By Christian Gampe


While biologic drugs currently dominate in immunology by sales, small molecule immunomodulators (SMIMs) are making a comeback fueled by better understanding of intracellular signaling pathways and new hit finding technologies. SMIMs can be grouped into two categories: those that engage large molecule (LM) targets in a new way, and those that engage targets that cannot be drugged by large molecules.

Depiction of a cross-section through a cell with large molecules drugging extracellular receptors and signaling molecules and small molecules drugging intracellular proteins
Figure 1. Large molecules are often the modality of choice for extracellular targets; however this recent dogma is being re-challenged by small molecules.

A Snapshot of Small Molecule Immunomodulators in H1 2021

A selection of interesting SMIMs that were published or otherwise in the news in the first half of 2021 are highlighted below. A more in depth discussion of the different molecules and mechanisms of action follows.

12 small molecule immunomodulators that have been in the literature or news in the first half of 2021  small molecule IL-17A inhibitor, antagonist, TLR7/8 antagonist, TNFa allosteric inhibitor, INCB086550, PD-L1 small molecule inhibitor, S1PR, ponesimod, voclosporin, calcineurin inhibitor, LYS006, LTA4H, PY-60, ANXA2, YAP inhibitor
 Figure 2. Highlights from small molecule immunomodulators in the literature or news in the first half of 2021.

Small Molecule Immunomodulators – H1 2021

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Category 1: Small Molecules Engaging Targets of Large Molecules

Small molecules that can be dosed orally are more convenient to take than injected large molecules, and convenience aids compliance, which increases efficacy. For example, the vast majority of rheumatoid arthritis patients would switch from their current injectable medication to a twice-daily oral drug, if safety and efficacy were equal. As illustrated below, SMIMs that engage classic targets of large molecules have to do so in a very different way than neutralizing antibodies, which may result in different beneficial drug effects. 

IL-17A antagonists

One hot area in immunology has been the race to a commercial small molecule IL-17A antagonist given the success of the marketed antibody drugs secukinumab (Cosentyx) and ixekizumab (Talz) in psoriasis, psoriatic arthritis, and ankylosing spondylitis. Back in 2013, Ensemble reported the identification of SM binders of IL-17A (WO2013116682). Three years later, a group at Pfizer published the co-cocrystal structure of closely related compounds with the IL-17A homodimer, revealing that these compounds bind at the interface of the monomers of the IL-17A dimer, thereby allosterically reducing its ability to engage the IL-17 receptor. In 2020, patent applications published by Lilly (WO2020146194), LEO Pharma (WO2020127685), and DiCE Molecules (20200247785A1) contained compounds that retain some of the features of the original Ensemble molecules (highlighted in color).

Ensemble Eli Lilly DiCE Molecules LEO Pharma small molecule IL-17A antagonists
Figure 3. SMIM IL-17A modulators.

While the exact structure of the clinical compounds has not been disclosed, Lilly and Leo both report to have begun phase I studies in H1 2021 with IL-17A SMs-antagonists (NCT04586920, NCT04883333). Two additional players may soon join: DiCE is poised for IND submission, and Sanofi entered the race by licensing C4Xs IL-17 SM antagonist. 

TNFα antagonists

Oral inhibitors of TNFα, the target of the mega-blockbuster injectables Humira and Enbrel, have been a holy grail for small molecule drug discovery. In December and January, BMS and AbbVie both disclosed the discovery of SM antagonist of TNFα  (both featured as Molecules of the Month). The BMS team identified their compound from a scaffold hop from the UCB starting point, which induces an inactive conformation of the TNFα trimer. The team rapidly fixed early compound 39’s low cellular potency (addition of F and CN, in color) and high CYP inhibition (addition of Me, in color) to arrive at the clinical candidate 42. This molecule showed a very good mouse PK profile (t1/2 = 6.2h, 58% F) and efficacy comparable to Enbrel in mouse collagen antibody-induced arthritis models at doses of 10 mpk, around 5 x whole blood in vitro EC50. Enabled by a robust protein crystallography platform, the AbbVie team identified fragment 6, which was optimized using structure-based drug design as well as insights from the SAR of the UCB molecule to arrive at compound 12. This TNF-inhibitor had a PK profile (t1/2 = 1.1 h, 50% F) conducive to further evaluation. Efficacy of 12 was seen in the glucose-6-phosphate isomerase induced arthritis model at 30 and 100 mpk (reaching Cmax of up to 6.19  μg/ml), albeit not at the same levels as the anti-TNF antibody control.

UCB Pharma, BMS, and AbbVie TNF inhibitors
Figure 4. A fragment screen, structure based drug design, and scaffold morphing was used to arrive at SM TNFα inhibitors with preclinical efficacy

PD-L1 inhibitors

Earlier this year, both Incyte (AACR meeting 2021) and Arbutus Biopharma (Nature Communications) disclosed their PD-L1 small molecule inhibitors. SM PD-L1 inhibition was kickstarted in 2015 by work at BMS that showed that BMS-202 induces PD-L1 dimerization and inhibits PD-1/PD-L1 signaling (20150291549A1). Shortly thereafter a cocrystal structure of the compound with the PD-L1 dimer was disclosed which opened the opportunity for structure-based drug design (SBDD). Incyte and Arbutus both took advantage of the inherent C2-symmetry in the PD-L1 dimer by symmetrizing their molecules (e.g. PDB: 6VQN shown in Figure 5). The symmetrization strategy brought potency at expense of PK properties, which had to be engineered back into the inhibitors in a medicinal chemistry tour de force. 

Notably, these molecules induce PD-L1 dimerization on the cell surface, which leads to PD-L1 internalization. Receptor internalization is dose dependent and correlates with CD8+-T-cell infiltration and efficacy in a syngeneic mouse model. It will be interesting to see whether the different MoA of the SMIM receptor internalizers, together with promising pre-clinical data and potential for higher tumor exposures, will translate into differentiated clinical benefits for patients from the marketed neutralizing antibodies.

Arbutus Biopharma and Incyte’s clinical molecules, BMS-202 molecule, and a co-crystal structure of a PD-L1 binder with the PD-L1 dimer
Figure 5. The (pseudo)-C2 symmetric clinical compounds bind the C2-symmetric PD-L1 dimer, resulting in receptor internalization and promising preclinical efficacy PDB: 6VQN

Category 2: Small Molecules Doing What Large Molecules Haven’t

Most SMIMs that have had a major impact on human health fall into this category including the classic NSAIDs and steroids, as well as modern kinase inhibitors for autoimmune diseases. A key differentiating factor between SMIMs and large molecules in this category is the small molecules’ passive permeability that allow them to reach intracellular targets that large molecules typically cannot. With increasing understanding of what drives disease and a large number of potential intracellular targets, SMIMs in this category are sure the keep playing an important role in immunology.

Approved Drugs

In the first half of 2021, the FDA approved two SMIM drugs, voclosporin and ponesimod. Voclosporin was approved for lupus nephritis, which is clinically managed by a combination of a slew of drugs including steroids, cyclophosphamide, mycophenolate mofetil, azathioprine, and calcineurin inhibitors (CNIs) such as tacrolimus or cyclosporin. CNIs have been suggested as long-term therapy; however, serious side effects and poorly predictable PK of these macrocylic drugs have further complicated clinical management of this disease. Thanks to an improved PK profile, voclosporin has shown a much-improved safety profile and thus an increased risk-benefit ratio.

A single methylene group difference from cyclosporin significantly changes the PK profile of voclosporin via target-mediated drug deposition and is likely responsible for the improved efficacy in lupus nephritis. The detailed account of the PK profiles of both cyclic peptides found here, and is a must read for PK aficionados. The 4-fold more potent voclosporin shows increased target-mediated drug deposition (more on TMDD here) in the target tissue at lower doses when compared to cyclosporin. This means that lower plasma drug concentrations are necessary to achieve similar efficacy in these tissues. In essence, introduction of a single methylene group dramatically improves the drug’s therapeutic index by driving the compound to where it’s supposed to be (increasing efficacy) and away from everywhere else (increasing the safety profile).

Ponesimod is an S1P1 agonist and like the other S1P modulators (fingolimod & siponimod (Novartis), ozanimod (BMS)), it effectively antagonizes S1P1 by inducing receptor internalization. It’s the fourth S1P1 modulator approved for MS and chemically stands out with its unusual 2-imino rhodamine core structure (highlighted in color). Rhodanines were once popular in screening decks because they are easily accessible and diversified via condensation of the parent rhodanine with aldehydes. After popping up as screening hits in the literature left and right, the community realized that the high reactivity of rhodanines as Michael acceptors make them PAINs (pan-assay interference compounds) that were to be generally avoided. The Janssen researchers’ success offers a helpful reminder that while heuristics like PAINs are helpful, they’re not a replacement for diligent follow-up.

structures of the drugs cyclosporin, voclosporin, and ponesimod
Figure 6. Voclosporin, a calcineurin inhibitor, and ponesimod, an S1P1 functional antagonist, have been FDA approved for lupus nephritis and multiple sclerosis, respectively

TPL2 Inhibitor

While tremendous progress has been made drugging various kinases for immunology, only one compound that targets the TPL2 kinase has moved into the clinic – GS-4875 (structure not disclosed). Unfortunately, at JP Morgan, Gilead reported that they had stopped development of their TPL2 inhibitor. As TPL2 is a kinase downstream of key inflammatory nodes including the TLRs, IL-1R, and TNF-R, this compound’s recent failure in ulcerative colitis is an unfortunate setback.


TLR7/8 Inhibitor

Earlier this year, EMD Serono disclosed the structure of their clinical TLR7/8 inhibitor (and recent Molecule of the Month) M5049, the most advanced TLR-antagonist SMIM currently in phase II trials for severe symptoms of Covid-19. TLRs are important sensors of pathogen associated molecular patterns (PAMPs) that launch an innate immune response upon activation. TLR overactivity is believed to be a driver of autoimmune diseases such as lupus. The paper does not outline the drug discovery efforts that resulted in identification of M5049 but describes in detail its mode of action, PK, and PD. Two molecules of M5049 (Figure 7, in red) have been found to glue together two TLR8 dimers, acting like hinges in a clam shell and stabilizing the inactive, i.e. open clam, conformer. The compound shows good PK properties (rat t1/2=5 h, 87% F) and high Vss (8.7 L/kg), in line with expectations for a basic, lipophilic amine. M5049 was shown to suppress production of cytokines IL-6 and TNF-α in mice at plasma exposures around 100 nM after IP dosing of a TLR agonist. Furthermore, the compound rescued the phenotype observed in a TLR7 overactive lupus mouse model when dosed 1 mpk QD. Interestingly, efficacy in this model was sustained at plasma exposures below the predicted in vivo IC50, suggesting that higher exposures may be observed in the target tissue as suggested by the high Vss. The authors also observed efficacy of M5049 in a rodent model that is not reported to be TLR7 dependent (IFN-a NZB/W model), which poses questions about our understanding of this disease model, the mode of action of the compound, and the relevance of these two rodent models in general. 

Inhibition and activation of Toll-like receptors (TLRs) and other PAMP sensors, such as NLRP3, cGAS, and STING, are compelling strategies for drug discovery for autoimmune diseases and cancer immunology, respectively. A recent review by Roche colleagues eloquently and concisely summarizes efforts in this field.

chemical structure of M5049 and cocrystal structure of M5049 with TLR8
Figure 7. M5049 stabilizes the inactive TLR8 dimer of dimers, shows efficacy in preclinical lupus models and is currently in the clinic for treatment of COVID

LTA4H inhibitor

The J. Med. Chem.Drug Annotation” for LYS006, Novartis’ clinical leukotriene A4 hydrolase (LTA4H) inhibitor and another recent Molecule of the Month, is full of great science. LTA4H is the enzyme that converts leukotriene A4 into B4, which is a potent chemokine and induced leukocyte activation and adhesion. The team reports a well-executed fragment-based campaign against this target. Because two identified fragments binding in adjacent parts of the active site overlapped nearly perfectly (Figure 8, in color), they could be swiftly merged to turn two micromolar fragments into a 4 nM hit. The team then addressed hERG inhibition by turning the amine in the molecule into a zwitterion (in blue). Given their similar in vitro properties, advanced lead compounds were then differentiated by an ex vivo duration of action PK/PD study, which indicated LYS006 as front runner. There were two particularly fascinating features about this reversible molecule:

  1. The potency measured in a cell-assay depends on the pre-incubation time of the compound, with longer pre-incubation leading to lower IC50’s. This may be attributed to slow distribution of the zwitterions into the cells to engage the target.
  2. PK experiments revealed a non-linear profile consistent with target-mediated drug deposition (more on TMDD here). The molecule preferentially distributes into leukocytes and erythrocytes which express high levels of LTA4H, driven by high binding affinity to the target.

PK/PD experiments showed almost complete depletion of LTB4 in plasma at 0.625 mpk in mice (147 nM plasma exposure) and excellent efficacy in skin inflammation and arthritis mouse models at doses from 1-10 mpk. LYS006 is currently in the clinic for a number of conditions, including UC and NASH.

chemical structures of the fragment hits and the clinical compound LYS006
Figure 8. A fragment screen enabled identification of the LTA4H inhibitor LYS006, which is currently investigated in a host of immunologic diseases.

Hippo Pathway Inhibitor

Finally, a team from Harvard and Scripps recently reported a new member of the Hippo pathway (ANXA2) along with a compound that can inhibit it. At the cellular level, Hippo pathway signaling mediates cell survival, and therefore ultimately controls organ size. Signaling is regulated by how dense the cellular environment is. Activation of Hippo signaling leads to transcription of genes associated with cell proliferation, via the transcription factor YAP. If the cell finds itself wedged between other cells, YAP gets phosphorylated and sequestered to prevent cell proliferation. Pharmacologic control over the Hippo pathway has been explored as strategy in oncology and for tissue regeneration, e.g. via modulation of TEAD or LATS.

The authors now describe the discovery of PY-60 as an activator of YAP-dependent gene expression. The team quickly confirmed efficacy in vivo by showing that topical treatment with PY-60 increases epidermal thickness and keratinocyte number in WT but not in YAP KO mice. Target ID using a photoaffinity probe was initiated and identified the protein ANXA2 as target for PY-60. This protein had previously been shown to interact with the cell-polarity complex and some Hippo signaling proteins. Further extensive experimentation showed that ANXA2 is a cell density-dependent interactor of YAP that can recruit YAP to the cell membrane, thus inactivating gene expression. PY-60 can liberate the YAP-ANXA2 complex from the membrane and increase YAP activity. 

chematic of YAP release from a multi-protein complex by binding of PY-60 to ANXA2
Figure 9. PY-60 was found to induce YAP-dependent gene expression, and target ID showed ANXA2 as cellular target and new member of the Hippo pathway

Conclusion

In H1 2021, we have seen a lot of activity in the “SM engaging LM-target” categories, e.g. IL-17a, TNFα, PD-L1, with several new molecules being disclosed and moving into the clinic. While this field initially had public activity from small biotech companies, we are now seeing big pharma investing in this area as well.

It was interesting to see that the majority of compounds shown here did not initially stem from a traditional high-throughput screening campaign. Instead, fragment screens and DEL screens featured prominently as successful hit-finding techniques. This nicely illustrated the current trend in the industry to cater hit-finding techniques to the target instead of using a one-size-fits-all approach. With these proofs-of-concept that large molecule targets can be drugged with small molecules and powerful hit-finding techniques in hand, it would seem that every LM-project would be well-served considering a small molecule approach as well.

About the Author – Christian Gampe

Christian Gampe received his Diploma in chemistry from the University of Würzburg (Germany) in 2006 and his Dr. Sc. from ETH Zürich under supervision of Prof. Carreira in 2011. After postdoctoral studies at Harvard with Prof. Kahne, Christian joined the Global Discovery Chemistry group at the Novartis Institutes for Biomedical Research in Cambridge, MA, in 2013, where he worked on small molecule drug discovery programs and new modalities alike, publishing on synthetic mRNA modification (here and here), synthetic methods, and medicinal chemistry isosteres. During his time in Boston, Christian was an active member of the Beacon VP biotech investment fund. In 2020, he moved to the west coast and joined the Discovery Chemistry group at Genentech in pursuit of drugs for the undruggable and finding new treatments for autoimmune diseases.


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