IV. Discovery of CXCR3 antagonists substituted with heterocycles as amide surrogates: improved PK, hERG and metabolic profiles.

The structure-human CXCR3 binding affinity relationship of a series of pyridyl/pyrazinyl-piperazinyl-piperidine derivatives were explored with a focus to improve PK, hERG and metabolic profiles. Several small heterocycles were identified as amide surrogates, which minimized many potential metabolite issues. During the course of SAR development, we have observed the additive effect of desirable functional groups to improve hERG and PK profiles which lead to the discovery of many clinically developable CXCR3 antagonists with excellent overall profile.

Multivalent bicyclic peptides are an effective antiviral modality that can potently inhibit SARS-CoV-2.

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses.

II. SAR studies of pyridyl-piperazinyl-piperidine derivatives as CXCR3 chemokine antagonists.

The structure-human CXCR3 binding affinity relationship of a series of pyridyl-piperazinyl-piperidine derivatives was explored. The optimization campaign highlighted the pronounced effect of 2'-piperazine substitution on CXCR3 receptor affinity. Analog 18j, harboring a 2'(S)-ethylpiperazine moiety, exhibited a human CXCR3 IC(50) of 0.2 nM.

III. Identification of novel CXCR3 chemokine receptor antagonists with a pyrazinyl-piperazinyl-piperidine scaffold.

The SAR of a novel pyrazinyl-piperazinyl-piperidine scaffold with CXCR3 receptor antagonist activity was explored. Optimization of the DMPK profile and reduction of hERG inhibition is described. Compound 16e with single-digit CXCR3 affinity, good rat PK and hERG profiles has been identified as a lead for further study.

Fully human antibody VH domains to generate mono and bispecific CAR to target solid tumors.

BACKGROUND: Chimeric antigen receptor (CAR) T cells are effective in B-cell malignancies. However, heterogeneous antigen expression and antigen loss remain important limitations of targeted immunotherapy in solid tumors. Therefore, targeting multiple tumor-associated antigens simultaneously is expected to improve the outcome of CAR-T cell therapies. Due to the instability of single-chain variable fragments, it remains challenging to develop the simultaneous targeting of multiple antigens using traditional single-chain fragment variable (scFv)-based CARs. METHODS: We used Humabody VH domains derived from a transgenic mouse to obtain fully human prostate-specific membrane antigen (PSMA) VH and mesothelin (MSLN) VH sequences and redirect T cell with VH based-CAR. The antitumor activity and mode of action of PSMA VH and MSLN VH were evaluated in vitro and in vivo compared with the traditional scFv-based CARs. RESULTS: Human VH domain-based CAR targeting PSMA and MSLN are stable and functional both in vitro and in vivo. VH modules in the bispecific format are capable of binding their specific target with similar affinity as their monovalent counterparts. Bispecific CARs generated by joining two human antibody VH domains can prevent tumor escape in tumor with heterogeneous antigen expression. CONCLUSIONS: Fully human antibody VH domains can be used to generate functional CAR molecules, and redirected T cells elicit antitumoral responses in solid tumors at least as well as conventional scFv-based CARs. In addition, VH domains can be used to generate bispecific CAR-T cells to simultaneously target two different antigens expressed by tumor cells, and therefore, achieve better tumor control in solid tumors.

Discovery of 2-aminoimidazopyridine adenosine A(2A) receptor antagonists.

A novel series of adenosine A(2A) receptor antagonists was identified by high-throughput screening of an encoded combinatorial compound collection. The initial hits were optimized for A(2A) binding affinity, A(1) selectivity, and in vitro microsomal stability generating orally available 2-aminoimidazo[4,5-b]pyridine-based A(2A) antagonist leads.

Discovery of novel quinolinone adenosine A2B antagonists.

A novel series of quinolinone-based adenosine A(2B) receptor antagonists was identified via high throughput screening of an encoded combinatorial compound collection. Synthesis and assay of a series of analogs highlighted essential structural features of the initial hit. Optimization resulted in an A(2B) antagonist (2i) which exhibited potent activity in a cAMP accumulation assay (5.1 nM) and an IL-8 release assay (0.4 nM).

Diverse human VH antibody fragments with bio-therapeutic properties from the Crescendo Mouse.

We describe the 'Crescendo Mouse', a human VH transgenic platform combining an engineered heavy chain locus with diverse human heavy chain V, D and J genes, a modified mouse Cγ1 gene and complete 3' regulatory region, in a triple knock-out (TKO) mouse background devoid of endogenous immunoglobulin expression. The addition of the engineered heavy chain locus to the TKO mouse restored B cell development, giving rise to functional B cells that responded to immunization with a diverse response that comprised entirely 'heavy chain only' antibodies. Heavy chain variable (VH) domain libraries were rapidly mined using phage display technology, yielding diverse high-affinity human VH that had undergone somatic hypermutation, lacked aggregation and showed enhanced expression in E. coli. The Crescendo Mouse produces human VH fragments, or Humabody® VH, with excellent bio-therapeutic potential, as exemplified here by the generation of antagonistic Humabody® VH specific for human IL17A and IL17RA.

Synthesis and SAR studies of trisubstituted purinones as potent and selective adenosine A2A receptor antagonists.

A series of trisubstituted purinones was synthesized and evaluated as A(2A) receptor antagonists. The A(2A) structure-activity relationships at the three substituted positions were studied and selectivity against the A(1) receptor was investigated. One antagonist 12o exhibits a K(i) of 9nM in an A(2A) binding assay, a K(b) of 18nM in an A(2A) cAMP functional assay, and is 220-fold selective over the A(1) receptor.

Novel CXCR3 antagonists with a piperazinyl-piperidine core.

High-throughput screening of an encoded combinatorial aryl piperazine library led to the identification of a novel series of potent piperazinyl-piperidine based CXCR3 antagonists. Analogs of the initial hit were synthesized via solid and solution phase methods to probe the influence of structure on the CXCR3 binding of these molecules. Various functional groups were found to contribute to the overall potency and essential molecular features were identified.

Intra-abdominal bacterial infection reactivates latent pulmonary cytomegalovirus in immunocompetent mice.

Critically ill surgery patients are susceptible to pulmonary reactivation of latent cytomegalovirus (CMV), but what triggers this reactivation is unknown. Immunosuppression and bacterial sepsis are thought to stimulate reactivation of CMV, and in this study it was hypothesized that immunosuppressive effects of surgery with or without concomitant bacterial infection may reactivate latent CMV. Mice infected with CMV were allowed to develop latent infections. Latently infected mice underwent a laparotomy with cecal ligation and puncture (CLP; n=30), a laparotomy alone (sham; n=10), or no surgery (control; n=5). Lung tissue homogenates were evaluated for viral activity, and, 2 and 3 weeks after CLP, lungs of 7 of 7 and 5 of 5 mice, respectively, showed reactivation of latent CMV. In contrast, lungs from all sham-operated animals and controls showed no viral reactivation. These findings demonstrate that surgery with subsequent intra-abdominal bacterial infection reactivated CMV in lungs of latently infected mice. The mechanism of this reactivation is unknown but likely involves cytokines induced by sepsis.

Occult herpes family viral infections are endemic in critically ill surgical patients.

OBJECTIVE: Herpes family viruses have been recognized as pathogens for many years in immunosuppressed transplant or human immunodeficiency virus patients, but they have garnered little attention as potential pathogens in the nonimmunosuppressed critically ill. The objective of this study was to define the prevalence of and risk factors for development of herpes family virus infection in chronic critically ill surgical patients. DESIGN: Prospective epidemiologic study. SETTING: A 38-bed surgical intensive care unit in a major university hospital. PATIENTS: Nonimmunosuppressed intensive care unit patients in intensive care unit for >/=5 days. INTERVENTIONS: None; patients received no antiviral treatment during the study. MEASUREMENTS AND MAIN RESULTS: Weekly cultures for cytomegalovirus (CMV) and herpes simplex virus, viral serologies, and T-cell counts were performed. The prevalence (95% confidence interval) of positive respiratory cultures for herpes simplex or CMV was 35% (22-49%); 15% (5-25%) cultured positive for CMV, 23% (11-35%) cultured positive for herpes simplex virus, and one patient's respiratory secretions culturing positive for both CMV and herpes simplex virus. The prevalence of CMV viremia was only 5.8% (1-10%). CMV+ patients had longer hospital admissions, intensive care unit admissions, and periods of ventilator dependence than CMV- patients, despite having comparable severity of illness scores. CMV+ patients also had significantly higher numbers of blood transfusions, prevalence of steroid exposure, and prevalence of hepatic dysfunction, and all were immunoglobulin G positive at the beginning of the study. In contrast, herpes simplex virus-positive patients had lengths of hospital admissions, lengths of intensive care unit admissions, and periods of ventilator dependence comparable with patients without viral infections (p >.05). CONCLUSIONS: There is a significant prevalence (22-49%) of occult active herpes family viruses in chronic critically ill surgical patients. The clinical significance of these viral infections is unknown, although CMV+ patients have significantly higher morbidity rates than CMV- patients. Several factors suggest pathogenicity, but further study is needed to define causality.

Small molecule biaryl FSH receptor agonists. Part 1: Lead discovery via encoded combinatorial synthesis.

High-throughput screening of two million compounds in 37 distinct encoded combinatorial libraries using FSH receptor transfected cells provided small molecule agonists such as 1 (EC(50)=3 microM) and 2 (EC(50)=3.9 microM), based on which a focused combinatorial library with a total of 31372 compounds was designed, synthesized, and screened to reveal 72 novel biaryl FSH receptor agonists such as 8a-c as well as a unique combinatorial SAR.