclosure of the target of the HJ compounds will follow once intellectual property filings are complete. Validation experiments in vivo have been completed in xenografts of non-small cell lung cancer and triple-negative breast cancer cell lines, confirming robust anticancer activity in both pre-clinical models.

Our expanding panel of pilot GUNS-DF libraries

ACB has now designed ten distinct GUNS-DF pilot libraries, the majority created with kinase inhibitory activity in mind. The human kinome is composed of 518 different protein kinase genes, which play a significant role in cellular processes contributing to the development and progression of diseases such as cancer5. Protein kinases are enzymes responsible for the transfer of a phosphate group from ATP to a substrate. Activating mutations in kinases can initiate tumorigenesis and drive the progression to metastasis. They represent perhaps, the most promising class of oncology targets and discovery programs that have focused on developing kinase inhibitory drugs significantly over the last two decades. Eight out of the ten core scaffolds of the GUNS-DF libraries inhibit kinases, motor proteins, and other ATP-binding proteins by binding to the hinge region of the adenine-binding pocket. Extension of each core scaffold with a hydrophobic binder is expected to convert scaffolds into type I or II kinase inhibitors. However, ACB has observed that the target spectrum is not limited to ATP-binding proteins because the hinge is a common motif found in other types of proteins.

The remaining two GUNS-DF libraries were modelled around a shared motif found in several polyploidy-synthetic lethal compounds that ACB has identified. The protein target(s) for these polyploidy-synthetic lethal compounds has yet to be identified. The diversity of all of ACB’s compound libraries will increase as lead optimisation is performed for any and all of the five concurrent drug-development streams ongoing at ACB. The application of these libraries to other drug development efforts should further enhance compound diversity. The repeated use for screening and directed expansion of molecules around the core scaffolds of the GUNS-DF libraries during different screening efforts reduces the effort needed to create NCEs with drug-like properties with future projects. 

Conclusions and perspectives

Scientific solutions often do not arise solely from just one discipline, so the exchange of scientific information inherent in ACB’s discovery process emphasises the value of collaboration. ACB has demonstrated the benefit of close collaboration in its small molecule drug discovery programs. It has resulted in the creation of the GUNS-DF libraries and success in the mechanism-informed phenotypic screening conducted by drug optimisation biologists. ACB’s HJ compounds are currently being assessed using ex vivo and small animal pharmacokinetic assays to determine the best candidate to advance from a pool of several interesting lead compounds.

We would like to emphasise that the utility of the GUNS-DF libraries is not limited to the discovery of oncology drugs. In theory, similar libraries and the phenotype-guided screening approach we have used with the GUNS-DF libraries should be applicable for discovering modulators for any druggable process where a screening readout can be developed. Since this approach does not require a high-throughput capacity, it may be conveniently adapted by individual laboratories and small start-up companies to combine with their expertise in disease biology to expand drug discovery.

Volume 22, Issue 3 – Summer 2021


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  2. Motati DR, Amaradhi R, Ganesh T. Azaindole therapeutic agents. Bioorg Med Chem 28(24):115830 (2020).
  3. Yang D, Liu H, Goga A, Kim S, Yuneva M, Bishop JM. Therapeutic potential of a synthetic lethal interaction between the MYC proto-oncogene and inhibition of aurora-B kinase. PNAS 107 (31): 13836-13841 (2010).
  4. Li J, Yan Z, Li H, Shi Q, Huang L, Nimishetti N, Allen TD, Yang D, Zhang J. A high-content screen for anti-mitosis and polyploidy-induction identifies an unknown activity of two benzophenanthridine alkaloids from Corydalis longicalcarata. Phytochemistry Letters 41:180–185 (2021).
  5. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome.Science 298:1912–1934 (2002).

Note – MBICR and ACB have applied for a patent on the GUNS-DF library method. There are restrictions on the GUNS-DF library’s commercial use due to a pending patent application (PCT/CN2021/071225). 

About the authors

Dr Dun Yang, PhD,  Founder, President and CEO of Anticancer Bioscience, is also the Director of J. Michael Bishop Institute of Cancer Research. He has an academic background in cancer cell biology, having undertaken graduate studies at Columbia University and completed post-doctoral training at the University of California, San Francisco.

Dr Naganna Nimishetti joined Anticancer Bioscience in 2019 as Chief of Small Molecule Drug Design. He has an academic background in medicinal chemistry and small molecule drug design, having completed post-doctoral training at the Centre for Drug Discovery at Purdue University.

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